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Heme binds to factor VIII and inhibits its interaction with activated factor IX
Abstract
Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme-binding proteins. Free heme has been proposed to affect coagulation and has been co-purified with the factor VIII (FVIII)-von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions.
To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro.
Heme bound to several sites on FVIII with high apparent affinity. Heme-binding inhibited FVIII procoagulant activity in a dose-dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme-mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme-binding protein known for its protective activity in hemolytic conditions.
Our data identify FVIII as a novel heme-binding protein. Occupation of high affinity heme-binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme-mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.
... Thus, it can interact with other plasma proteins and affect their stability and/or function, which is described for several proinflammatory proteins (e.g., C3, TNF, and RAGE [22][23][24]) [19,25]. Although various effects of heme on the coagulation system were demonstrated, direct heme binding has only been reported for factor VIII (FVIII), fibrinogen, and activated protein C [7,8,[26][27][28][29]. Among these, FVIII has the highest heme-binding affinity with a K D of ~12 nM and ~ 1.9 nM for full-length (FL-FVIII) and Bdomain-deleted FVIII (BDD-FVIII), respectively [27]. ...
... Although various effects of heme on the coagulation system were demonstrated, direct heme binding has only been reported for factor VIII (FVIII), fibrinogen, and activated protein C [7,8,[26][27][28][29]. Among these, FVIII has the highest heme-binding affinity with a K D of ~12 nM and ~ 1.9 nM for full-length (FL-FVIII) and Bdomain-deleted FVIII (BDD-FVIII), respectively [27]. The glycoprotein FVIII circulates as a heterodimer, consisting of a heavy and a light chain, in plasma in a concentration of ~0.5-1 nM [30,31]. ...
... In hemolytic disorders, upregulation of FVIII was reported, thus highlighting FVIII as an interesting target for heme binding [36]. With the proposed 10 heme-binding sites, its heme-binding capacity is the highest among the transiently heme-binding proteins ever reported [25,27]. By applying a combination of spectroscopic methods a potential physiological effect of heme on FVIII was discussed as well [27]. ...
... Both pathways culminate in the common pathway, which finally leads to the formation of a fibrin clot. Superimposition of the HemeThrombKG network and the intrinsic pathway (Reactome, R-HSA-140837) revealed the coagulation factors VIII, IX, and XII, the coagulation inhibitor activated protein C (APC), the adhesive protein von Willebrand factor (VWF), and plasma kallikrein as common nodes, which were described in the past to bind and/or to be affected by heme in vitro and partially in vivo [20,[35][36][37][38][39] (Figure 3A). ...
... Apart from potential procoagulant effects on these proteins of the intrinsic pathway, high-affinity heme binding [K D~1 .9 nM (FVIII) and K D~1 2.7 nM (FVIIIa)] to FVIII(a) abolishes its interaction with FIX, which ultimately leads to the inhibition of the clotting process in vitro [37]. In contrast, persistent FVIII-VWF complex formation and increased binding of this complex to human platelets in the presence of heme directs again towards procoagulant signaling [38]. ...
... VWF itself has been found to show higher expression levels, string formation, and secretion from Weibel-Palade bodies upon incubation of endothelial cells with heme (up to 100 µM; in vitro) and/or heme injection (3.2 µmol/kg) into mice (in vivo) [20,23]. In vitro, VWF was also capable of the protection of FVIII(a) from its heme-driven inhibition [37]. Furthermore, heme upregulates proteases (e.g., MMP9), which regulate VWF digestion, as has been observed in plasma from SCD patients and with heme (up to 60 µM) incubated endothelial cells [39]. ...
Excess labile heme, occurring under hemolytic conditions, displays a versatile modulator in the blood coagulation system. As such, heme provokes prothrombotic states, either by binding to plasma proteins or through interaction with participating cell types. However, despite several independent reports on these effects, apparently contradictory observations and significant knowledge gaps characterize this relationship, which hampers a complete understanding of heme-driven coagulopathies and the development of suitable and specific treatment options. Thus, the computational exploration of the complex network of heme-triggered effects in the blood coagulation system is presented herein. Combining hemostasis- and heme-specific terminology, the knowledge available thus far was curated and modeled in a mechanistic interactome. Further, these data were incorporated in the earlier established heme knowledge graph, "HemeKG", to better comprehend the knowledge surrounding heme biology. Finally, a pathway enrichment analysis of these data provided deep insights into so far unknown links and novel experimental targets within the blood coagulation cascade and platelet activation pathways for further investigation of the prothrombotic nature of heme. In summary, this study allows, for the first time, a detailed network analysis of the effects of heme in the blood coagulation system.
... Approximately at the same time, direct binding of heme in form of hemin was confirmed by UV/vis spectroscopy, revealing a Soret band shift to ~412 nm as well as rather high heme-binding affinities for the full-length (Helixate ® ; KD ~12.7 nM) and the B-domain-deleted (ReFacto ® ; KD ~1.9 nM) version of recombinant FVIII (rFVIII) [241]. Furthermore, a total number of ~10 heme-binding sites with heterogeneous affinity was estimated. ...
... The procoagulant activity of full-length (up to 50%) and B-domain-deleted (up to 51%) FVIII was impaired by heme (5 min preincubation) in a dose-dependent manner, as determined by routine factor X generation assay. In contrast to earlier reports [174], hematoporphyrin showed no impact on FVIII's procoagulant activity [241]. The procoagulant activity of FVIII is caused by its cofactor properties towards factor IXa and thus, the ability to support the generation of active factor X. Repessé and colleagues demonstrated that the interaction of rFVIII with FIX is impaired in the presence of heme (100-fold molar excess; by ~52%), thereby explaining the inhibition of FVIII's procoagulant activity [241]. ...
... In contrast to earlier reports [174], hematoporphyrin showed no impact on FVIII's procoagulant activity [241]. The procoagulant activity of FVIII is caused by its cofactor properties towards factor IXa and thus, the ability to support the generation of active factor X. Repessé and colleagues demonstrated that the interaction of rFVIII with FIX is impaired in the presence of heme (100-fold molar excess; by ~52%), thereby explaining the inhibition of FVIII's procoagulant activity [241]. In contrast, the interaction of FVIII with VWF, platelets and phosphatidyl serine was not altered. ...
Thrombosis is one of the leading causes of death worldwide. As such, it also occurs as one of the major complications in hemolytic diseases, like hemolytic uremic syndrome, hemorrhage and sickle cell disease. Under these conditions, red blood cell lysis finally leads to the release of large amounts of labile heme into the vascular compartment. This, in turn, can trigger oxidative stress and proinflammatory reactions. Moreover, the heme-induced activation of the blood coagulation system was suggested as a mechanism for the initiation of thrombotic events under hemolytic conditions. Studies of heme infusion and subsequent thrombotic reactions support this assumption. Furthermore, several direct effects of heme on different cellular and protein components of the blood coagulation system were reported. However, these effects are controversially discussed or not yet fully understood. This review summarizes the existing reports on heme and its interference in coagulation processes, emphasizing the relevance of considering heme in the context of the treatment of thrombosis in patients with hemolytic disorders.
... In vitro experiments using radiolabeled heme revealed that heme interacts with purified human FVIII [37]. Further binding analyses revealed that this interaction [ 1 9 _ T D $ D I F F ] occurs at high affinity (K D 0.1 nM), exceeding that of heme binding to albumin (K D 10 nM) [42]. Heme significantly reduced FVIII cofactor activity by inhibiting its interaction with activated factor IX (FIXa) in vitro. ...
... and mice, VWF protects FVIII from degradation by proteases in the circulation and controls FVIII catabolism [43]. In vitro studies have shown that heme binding does not impair the interactions of human FVIII with VWF or phospholipids [42]. In addition, upon thrombin activation, heme binding to the FVIII-VWF complex prevented dissociation VWF from FVIII [37]. ...
... In addition, upon thrombin activation, heme binding to the FVIII-VWF complex prevented dissociation VWF from FVIII [37]. Human VWF, however, protects human FVIII very efficiently from in vitro inactivation by heme [42]. ...
Hemolytic diseases such as sickle-cell disease, β-thalassemia, malaria, and autoimmune hemolytic anemia continue to present serious clinical hurdles. In these diseases, lysis of erythrocytes causes the release of hemoglobin and heme into plasma. Extracellular heme has strong proinflammatory potential and activates immune cells and endothelium, thus contributing to disease pathogenesis. Recent studies have revealed that heme can interfere with the function of plasma effector systems such as the coagulation and complement cascades, in addition to the activity of immunoglobulins. Any perturbation in such functions may have severe pathological consequences. In this review we analyze heme interactions with coagulation, complement, and immunoglobulins. Deciphering such interactions to better understand the complex pathogenesis of hemolytic diseases is pivotal.
... In SCD patients, high levels of free heme have been associated with the risk of acute chest syndrome and vasocclusion [12]. On the other hand, heme has also been shown to have anticoagulant effects in patients with porphyria [13], and to inhibit the activity of specific coagulation factors in in vitro studies [14,15]. Accordingly, it was recently hypothesized that heme could trigger coagulation initiation, while also limiting its propagation [16]. ...
... In contrast, intravenous hematin (which is very similar to heme preparations used in the former experiments) used in patients with porphyria is not associated with a systemic hypercoagulable state, and has been previously associated with prolongation of coagulation times and to reduced levels of coagulation factors V and VIII [41,42]. Moreover, heme presents complex effects on different compartments of hemostasis that include platelet activation [43] and interactions with VIII [15] and fibrinogen [44] of yet unknown physiological significance. Together, these results illustrate the challenges of addressing the effects of heme on hemostasis, and highlight the importance of methods able to explore its net global effect on this process. ...
Heme has been characterized as potent trigger of inflammation. In hemostasis, although heme has been shown to both induce and inhibit different compartments of hemostasis, its net effect on the hemostatic balance, and the biological relevance of these effects remain to be determined. Herein we evaluated the effect of heme on hemostasis using a global assay able to generate clinically relevant data in several other complex hemostatic diseases. Citrated whole blood samples from healthy participants were stimulated by heme or vehicle and incubated for 4h at 37°C. Rotational thromboelastometry was immediately performed. The participation of tissue factor in coagulation activation was evaluated using inhibitory antibody. Heme was able of inducing ex vivo coagulation activation in whole blood, affecting predominantly parameters associated with the initial phases of clot formation. This activation effect was at least partially dependent on hematopoietic tissue factor, since the effects of heme were partially abrogated by the inhibition of human tissue factor. In conclusion, using a global hemostasis assay, our study confirmed that heme is able to activate coagulation in whole blood, in a tissue factor-dependent way. These findings could explain the disturbance in hemostatic balance observed in conditions associated with the release of heme such as sickle cell disease.
... Recently, it has been shown that heme can bind FVIII in vitro and inhibit its procoagulant properties. 34 However, heme could only inhibit the procoagulant functions of FVIII when administered in a molar excess and in the absence of von Willebrand Factor (VWF) which is a natural chaperone of FVIII. 34 This suggests that an excess of heme might have an anticoagulant effect, but this remains to be tested in vivo. ...
... 34 However, heme could only inhibit the procoagulant functions of FVIII when administered in a molar excess and in the absence of von Willebrand Factor (VWF) which is a natural chaperone of FVIII. 34 This suggests that an excess of heme might have an anticoagulant effect, but this remains to be tested in vivo. However, Belcher and colleagues recently demonstrated that heme rapidly triggers the release of VWF from EC both in vitro and in vivo, 20 which would diminish the anticoagulant properties of heme and support the procoagulant role that we observed. ...
An excess of free heme is present in the blood during many types of hemolytic anemia. This has been linked to organ damage caused by heme-mediated oxidative stress and vascular inflammation. We investigated the mechanism of heme-induced coagulation activation in vivo. Heme caused coagulation activation in wild type mice that was attenuated by an anti-tissue factor antibody and in mice expressing low levels of tissue factor. In contrast, neither factor XI deletion nor inhibition of factor XIIa-mediated factor XI activation reduced heme-induced coagulation activation, suggesting that the intrinsic coagulation pathway is not involved. We investigated the source of tissue factor in heme-induced coagulation activation. Heme increased the procoagulant activity of mouse macrophages and human PBMCs. Tissue factor-positive staining was observed on leukocytes isolated from the blood of heme-treated mice but not on endothelial cells in the lungs. Furthermore, heme increased vascular permeability in the mouse lungs, kidney and heart. Deletion of tissue factor from either myeloid cells, hematopoietic and endothelial cells, or inhibition of tissue factor expressed by non-hematopoietic cells did not reduce heme-induced coagulation activation. However, heme-induced activation of coagulation was abolished when both non-hematopoietic and hematopoietic cell tissue factor was inhibited. Finally, we demonstrated that coagulation activation was partially attenuated in sickle cell mice treated with recombinant hemopexin to neutralize free heme. Our results indicate that heme promotes tissue factor-dependent coagulation activation and induces tissue factor expression on leukocytes in vivo. We also demonstrated that free heme may contribute to thrombin generation in a mouse model of sickle cell disease.
Copyright © 2015, Ferrata Storti Foundation.
... Spontaneous autooxidation of ferrous (Fe2+) HGB to ferric (Fe3+) HGB, superoxide, and ferryl (Fe4+) HGB releases free heme and ROS, which in turn induce bilirubin generation as a result of heme degradation by heme oxygenase and biliverdin reductase (Gram et al., 2013). HGB and free heme are involved in the humoral compartment of the innate immune system, immunoglobulins, and complement system regulation (Repessé et al., 2012;Anderson et al., 2018), and their accumulation in plasma enhances the production of ROS by activating the Toll-like receptor 4 signaling pathway (Nader et al., 2020). In this regard, we have previously reported that the plasma concentration of bilirubin was at the highest level in early lactation dairy cows . ...
Oxidative stress and inflammation, as natural parts of metabolic adaptations during the transition from late gestation to early lactation, are critical indicators of dairy cows' metabolic health. This study was designed to investigate the effects of abomasal infusion of essential fatty acids (EFA), particularly α-linolenic acid, and conjugated linoleic acid (CLA) on plasma, erythrocyte, and liver markers of oxidative stress in dairy cows during the transition period. Rumen-cannulated German Holstein cows (n = 38) in their second lactation (11,101 ± 1,118 kg milk/305 d, mean ± standard deviation) were abomasally infused with one of the following treatments from d -63 antepartum until d 63 postpartum (PP): CTRL (n = 9; 76 g/d coconut oil); EFA (n = 9; 78 g/d linseed plus 4 g/d safflower oil); CLA (n = 10; isomers cis-9,trans-11 and trans-10,cis-12 CLA; 38 g/d); and EFA+CLA (n = 10; 120 g/d). Hematological parameters as well as markers of oxidative status were measured in plasma, erythrocytes, and liver before and after calving. Immunohematological parameters, including erythrocyte number, hematocrit, hemoglobin, mean corpuscular hemoglobin, leukocytes, and basophils, were affected by time, and their peak levels were observed on the day after calving. The oxidative stress markers glutathione peroxidase 1 and reactive oxygen metabolites in plasma and erythrocytes were both affected by time, exhibiting the highest levels on d 1 PP, whereas β-carotene, retinol, and tocopherol were at their lowest levels at the same time. Immunohematological parameters were only marginally affected by fatty acid treatment in a time-dependent manner. As such, lymphocyte and atypical lymphocyte counts were both significantly highest in the groups that received EFA at d 1 PP. Moreover, EFA supplementation increased the mean corpuscular volume and showed a trend for induction of mean corpuscular hemoglobin compared with the CLA group during the transition period. The PP mean thrombocyte volume was higher in the EFA than in the CLA group (except for d 28) and both EFA and CLA reduced number of thrombocytes and thrombocrit at distinct time points. Hepatic mRNA abundance of markers related to oxidative status, including glutathione peroxidase (GPX-1) and catalase (CAT), was lower (P < 0.05) in EFA-treated than non-EFA-treated cows at d 28 PP. Dairy cows at the onset of lactation were characterized by induced markers of both oxidative stress and inflammation. Supplementing EFA and CLA had minor and time-dependent effects on markers of oxidative stress in plasma, erythrocytes, and liver. A comparison of EFA supplementation with CLA or CTRL showed higher immunohematological response at d 1 PP and lower hepatic antioxidant levels by d 28 PP. Supplementation with EFA+CLA had only a minor effect on oxidative markers, which were more similar to those with the EFA treatment. Altogether, despite the time-dependent differences, the current findings show only minor effects of EFA and CLA supplementation in the prevention of early lactation-induced oxidative stress.
... 83 In addition to its aforementioned stimulation of TF expression by endothelial cells, in vivo administration of heme also stimulates TF production in leukocytes 33 and may activate coagulation, at high concentrations, via stimulation of TF and the extrinsic pathway. 33,84 In contrast, free heme binds to several sites on FVIII with high affinity and at high concentrations can inhibit its activity in vitro; 85 FVIII-heme interactions can, however, be inhibited by vWF, whose release from endothelial cells is also stimulated by heme. 68 In turn, multimeric vWF can be bound by extracellular hemoglobin and bilirubin (a degradation product of heme), impairing its enzymatic cleavage by the ADAMTS-13 protein, resulting in increased prothombotic activity. ...
Sickle cell disease (SCD) is an inherited hemoglobinopathy that is caused by the presence of abnormal hemoglobin S (HbS) in red blood cells, leading to alterations in red cell properties and shape, as the result of HbS dexoygenation and subsequent polymerization. SCD pathophysiology is characterized by chronic inflammatory processes, triggered by hemolytic and vaso-occlusive events, which lead to the varied complications, organ damage and elevated mortality seen in individuals with the disease. In association with activation of the endothelium and leukocytes, hemostatic alterations and thrombotic events are well-documented in SCD. Here we discuss the role for inflammatory pathways in modulating coagulation and inducing platelet activation in SCD, due to tissue factor activation, adhesion molecule expression, inflammatory mediator production and the induction of innate immune responses, amongst other mechanisms. Thromboinflammatory pathways may play a significant role in some of the major complications of SCD, such as stroke, venous thromboembolism and possibly acute chest syndrome, besides exacerbating the chronic inflammation and cellular interactions that trigger vaso-occlusion, ischemia-reperfusion processes, and eventually organ damage.
... The latter applies to C3 as well, yet molecular docking suggested a binding site close to the functionally important thioester bond [24]. Furthermore, heme influences factor VIII and fibrinogen in seemingly contradictory fashion, but partially due to the lack of structural information this dissonance has not been unraveled yet [26][27][28]. ...
Background:
The notion of heme as a regulator of many physiological processes via transient binding to proteins is one that is recently being acknowledged. The broad spectrum of the effects of heme makes it important to identify further heme-regulated proteins to understand physiological and pathological processes. Moreover, several proteins were shown to be functionally regulated by interaction with heme, yet, for some of them the heme-binding site(s) remain unknown. The presented application HeMoQuest enables identification and qualitative evaluation of such heme-binding motifs from protein sequences.
Results:
We present HeMoQuest, an online interface (http://bit.ly/hemoquest) to algorithms that provide the user with two distinct qualitative benefits. First, our implementation rapidly detects transient heme binding to nonapeptide motifs from protein sequences provided as input. Additionally, the potential of each predicted motif to bind heme is qualitatively gauged by assigning binding affinities predicted by an ensemble learning implementation, trained on experimentally determined binding affinity data. Extensive testing of our implementation on both existing and new manually curated datasets reveal that our method produces an unprecedented level of accuracy (92%) in identifying those residues assigned "heme binding" in all of the datasets used. Next, the machine learning implementation for the prediction and qualitative assignment of binding affinities to the predicted motifs achieved 71% accuracy on our data.
Conclusions:
Heme plays a crucial role as a regulatory molecule exerting functional consequences via transient binding to surfaces of target proteins. HeMoQuest is designed to address this imperative need for a computational approach that enables rapid detection of heme-binding motifs from protein datasets. While most existing implementations attempt to predict sites of permanent heme binding, this application is to the best of our knowledge, the first of its kind to address the significance of predicting transient heme binding to proteins.
... The immunogenicity of the oxidized FVIII was, however, not reduced when mice were treated with NAC, suggesting that NAC does not merely affect the immune response but may act directly by preventing FVIII oxidation. An earlier study also identified FVIII as a heme-binding protein (122). The binding of heme to FVIII resulted in a partial loss of pro-coagulant activity, which was at least in part consecutive to a reduced capacity of FVIII to interact with activated FIX. ...
Development of neutralizing antibodies against biotherapeutic agents administered to prevent or treat various clinical conditions is a longstanding and growing problem faced by patients, medical providers and pharmaceutical companies. The hemophilia A community has deep experience with attempting to manage such deleterious immune responses, as the lifesaving protein drug factor VIII (FVIII) has been in use for decades. Hemophilia A is a bleeding disorder caused by genetic mutations that result in absent or dysfunctional FVIII. Prophylactic treatment consists of regular intravenous FVIII infusions. Unfortunately, 1/4 to 1/3 of patients develop neutralizing anti-FVIII antibodies, referred to clinically as “inhibitors,” which result in a serious bleeding diathesis. Until recently, the only therapeutic option for these patients was “Immune Tolerance Induction,” consisting of intensive FVIII administration, which is extraordinarily expensive and fails in ~30% of cases. There has been tremendous recent progress in developing novel potential clinical alternatives for the treatment of hemophilia A, ranging from encouraging results of gene therapy trials, to use of other hemostatic agents (either promoting coagulation or slowing down anti-coagulant or fibrinolytic pathways) to “bypass” the need for FVIII or supplement FVIII replacement therapy. Although these approaches are promising, there is widespread agreement that preventing or reversing inhibitors remains a high priority. Risk profiles of novel therapies are still unknown or incomplete, and FVIII will likely continue to be considered the optimal hemostatic agent to support surgery and manage trauma, or to combine with other therapies. We describe here recent exciting studies, most still pre-clinical, that address FVIII immunogenicity and suggest novel interventions to prevent or reverse inhibitor development. Studies of FVIII uptake, processing and presentation on antigen-presenting cells, epitope mapping, and the roles of complement, heme, von Willebrand factor, glycans, and the microbiome in FVIII immunogenicity are elucidating mechanisms of primary and secondary immune responses and suggesting additional novel targets. Promising tolerogenic therapies include development of FVIII-Fc fusion proteins, nanoparticle-based therapies, oral tolerance, and engineering of regulatory or cytotoxic T cells to render them FVIII-specific. Importantly, these studies are highly applicable to other scenarios where establishing immune tolerance to a defined antigen is a clinical priority.
... This could be related to the mobilization of the Weibel-Palade bodies, resulting in surface expression of P-selectin and release of ultra-large VWF multimers [79,130]. Heme induces endothelial tissue factor expression [148,149]; it activates platelets, but inhibits thrombin and plasmin; heme also binds to factor VIII and inhibits its interaction with activated coagulation factor IX [150]. Taken together, then, the data in the literature suggest that the release of heme initiates the extrinsic coagulation pathway via the upregulation of tissue factor in ECs and leukocytes, but subsequently blocks the propagation of coagulation by inhibiting FVIII and FV, and by inhibiting the conversion of fibrinogen into fibrin and hence the formation of fibrin clots. ...
Vascular diseases are multifactorial, often requiring multiple challenges, or ‘hits’, for their initiation. Intra-vascular hemolysis illustrates well the multiple-hit theory where a first event lyses red blood cells, releasing hemolysis-derived products, in particular cell-free heme which is highly toxic for the endothelium. Physiologically, hemolysis derived-products are rapidly neutralized by numerous defense systems, including haptoglobin and hemopexin which scavenge hemoglobin and heme, respectively. Likewise, cellular defense mechanisms are involved, including heme-oxygenase 1 upregulation which metabolizes heme. However, in cases of intra-vascular hemolysis, those systems are overwhelmed. Heme exerts toxic effects by acting as a damage-associated molecular pattern and promoting, together with hemoglobin, nitric oxide scavenging and ROS production. In addition, it activates the complement and the coagulation systems. Together, these processes lead to endothelial cell injury which triggers pro-thrombotic and pro-inflammatory phenotypes. Moreover, among endothelial cells, glomerular ones display a particular susceptibility explained by a weaker capacity to counteract hemolysis injury. In this review, we illustrate the ‘multiple-hit’ theory through the example of intra-vascular hemolysis, with a particular focus on cell-free heme, and we advance hypotheses explaining the glomerular susceptibility observed in hemolytic diseases. Finally, we describe therapeutic options for reducing endothelial injury in hemolytic diseases.
... Results from our laboratory have demonstrated that heme is also capable of interacting with FVIII. This interaction results in a decreased pro-coagulant activity of FVIII due to the inhibition of the interaction of FVIII with activated FIX (FIXa) (annexe 1, (Repesse et al., 2012)). However, the presence of heme on the FVIII molecule did not alter the immunogenicity of FVIII. ...
Hemophilia A is a rare X-linked hemorrhagic disease consecutive to the lack of functional pro-coagulant factor VIII (FVIII). In patients with the severe form of hemophilia A, uncontrolled hemorrhages increase the morbidity and affect the quality of life. To prevent or treat bleeding episodes, therapeutic FVIII is administered intravenously. However, an anti-FVIII immune response develops in 5 to 30% of the patients. The anti-FVIII immune response is characterized by the development of anti-FVIII IgG antibodies that inhibit FVIII activity. These antibodies, referred to as “FVIII inhibitors” represent the major complication in the treatment of hemophilia A patients. Several risk factors have been suggested or proposed to predispose to the development of FVIII inhibitors. Amongst them, inspired by the “danger” theory, the presence of inflammatory mediators released upon bleeding episodes is thought to adjuvant the anti-FVIII immune response. Bleeding episodes trigger several pro and anti-inflammatory mechanisms. Thus, damage to the endothelium triggers the recruitment and activation of effector cells from the innate and adaptive compartments of the immune system. During my PhD, I investigated the role of bleedings in the development of the anti-FVIII immune response. Thus, I discovered an association between a polymorphism in the promoter region of a gene that is induced in response to bleedings, and the presence of FVIII inhibitors in a cohort of patients with severe hemophilia A. I followed the development of the immune response to therapeutic FVIII in a mouse model that mimics the localized bleedings found in patients with severe hemophilia A. Ultimately, I characterized the effects of reactive oxygen species (ROS) that are potentially released at the bleeding site in view of the structure, function and immunogenicity of the FVIII molecule. The results I obtained during my PhD demonstrate that bleedings are not associated with a higher risk for FVIII inhibitor development in the commonly used mouse model of severe FVIII deficiency. Additionally, I demonstrated an association between a polymorphism in the promoter of the HMOX1 gene and the presence of FVIII inhibitors. In parallel, I report for the first time an exacerbated oxidative status in FVIII-deficient mice as compared to control mice, and demonstrate that the control of the oxidative status in vivo reduces the anti-FVIII immune response. Conversely, the exposure of FVIII to ROS ex vivo increases the immunogenicity of the FVIII molecule. Taken together, my results suggest that, although the pro-inflammatory molecules released upon bleeding may positively affect the anti-FVIII immune response, several strong anti-inflammatory compounds are generated in vivo that dampen the potential adjuvant effect of bleedings. Ultimately, these results highlight the complex balance between the pro and anti-inflammatory mediators generated upon bleeding and their effect on the anti-FVIII immune response.
... A useful approach, therefore, is to prevent inhibitor development in the first phase of the immune response. Some potential strategies for inhibitor prevention may include [38][39][40][41][42] as not simple to achieve in practice). ...
Immune tolerance induction (ITI) therapy in patients with haemophilia A and inhibitors constitutes a huge burden for affected patients and families and poses a large economic burden for a chronic disease. Concerted research efforts are attempting to optimize the therapeutic approach to the prevention and eradication of inhibitors. The Italian ITI Registry has provided data on 110 patients who completed ITI therapy as at July 2013. Analysis of independent predictors of success showed that, together with previously recognized factors – namely inhibitor titre prior to ITI, historical peak titre and peak titre on ITI – the type of causative FVIII gene mutation also contributes to the identification of patients with good prognosis and may be useful to optimize candidate selection and treatment regimens. Numerous studies have demonstrated that inhibitor reactivity against different FVIII products varies and is lower against concentrates containing von Willebrand factor (VWF). An Italian study compared inhibitor titres against a panel of FVIII concentrates in vitro and correlated titres with the capacity to inhibit maximum thrombin generation as measured by the thrombin generation assay (TGA). Observations led to the design of the PredictTGA study which aims to correlate TGA results with epitope specificity, inhibitor reactivity against different FVIII concentrates and clinical data in inhibitor patients receiving FVIII in the context of ITI or as prophylactic/on demand treatment. At the immunological level, it is known that T cells drive inhibitor development and that B cells secrete FVIII-specific antibodies. As understanding increases about the immunological response in ITI, it is becoming apparent that modulation of T-cell- and B-cell-mediated responses offers a range of potential new and specific approaches to prevent and eliminate inhibitors as well as individualize ITI therapy.
Heme is a primordial macrocycle upon which most aerobic life on Earth depends. It is essential to the survival and health of nearly all cells, functioning as a prosthetic group for oxygen‐carrying proteins and enzymes involved in oxidation/reduction and electron transport reactions. Heme is essential for the function of numerous hemoproteins and has numerous other roles in the biochemistry of life. In mammals, heme is synthesised from glycine, succinyl‐CoA, and ferrous iron in a series of eight steps. The first and normally rate‐controlling step is catalysed by 5‐aminolevulinate synthase (ALAS), which has two forms: ALAS1 is the housekeeping form with highly variable expression, depending upon the supply of the end‐product heme, which acts to repress its activity; ALAS2 is the erythroid form, which is regulated chiefly by the adequacy of iron for erythroid haemoglobin synthesis. Abnormalities in the several enzymes of the heme synthetic pathway, most of which are inherited partial enzyme deficiencies, give rise to rare diseases called porphyrias. The existence and role of heme importers and exporters in mammals have been debated. Recent evidence established the presence of heme transporters. Such transporters are important for the transfer of heme from mitochondria, where the penultimate and ultimate steps of heme synthesis occur, and for the transfer of heme from cytoplasm to other cellular organelles. Several chaperones of heme and iron are known and important for cell health. Heme and iron, although promoters of oxidative stress and potentially toxic, are essential cofactors for cellular energy production and oxygenation.
Heme is a double-edged sword. On the one hand, it has a pivotal role as a prosthetic group of hemoproteins in many biological processes ranging from oxygen transport and storage to miRNA processing. On the other hand, heme can transiently associate with proteins, thereby regulating biochemical pathways. During hemolysis, excess heme, which is released into the plasma, can bind to proteins and regulate their activity and function. The role of heme in these processes is under-investigated, with one problem being the lack of knowledge concerning recognition mechanisms for the initial association of heme with the target protein and the formation of the resulting complex. A specific heme-binding sequence motif is a prerequisite for such complex formation. Although numerous short signature sequences indicating a particular protein function are known, a comprehensive analysis of the heme-binding motifs (HBMs) which have been identified in proteins, concerning specific patterns and structural peculiarities, is missing. In this report, we focus on the evaluation of known mammalian heme-regulated proteins concerning specific recognition and structural patterns in their HBMs. The Cys-Pro dipeptide motifs are particularly emphasized because of their more frequent occurrence. This analysis presents a comparative insight into the sequence and structural anomalies observed during transient heme binding, and consequently, in the regulation of the relevant protein.
Independent of etiology, hemolytic diseases are associated with thrombosis, inflammation and immune dysregulation, all together contributing to organ damage and poor outcome. Beyond anemia and the loss of the anti-inflammatory functions of red blood cells, hemolysis leads to the release of damage-associated molecular patterns including ADP, hemoglobin, and heme, which act through multiple receptors and signaling pathways fostering a hyperinflammatory-y and hypercoagulable state. Extracellular-free heme is promiscuous damage-associated molecular pattern capable of triggering oxido-inflammatory and thrombotic events by inducing the activation of platelets, endothelial and innate cells as well as the coagulation and complement cascades. In this review, we discuss the main mechanisms by which hemolysis and, in particular, heme, drive this thrombo-inflammatory milieu and discuss the consequences of hemolysis on the host response to secondary infections.
Intravascular hemolysis occurs in diverse pathological conditions. Extracellular hemoglobin and heme have strong pro-oxidative and pro-inflammatory potentials that can contribute to the pathology of hemolytic diseases. However, many of the effects of extracellular hemoglobin and heme in hemolytic diseases are still not well understood. Here we demonstrate that oxidized hemoglobin (methemoglobin) can modify the antigen-binding characteristics of human immunoglobulins. Thus, incubation of polyclonal or some monoclonal human IgG in the presence of methemoglobin results in an appearance of binding reactivities towards distinct unrelated self-proteins, including the protein constituent of hemoglobin i.e., globin. We demonstrate that a transfer of heme from methemoglobin to IgG is indispensable for this acquisition of antibody polyreactivity. Our data also show that only oxidized form of hemoglobin have the capacity to induce polyreactivity of antibodies. Site-directed mutagenesis of a heme-sensitive human monoclonal IgG1 reveals details about the mechanism of methemoglobin-induced antigen-binding polyreactivity. Further here we assess the kinetics and thermodynamics of interaction of a heme-induced polyreactive human antibody with hemoglobin and myoglobin. Taken together presented data contribute to a better understanding of the functions of extracellular hemoglobin in the context of hemolytic diseases.
Heme regulates important biological processes by transient interactions with many human proteins. The goal of the present study was to assess extends of protein binding promiscuity of heme. To this end we evaluated interaction of heme with >9000 human proteins. Heme manifested high binding promiscuity by binding to most of the proteins in the array. Nevertheless, some proteins have outstanding heme binding capacity. Bioinformatics analyses revealed that apart from typical haemoproteins, these proteins are frequently involved in metal binding or have the potential to recognize DNA. This study can contribute for understanding the regulatory functions of labile heme.
Aims:
In hemolysis, which is accompanied by increased levels of labile redox-active heme and is often associated with hemostatic abnormalities, a decreased activity of activated protein C (APC) is routinely detected. APC is a versatile enzyme that exerts its anticoagulant function through inactivation of clotting factors Va and VIIIa. APC has not been demonstrated to be affected by heme as described for other clotting factors and, thus, is a subject of investigation.
Results:
We report the interaction of heme with APC and its impact on the protein function by employing spectroscopic and physiologically relevant methods. Binding of heme to APC results in inhibition of its amidolytic and anticoagulant activity, increase of the peroxidase-like activity of heme, and protection of human umbilical vein endothelial cells from heme-induced hyperpermeability. To define the sites that are responsible for heme binding, we mapped the surface of APC for potential heme-binding motifs. T285GWGYHSSR293 and W387IHGHIRDK395, both located on the basic exosite, turned out as potential heme-binding sites. Molecular docking employing a homology model of full-length APC indicated Tyr289 and His391 as the Fe(III)-coordinating amino acids.
Innovation:
The results strongly suggest that hemolysis-derived heme may directly influence the protein C pathway through binding to APC, conceivably explaining the decreased activity of APC under hemolytic conditions. Further, these results extend our understanding of heme as a multifaceted effector molecule within coagulation and may allow for an improved understanding of disease development in hemostasis under hemolytic conditions.
Conclusion:
Our study identifies APC as a heme-binding protein and provides insights into the functional consequences.
Deviant levels of available heme and related molecules can result from pathological situations such as impaired heme biosynthesis or increased hemolysis as a consequence of vascular trauma or bacterial infections. Heme-related biological processes are affected by these situations, and it is essential to fully understand the underlying mechanisms. While heme has long been known as an important prosthetic group of various proteins, its function as a regulatory and signaling molecule is poorly understood. Diseases such as porphyria are caused by impaired heme metabolism, and heme itself might be used as a drug in order to downregulate its own biosynthesis. In addition, heme-driven side effects and symptoms emerging from heme-related pathological conditions are not fully comprehended and thus impede adequate medical treatment. Several heme-regulated proteins have been identified in the past decades, however, the molecular basis of transient heme-protein interactions remains to be explored. Herein, we summarize the results of an in-depth analysis of heme binding to proteins, which revealed specific binding modes and affinities depending on the amino acid sequence. Evaluating the binding behavior of a plethora of heme-peptide complexes resulted in the implementation of a prediction tool (SeqD-HBM) for heme-binding motifs, which eventually led and will perspectively lead to the identification and verification of so far unknown heme-regulated proteins. This systematic approach resulted in a broader picture of the alternative functions of heme as a regulator of proteins. However, knowledge on heme regulation of proteins is still a bottomless barrel that leaves much scope for future research and development.
Hemoglobin (Hb), a metalloprotein in Red Blood Cells (RBC), is highly
conserved across all vertebrates and in some invertebrates. Each RBC
houses approximately 250 million Hb molecules which serve as transporters
of oxygen from the lungs to the tissues and carbon dioxide from the tissues
to the lungs. However in the event of intravascular hemolysis, lysed RBCs
release Hb into the circulation. A large fraction of this extracellular Hb forms
complex with Haptoglobin (Hp) and is cleared from circulation by CD163,
a macrophage surface receptor. Heme, a by-product of Hb oxidation, upon
binding to Hemopexin (Hpx) is cleared by phagocytes in a similar manner. Once
the scavenging capacity of Hp and Hpx reaches the saturation point, residual
cell-free Hb and heme ensue into vascular dysfunctions and thrombogenic
complications. Cell-free Hb also limits the bioavailability of Nitric Oxide (NO)
and Carbon monoxide (CO), signalling molecules crucial for the maintainance
of vascular architecture and hemostasis. Besides, studies have described that
the oxidized form of Hb such as metHb or by-product(s) of heme are highly
redox reactive and cause oxidative damage to surrounding tissues, resulting in
initiation of pro-inflammatory and pro-coagulative cascades. On the other hand,
cell-free Hb also manifests cytoprotective effect through modulation of Heme
Oxygenase 1 (HO-1), ferritin and anti-oxidative response gene(s). In hemolytic
diseases, accumulation of excess free Hb in plasma triggers pathophysiological
events that are associated with adverse clinical outcomes such as acute and
chronic vascular disease, inflammation, thrombosis and renal impairment.
Cell-free Hb also activates platelets via both direct and/or indirect mechanisms
and promotes clinical events such as thrombosis and hypercoagulation. Apart
from these complications, cell-free Hb also modulates phenotype and function
of cells of both arms of the immune system- innate and adaptive. Monocytes,
macrophages and neutrophils, which play important role in the first line of
defense, are significantly affected when exposed to free Hb or heme. Similarly,
the adaptive immune cells such as T and B lymphocytes also show altered
response under hemolytic conditions. It has been demonstrated that patients
with haemolytic disorders harbour altered immune cytome and response. This
review briefly describes the current understanding of the effect of cell-free Hb on
immune response in haemolytic disorders.
Keywords: Free haemoglobin; Heme; Monocytes; Macrophages;
Neutrophils; B and T lymphocytes; Hemolytic diseases
More than 20 years of research on heme as a temporary effector molecule of proteins have revealed its widespread impact on virtually all primary functions in the human organism. As our understanding of this influence is still growing, a comprehensive overview of compiled data will give fresh impetus for creativity and developing new strategies in heme-related research. From known data concerning heme-regulated proteins and their involvement in the development of diseases, we provide concise information of FeII/III heme as a regulator and the availability of “regulatory heme”. The latter is dependent on the balance between free and bound FeII/III heme, here termed “hemeostasis”. Imbalance of this system can lead to the development of diseases that were not always attributed to this small molecule. Diseases such as cancer or Alzheimer's disease highlight the reawakened interest in heme, whose function was previously believed to be completely understood.
Polyreactive antibodies represent a significant fraction of immune repertoires and play an important role in the immune defense and immune homeostasis. Polyreactive B-cell receptors (BCR), however, are frequently expressed by B-cell lymphomas. It was suggested that polyreactive BCR on lymphoma cells might deliver stimulation signals by binding to various endogenous or exogenous antigens, thus promoting the survival of the malignant cells. In addition to natural polyreactive antibodies, immune repertoires contain antibodies that acquire polyreactivity after exposure to different redox-active substances such as reactive oxygen species, iron ions and heme. Here, we demonstrate that an antibody cloned from a patient's splenic marginal zone B-cell lymphoma acquires physiologically relevant binding affinity to various autoantigens following exposure to heme. We elucidated the mechanisms underlying polyreactive antigen binding. The results obtained in this study imply that antigen-binding receptors expressed on some malignant cells acquire polyreactivity after exposure to redox substances that are released at sites of inflammation or as a result of cellular damage. The acquisition of novel BCR specificities under hemolytic or inflammatory conditions may play an important role in the physiopathology of certain B-cell malignancies.
AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6(th) coordination bond with His(E7).
Recombinant AHSP, WT α-Hb:AHSP and α-Hb(HE7Q):AHSP complexes were expressed in E. coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex, were measured by time-resolved absorption spectroscopy.
AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex, reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.
α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis. Significance The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.
Von Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life.
Standardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15-44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated.
FVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5+/-2.6 h versus 14.3+/-3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%).
Our approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.
Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by approximately 95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
The effective activation of factor X by factor IXa requires the co-factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303-2317, 2305-2332, and 2308-2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318-2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid.
Hematin, the drug used for acute porphyric attacks, has been shown to cause disturbances in hemostasis, mainly because of its degradation products. Lately a new heme compound, heme arginate, has been developed for the treatment of porphyrias. In experimental animal studies as well as in clinical use it has proved to be well tolerated. To find out whether heme arginate has any effects on hemostasis we have studied a number of parameters of coagulation and fibrinolysis after a heme arginate infusion in seven healthy volunteers. All parameters studied remained practically unchanged except the coagulation factor X, which showed a transient, insignificant decrease during the maximal heme concentration. We believe that the lack of side effects is due to a better stability of heme arginate, the degradation rates being 1% for heme arginate and 61% for hematin in four hours. Our data favor the use of heme arginate in acute porphyrias as well as in other deficiency states of heme.
Hemin, in the presence of 2-mercaptoethanol and oxygen, catalyzes the selective degradation of heme-binding proteins to small peptide fragments. Among the proteins examined, the heme-binding protein of rabbit serum (HBP-93) proved to be unusually sensitive. Myoglobin also exhibited considerable sensitivity whereas hemopexin and bovine serum albumin were only slightly susceptible to this degradative action of hemin. The reaction with HBP-93 depended upon coordination of the protein with hemin, was optimal at pH 6.5 and increased 4-fold as the temperature was elevated from 10 to 60 degrees C. The requirement for both oxygen and the reducing agent, 2-mercaptoethanol, and the partial protection of HBP-93 to degradation by catalase, superoxide dismutase, mannitol, and thiourea suggest the involvement of reduced oxygen species in the reaction. A possible role for the heme-mediated degradation of proteins in cell differentiation and other biological responses is discussed.
The deduced amino acid sequence of human factor VIII, obtained from the DNA sequence, predicts a mature polypeptide of 2,332 amino acids containing a triplicated domain structure. The polypeptide has 35% sequence homology with the copper-binding plasma protein, ceruloplasmin. Determination of the thrombin cleavage sites in plasma-derived factor VIII polypeptides allows prediction of the domains involved in the associated activation and inactivation of the protein.
Synthetic membranes of phosphatidylcholine require inclusion of at least 5% phosphatidylserine (Ptd-L-Ser) to form binding
sites for factor VIII. The relatively high requirement for Ptd-L-Ser suggests that stimulated platelets may contain another
membrane constituent that enhances expression of factor VIII-binding sites. We report that phosphatidylethanolamine (PE),
which is exposed in concert with Ptd-L-Ser in the course of platelet stimulation, induces high affinity binding sites for
factor VIII on synthetic membranes containing 1-15% Ptd-L-Ser. The affinity of factor VIII for binding sites on membranes
of Ptd-L-Ser/PE/phosphatidylcholine in a 4:20:76 ratio was 10.2 ± 3.5 nM with 180 ± 33 phospholipid molecules/site. PE did
not induce binding sites on membranes of 4% Ptd-D-Ser, indicating that the induced binding sites require the correct stereochemistry
of Ptd-L-Ser as well as PE. Egg PE and dimyristoyl-PE were equivalent for inducing factor VIII-binding sites, indicating that
hexagonal phase-inducing properties of PE are not important. We conclude that PE induces high affinity factor VIII-binding
sites on membranes with physiologic mole fractions of Ptd-L-Ser, possibly including those of stimulated platelets.
The interaction between human factor IXa and factor VIII or its constituent units was investigated. Equilibrium binding studies were performed employing factor VIII light chain that was immobilized on a monoclonal antibody. Factor VIII light chain was observed to bind factor IXa with high affinity (Kd = 14.8 +/- 3.2 nM) and approximately 1:1 stoichiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution effectively inhibited binding of factor IXa to the immobilized light chain (Ki = 10.9 +/- 1.9 nM). The isolated factor VIII light chain and the factor VIII heterodimer were equally effective in factor IXa binding, demonstrating that this interaction did not require the factor VIII heavy chain. Factor Xa and activated Protein C were found to be inefficient (Ki > or = 1.2 microM) in competing with factor IXa, indicating that the high affinity for factor VIII light chain was unique for factor IXa. The factor IXa-factor VIII light chain interaction was inhibited by von Willebrand factor, but this effect was abolished by cleavage of the factor VIII light chain by thrombin. An antibody that inhibits von Willebrand factor-factor VIII complex formation did not compete for factor IXa binding. In contrast, association of factor IXa with the factor VIII light chain was inhibited by an antibody directed against the factor VIII region Gln1778-Asp1840. We propose that this sequence provides a factor IXa binding site and that its exposure requires dissociation of the factor VIII-von Willebrand factor complex.
Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. Heme serves as the prosthetic group of numerous hemoproteins (eg, hemoglobin, myoglobin, cytochromes, guanylate cyclase, and nitric oxide synthase) and plays an important role in controlling protein synthesis and cell differentiation. Cellular heme levels are tightly controlled; this is achieved by a fine balance between heme biosynthesis and catabolism by the enzyme heme oxygenase. On a per-cell basis, the rate of heme synthesis in the developing erythroid cells is at least 1 order of magnitude higher than in the liver, which is in turn the second most active heme producer in the organism. Differences in iron metabolism and in genes for 5-aminolevulinic acid synthase (ALA-S, the first enzyme in heme biosynthesis) are responsible for the differences in regulation and rates of heme synthesis in erythroid and nonerythroid cells. There are 2 different genes for ALA-S, one of which is expressed ubiquitously (ALA-S1), whereas the expression of the other (ALA-S2) is specific to erythroid cells. Because the 5'-untranslated region of the erythroid-specific ALA-S2 mRNA contains the iron-responsive element, a cis-acting sequence responsible for translational induction of erythroid ALA-S2 by iron, the availability of iron controls protoporphyrin IX levels in hemoglobin-synthesizing cells. In nonerythroid cells, the rate-limiting step of heme production is catalyzed by ALA-S1, whose synthesis is feedback-inhibited by heme. On the other hand, in erythroid cells, heme does not inhibit either the activity or the synthesis of ALA-S but does inhibit cellular iron acquisition from transferrin without affecting its utilization for heme synthesis. This negative feedback is likely to explain the mechanism by which the availability of transferrin iron limits heme synthesis rate. Moreover, in erythroid cells heme seems to enhance globin gene transcription, is essential for globin translation, and supplies the prosthetic group for hemoglobin assembly. Heme may also be involved in the expression of other erythroid-specific proteins. Furthermore, heme seems to play a role in regulating either transcription, translation, processing, assembly, or stability of hemoproteins in nonerythroid cells. Heme oxygenase, which catalyzes heme degradation, seems to be an important enzymatic antioxidant system, probably by providing biliverdin, which is an antioxidant agent.
Of the various types of hemophilia, the most common of these lifelong bleeding disorders are due to an inherited deficiency of factor VIII or factor IX (Table 1). The genes for these blood coagulation factors lie on the X chromosome, and when mutated, they cause the X-linked recessive traits hemophilia A and B. Since these disorders are X-linked, they usually occur in males. Usually, the affected boy has inherited the mutant gene (XH ) from his carrier mother (X H/X ), but about 30 percent of cases arise from a spontaneous mutation, and there is . . .
The heme-heme oxygenase system has recently been recognized to possess important regulatory properties. It is tightly involved in both physiological as well as pathophysiological processes, such as cytoprotection, apoptosis, and inflammation. Heme functions as a double-edged sword. In moderate quantities and bound to protein, it forms an essential element for various biological processes, but when unleashed in large amounts, it can become toxic by mediating oxidative stress and inflammation. The effect of this free heme on the vascular system is determined by extracellular factors, such as hemoglobin/heme-binding proteins, haptoglobin, albumin, and hemopexin, and intracellular factors, including heme oxygenases and ferritin. Heme oxygenase (HO) enzyme activity results in the degradation of heme and the production of iron, carbon monoxide, and biliverdin. All these heme-degradation products are potentially toxic, but may also provide strong cytoprotection, depending on the generated amounts and the microenvironment. Pre-induction of HO activity has been demonstrated to ameliorate inflammation and mediate potent resistance to oxidative injury. A better understanding of the complex heme-heme
Procoagulant factor VIII (FVIII) is either produced endogenously under physiologic conditions, or administered exogenously as a therapeutic hemostatic drug in patients with hemophilia A. In the circulation, FVIII interacts with a multitude of glycoproteins, and may be used for coagulation at the sites of bleeding, eliminated by scavenger cells, or processed by the immune system, either as a self-constituent or as a foreign antigen. The fate of FVIII is dictated by the immune status of the individual, the location of FVIII in the body at a given time point, and the inflammatory microenvironment. It also depends on the local concentration of FVIII and of each interacting partner, and on the affinity of the respective interactions. FVIII, by virtue of its promiscuity, thus constitutes the core of a dynamic network that links the coagulation cascade, cells of the immune system, and, presumably, the inflammatory compartment. We describe the different interactions that FVIII is prone to establish during its life cycle, with a special focus on players of the innate and adaptive immune response. Lessons can be learned from understanding the dynamics of FVIII interactions--lessons that should pave the way to the conception of long-lasting hemostatic drugs devoid of iatrogenic immunogenicity.
The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4κ monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass ∼7.4 × 105M−1 s−1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss ≤1 × 10−5s−1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.
The effective activation of factor X by factor IXa requires the co- factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303–2317, 2305- 2332, and 2308–2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318–2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid.
Hematin, the drug used for acute porphyric attacks, has been shown to cause disturbances in hemostasis, mainly because of its degradation products. Lately a new heme compound, heme arginate, has been developed for the treatment of porphyrias. In experimental animal studies as well as in clinical use it has proved to be well tolerated. To find out whether heme arginate has any effects on hemostasis we have studied a number of parameters of coagulation and fibrinolysis after a heme arginate infusion in seven healthy volunteers. All parameters studied remained practically unchanged except the coagulation factor X, which showed a transient, insignificant decrease during the maximal heme concentration. We believe that the lack of side effects is due to a better stability of heme arginate, the degradation rates being 1% for heme arginate and 61% for hematin in four hours. Our data favor the use of heme arginate in acute porphyrias as well as in other deficiency states of heme.
Hematin is clinically useful in the treatment of acute intermittent porphyria. Recently, hematin-induced coagulopathy has been reported, and a patient we treated bled during hematin therapy. On 3 separate occasions, infusions of hematin (4 mg/kg) induced thrombocytopenia, prolongation of the prothrombin time, partial thromboplastin time. Reptilase time, and apparent decreases in fibrinogen and increases in fibrin(ogen) degradation products (FDP). However, fibrinogen assayed by heat precipitation was unchanged, the protamine paracoagulation test was negative, there was no red blood cell fragmentation, and plasminogen and antithrombin III remained normal, excluding the presence of disseminated intravascular coagulation. Furthermore, premedication with heparin, 5000 U i.v., failed to prevent the lengthening of the Reptilase time and exacerbated the thrombocytopenia. In vitro studies revealed that hematin, 0.1 mg/ml, aggregated platelets and induced the release of 14C-serotonin and adenosine triphosphate (ATP). Hematin also aggregated washed or gel-filtered platelets but had no effect on formalin-fixed platelets. Aggregation was inhibited by aspirin (0.12 mg/ml), adenosine triphosphate, and apyrase, suggesting that hematin aggregated platelets by inducing adenosine diphosphate (ADP) release. Hematin (0.07 mg/ml) progressively inactivated thrombin and 0.1 mg/ml prolonged the Reptilase time. Thus, hematin is unique in that it both induces platelet aggregation and inhibits coagulation.
Hematin, the drug used for acute porphyric attacks, has been shown to cause disturbances in hemostasis, mainly because of its degradation products. Lately a new heme compound, heme arginate, has been developed for the treatment of porphyrias. In experimental animal studies as well as in clinical use it has proved to be well tolerated. To find out whether heme arginate has any effects on hemostasis we have studied a number of parameters of coagulation and fibrinolysis after a heme arginate infusion in seven healthy volunteers. All parameters studied remained practically unchanged except the coagulation factor X, which showed a transient, insignificant decrease during the maximal heme concentration. We believe that the lack of side effects is due to a better stability of heme arginate, the degradation rates being 1% for heme arginate and 61% for hematin in four hours. Our data favor the use of heme arginate in acute porphyrias as well as in other deficiency states of heme.
Heme cofactors are found in a wide range of proteins, where they play various roles in e.g., steroid1 and bioactive lipid synthesis,2 energy transduction,3 gene regulation,4 cellular signaling,5 oxygen transport6 and antibiotic biosynthesis.7 The diversity of physiological functions performed by hemoproteins means that heme is among the most versatile of protein
cofactors.8 Aside from electron transferase functions observed in respiratory cytochromes (e.g., mitochondrial cytochrome c ref. 9), several hemoprotein sensory or catalytic functions are recognized that involve the binding of gaseous ligands to
the heme iron and/or the dissociation or switching of amino acid side chains as axial ligands to the iron.4,10 Moreover, heme-dependent activation of iron-bound dioxygen (as seen in e.g., nitric oxide synthase and the cytochromes P450)
enables a broad repertoire of reactions, including hydroxylation, epoxidation, demethylation and carbon-carbon bond cleavage.11–13 This chapter reviews (i) the basic properties and synthesis of heme cofactors, (ii) the nature of their attachment to hemoproteins
and the various types of protein scaffolds in which hemes are incorporated, (iii) exemplary functions of hemoproteins that
demonstrate their broad range of biochemical functions, and (iv) analytical techniques that facilitate the understanding of
the structural and redox characteristics of the protein-bound heme cofactor, and the mode of its ligation to the protein.
Administration of carbon monoxide derived from carbon monoxide-releasing molecules (CORMs) have been demonstrated to enhance coagulation and diminish fibrinolysis in vitro at small concentrations (100-200 μmol/l) in human and rabbit plasma, whereas in vivo administration of large concentrations (>1400 μmol/l) of carbon monoxide has mildly increased bleeding time in vivo in rats. We sought to determine whether CORM-2 [tricarbonyldichlororuthenium (II) dimer] would improve coagulation and attenuate tissue-type plasminogen activator (tPA)-mediated fibrinolysis in rabbit whole blood as determined in vitro by thrombelastography and in an in vivo preclinical rabbit model of ear bleeding time administered intravenous tPA (1 mg/kg). Addition of 200, 400 and 600 μmol/l CORM-2 to whole blood significantly improved coagulation and attenuated fibrinolysis compared with blood without CORM-2. Rabbits administered CORM-2 (10 mg/kg, 279 μmol/l) had a small but significant decrease in bleeding time before tPA administration. Administration of tPA resulted in bleeding times more than six-fold greater than baseline in animals not exposed to CORM-2, whereas rabbits administered CORM-2 had significantly smaller (more than five-fold less) bleeding time values after tPA administration. CORM-2 administration significantly decreases fibrinolytic bleeding in the rabbit in vivo. Additional preclinical investigation of the effects of CORM-2 on coagulopathy (e.g. heparin-mediated or clopidogrel-mediated) utilizing this rabbit model are planned.
Cigarette smoking is associated with plasmatic hypercoagulability, and carbon monoxide has been demonstrated to enhance coagulation by binding to a fibrinogen-bound heme. Our objective was to design and test a redox-based method to detect carboxyhemefibrinogen. Normal, pooled, citrated plasma was exposed to 0-100 μmol/l carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2) before or after exposure to the organic reductant phenylhydroxylamine (PHA, 0-30 mmol/l), a compound that rapidly converts Fe(+2) to Fe(+3) in heme. Addition of calcium and tissue factor activation in disposable thrombelastographic cups was performed, followed by data collection at 37°C for 15 min. Elastic modulus (G, dynes/cm(2)) was the primary endpoint. CORM-2 significantly increased G values by 67.8% compared to unexposed plasma; pretreatment with 10 mmol/l PHA significantly decreased G values in CORM-2-exposed plasma by 77.1%, whereas 30 mmol/l PHA was required to significantly decrease G values by 64.0% in plasma following CORM-2 pre-exposure. G values were not significantly different between unexposed plasma and plasma exposed to CORM-2 followed by 30 mmol/l PHA addition. Conversion of fibrinogen-bound to the metheme state alone decreased G by 34.3-38.9% following exposure to 10-30 mmol/l PHA. Conversion of fibrinogen-bound heme Fe(+2) to Fe(+3) with PHA abrogated carbon monoxide-mediated increases in clot strength. Clinical trials are planned to investigate smoking individuals to mechanistically link carboxyhemefibrinogen formation with in-vitro hypercoagulability.
The objective of this study was to determine how carbon monoxide directly modifies fibrinogen utilizing liquid chromatography-mass spectrometry (LC-MS/MS) by examining fibrinogen exposed to carbon monoxide releasing molecule-2 [tricarbonyldichlororuthenium (II) dimer; CORM-2]. Purified fibrinogen was exposed to 0, 25, 50 or 100 μmol/l CORM-2 for 5 min at 37°C and then stored at -80°C before analyses with LC-MS/MS. In a second series of experiments, normal plasma was exposed to 0 or 100 μmol/l CORM-2 in the absence or presence of the nitric oxide donor sodium nitroprusside and hydroquinone (an organic reductant) to compete with carbon monoxide binding to a putative heme group found on fibrinogen. Coagulation was activated with tissue factor (n=8 per condition). Thrombus growth was monitored with thrombelastography for 15 min. LC-MS/MS did not detect any direct modifications of amino acids in fibrinogen, but detection of small regions of both the alpha and gamma chains was lost following exposure to CORM-2 and endoproteinase digestion with trypsin and Glu-C. An ion with the same m/z and expected retention time as heme was found in the purified fibrinogen. Exposure of plasma to nitric oxide/hydroquinone significantly decreased CORM-2-mediated enhancement of coagulation without affecting the coagulation kinetics of plasma not exposed to CORM-2. Carbon monoxide derived from CORM-2 likely modifies fibrinogen via modulation of a fibrinogen-associated heme group(s). Whereas the precise molecular location of heme attachment and three-dimensional conformational change secondary to carbon monoxide exposure remain to be determined, fibrinogen appears to be a carbon monoxide sensing molecule.
Replacement therapy in hemophilia A with exogenous coagulation factor VIII (FVIII) often results in the development of FVIII-neutralizing antibodies, referred to as inhibitors. Despite of large number of studies on the functional properties of FVIII inhibitors, detailed physicochemical characterization of their interactions is not available. Here we studied the biophysical mechanism of the interaction between a human pathogenic antibody--BO2C11 and its target antigen--FVIII. Kinetic and thermodynamic analyses implied that this interaction is not accompanied by significant conformational changes in the proteins. The data also suggested that association of BO2C11 to FVIII is driven mainly by a hydrophobic effect. The protein electrostatics however played a decisive role in this association. Thus, a gradual increase in ionic strength resulted in a considerable increase in the association rate of binding of BO2C11 to FVIII. Such an ionic strength-dependency is uncommon for other antibody-antigen interactions. Our data suggest that electrostatic effects observed for BO2C11-FVIII association may arise from high-energy penalty of desolvation of the charged residues at the binding interfaces. We hypothesize that untypical ionic strength dependence of association of BO2C11 to FVIII reflects the nature of the recognized epitope, namely a molecular surface involved in the binding of FVIII to phospholipids. The presented data provide mechanistic information about FVIII neutralization by an inhibitory antibody and also contribute to the understanding of the general mechanisms of antibody-antigen interactions.
This article has no abstract; the first 100 words appear below.
The discovery that hematin represses induction of δ-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme for hepatic porphyrin and heme synthesis,¹ is central to recent studies seeking to provide a means of terminating or ameliorating the acute attack of acute intermittent porphyria, porphyria variegata or hepatic coproporphyria. The induction of ALA-S in the acute attack of AIP, acute intermittent porphyria, porphyria variegata or hepatic coproporphyria markedly increases the porphyrin precursors, ALA and porphobilinogen (PBG), in liver, blood plasma and urine. The enzyme is induced because of heme deficiency in the liver secondary to the genetic partial lack of uroporphyrinogen synthetase (UPG-S) . . .
C.J. Watson, M.D.
Northwestern Hospital Minneapolis, MN 55407
Hemophiliacs often have mild anemia, and hemolysis has been suggested as the likely mechanism on the basis of the reduced serum haptoglobin values frequently observed in these patients. It has been suggested that hypohaptoglobinemia results from isohemagglutinins or other contaminating proteins in the infused factor concentrates. The advent and increased utilization of Factor VIII concentrates that are highly purified by use of monoclonal antibodies have provided the opportunity to study whether proteins other than Factor VIII contained in the concentrate induce hemolysis. Of 49 consecutively studied Factor VIII-deficient hemophiliacs, 19 (39%) had a reduced serum haptoglobin level (less than 27 mg/dl). In particular, 16 of 35 (46%) of patients receiving only monoclonally purified Factor VIII products (Monoclate or Hemofil-M) had a reduced serum haptoglobin value. Haptoglobin measurements were variable on repeat measurement in 8 patients. Haptoglobin levels did not correlate with type or severity of hemophilia, hemoglobin value, or alterations in liver function. Low serum haptoglobin values were also observed in children with leukemia, without apparent hemolysis, who had extensive cutaneous hemorrhage associated with thrombocytopenia. We propose that reduced serum haptoglobin values in hemophiliacs do not result from immune-mediated hemolysis due to contaminating proteins in the concentrate. Moreover, hypohaptoglobinemia may not be due to hemolysis at all but may instead result from dissolution of hematomas and other foci of internal hemorrhage.
Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 U/ml) with 0.1 microgram/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 micrograms/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 micrograms/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12, 20/21, 23, 27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.
Intravenous infusion of hematin, used in the treatment of acute porphyria, induces a decline in the plasma factor VIII/von Willebrand factor complex (VIII/vWF) and thrombocytopenia. We investigated this problem by studying the interaction between hematin, purified VIII/vWF, and platelets in vitro. Hematin was labeled with either 59Fe or 3H and characterized by gel chromatography. Hematin self-aggregated, forming a complex with an average molecular weight of approximately 10,000 daltons. When incubated with VIII/vWF for 30 min at 37 degrees C and applied to Sepharose CL-4B, the hematin eluted with the VIII/vWF in the void volume. Hematin inhibited the dissociation of factor VIII antigen (VIII:Ag) from the von Willebrand antigen (vWF:Ag) in 0.25 M CaCl2, and reversed the aggregation of VIII:Ag induced by 0.1 M 6-aminocaproic acid. Both hematin and the hematin-VIII/vWF complex bound to washed normal platelets and to platelets from a patient with Bernard-Soulier syndrome. Thrombasthenic platelets were not aggregatable by hematin, and bound significantly less hematin-VIII/vWF than normal platelets suggesting that hematin-induced platelet activation was required for binding. Likewise, binding was inhibited by PGE1 which also prevented aggregation. We conclude that hematin forms complexes with VIII/vWF, alters the functional activity and dissociation of this compound, and participates in the binding of VIII/vWF to platelets.
Mild leukopenia and thrombocytopenia are common in multitransfused hemophiliacs. Because little attention has previously been directed to measurements of erythropoiesis in these patients, we prospectively examined hemoglobin concentration and RBC indices in 94 children and young adults with hemophilia during comprehensive clinic visits. Additional studies performed in many included serum transferrin saturation, ferritin, haptoglobin, and free erythrocyte protoporphyrin measurements. Hemoglobin concentrations were recorded as age-related percentile values. Hemophiliacs of all ages and degrees of severity often had lower than average values for hemoglobin; 31% had values less than the third percentile, 46% less than the tenth percentile, and 83% less than the mean value. Reduced hemoglobin percentile values were unrelated to age, severity of disease, or human immunodeficiency virus antibody status. Only five patients had an obvious cause for anemia. Serum ferritin, transferrin saturation, and erythrocyte protoporphyrin values were usually normal, indicating that iron deficiency and anemia of chronic disease were uncommon. Although serum haptoglobin was reduced in 44% of the patients, reticulocyte count was infrequently increased. We conclude that hemoglobin values are frequently less than the mean normal values for age in hemophiliacs. Although frank anemia is common, it is usually mild and without obvious cause. Hemophiliac individuals with slightly reduced hemoglobin values probably do not routinely require detailed investigation for occult blood loss, iron deficiency, or inflammation.
The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron-free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 μM−1. From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long-chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open-chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.
Plasma concentrations of hemopexin, haptoglobin and heme were determined in patients with various hemolytic diseases. Diminished concentrations of hemopexin were found only when the concentrations of haptoglobin were decreased; the former were not in all instances lowered to the same extent as the haptoglobin levels. Invariably, high concentrations of heme were associated with low concentrations of hemopexin.
Prolonged clotting times and reduced levels of clotting factors have been reported in hematin-treated patients. This effect persists for up to 5 hr after hematin infusion, associated with plasma levels ranging from 0.01 to 0.04 mg/ml. Therefore we performed in vitro studies to investigate the effects of hematin on fibrinogen, thrombin, factor VIII:C, and plasmin. Hematin in a final concentration of 0.01 mg/ml inhibited the clotting of bovine fibrinogen (1.3 to 2.6 mg/ml) by bovine thrombin (0.12 U/ml) and inhibited the hydrolysis of a synthetic substrate by human thrombin. However, if the hematin was first mixed with albumin (25 mg/ml), fourfold higher concentrations were required to prolong the thrombin clotting time. Hematin, 0.035 mg/ml, reduced VIII:C activity from 0.88 to 0.40 U/ml as measured by two-stage assay. Hematin (0.05 mg/ml) also inhibited the activation of VIII:C by thrombin (0.04 U/ml): baseline activity, 0.84 U/ml; thrombin-activated, 2.94 U/ml; with hematin added, 1.33 U/ml. Hematin also inhibited clot lysis. The inclusion of hematin (0.03 mg/ml) in the diluting buffer reduced the lysis of whole blood clots from 86% +/- 5 to 23% +/- 5 (p less than 0.001, mean +/- S.D. of four determinations) and decreased the lysis of 125I-fibrin clots induced by plasmin (0.02 CTA U/ml) from 100% to 27%. In concentrations as low as 0.09 microgram/ml, hematin inhibited the hydrolysis of a synthetic substrate by plasmin. Hematin was mixed with fibrinogen, albumin, or thrombin, and the mixtures applied to Sephadex G-200 columns. Adherence of the hematin to Sephadex was prevented by either prerinsing the column with albumin or using borate buffer at pH 9.2. Hematin co-eluted with each protein applied to the column and, in the case of fibrinogen, altered its electrophoretic mobility and markedly prolonged the thrombin clotting time of the eluted fibrinogen. We conclude that hematin binds to a variety of hemostatic proteins, inhibiting their biologic activity.
The adhesion of platelets to subendothelium exposed to flowing blood involves two distinct morphological stages: (1) platelet contact (C), the initial attachment of unspread, discoid platelets to the subendothelium, and (2) spread platelets (S), the attachment that results as contact platelets spread on the surface and become more firmly bound to it. A defect in either initial platelet attachment or platelet spreading can result in reduced levels of platelet adhesion (C + S). The combined observation of decreased platelet adhesion (C + S) and increased platelet contact (C) has been previously utilized to conclude that a defect exists in the ability of platelets to spread on subendothelium in von Willebrand's disease. In this present investigation, we demonstrate, by modeling the contact and spreading stages of platelet adhesion as a classic set of reactions in series, that the combination of reduced adhesion (C + S) and increased contact (C) is inconclusive with regard to the nature of the adhesion defect in von Willebrand's disease. Decreased adhesion (C + S) coupled with increased platelet contact (C) can result from either decreased rates of initial attachment or decreased rates of spreading. In fact, given the complexity of the temporal behavior of platelet contact (C) and platelet spreading (S), and the relatively small fraction (less than 10%) of the platelets that are in contact (C) at any time, we conclude that a determination of the nature of the adhesion (C + S) defect in von Willebrand's disease is not statistically feasible under conditions in which both contact and spreading occur simultaneously. Experiments were conducted in which blood anticoagulated with EDTA was exposed to subendothelium digested with alpha-chymotrypsin for periods of 10 and 40 min. Under such conditions, platelet spreading (S) was substantially inhibited so that the predominant platelet interaction (greater than 80%) on the subendothelium was platelet contact (C). Values of platelet adhesion (C + S) in von Willebrand's disease were significantly reduced (p less than 0.05) compared with normal values at both exposure times. Thus we conclude that the defect in platelet adhesion (C + S) in von Willebrand's disease appears to be associated with a reduced ability of platelets to attach to the surface rather than their inability to spread on the surface.
Hematin is clinically useful in the treatment of acute intermittent porphyria. Recently, hematin-induced coagulopathy has been reported, and a patient we treated bled during hematin therapy. On 3 separate occasions, infusions of hematin (4 mg/kg) induced thrombocytopenia, prolongation of the prothrombin time, partial thromboplastin time. Reptilase time, and apparent decreases in fibrinogen and increases in fibrin(ogen) degradation products (FDP). However, fibrinogen assayed by heat precipitation was unchanged, the protamine paracoagulation test was negative, there was no red blood cell fragmentation, and plasminogen and antithrombin III remained normal, excluding the presence of disseminated intravascular coagulation. Furthermore, premedication with heparin, 5000 U i.v., failed to prevent the lengthening of the Reptilase time and exacerbated the thrombocytopenia. In vitro studies revealed that hematin, 0.1 mg/ml, aggregated platelets and induced the release of 14C-serotonin and adenosine triphosphate (ATP). Hematin also aggregated washed or gel-filtered platelets but had no effect on formalin-fixed platelets. Aggregation was inhibited by aspirin (0.12 mg/ml), adenosine triphosphate, and apyrase, suggesting that hematin aggregated platelets by inducing adenosine diphosphate (ADP) release. Hematin (0.07 mg/ml) progressively inactivated thrombin and 0.1 mg/ml prolonged the Reptilase time. Thus, hematin is unique in that it both induces platelet aggregation and inhibits coagulation.
1.1. The interaction of albumin with haeme was studied by measuring the absorption spectra in the Soret region. Albumin was confirmed to have one strong binding center besides some weaker ones; from the latters haeme can be removed by ion exchange chromatography on DEAE cellulose thus forming an equimolar haeme-albumin complex,2.2. Haeme transfer within the ferrihaemoglobin-albumin system was studied using CD spectra measurements in the Soret region. The transfer mechanism was described by the four consecutive reactions: (i = 4.3.2.1). characterized by equilibrium constants K1 = 0.39; K2 = 0.22; K3 = 0.032 and K4 = 0.15.3.3. The transfer of haeme in the haeniealbumin-apohaemopexin system was investigated by means of difference absorption spectra in the Soret region. The system can be defined by a simple stechiometrie equation characterized by the equilibrium constant K = 75.
In previous studies have shown that the interaction between factor IXa and VIII involves the light chain of factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of factor IXa to immobilized factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated factor IX.
Recent evidence indicates that there are significant tissue-specific expression and heme-mediated regulation of enzymes in the heme biosynthetic pathway. In particular, delta-aminolevulinate synthase (ALAS) [EC 2.3.1.37] has been shown to exist as tissue-specific isozymes, i.e. the erythroid and non-specific ALAS, which are coded by two separate genes. In mammals, ALAS activity can be found in many tissues, the highest activity being found in the Harderian gland in rodents, the liver of chemically induced porphyric animals, and developing erythroblasts. In the liver, ALAS expression is under negative control by heme, while in the developing erythroblasts it is positively influenced by heme. In contrast to these tissues, ALAS is maximally expressed in the Harderian gland and is not influenced by heme. In addition to the tissue-specific regulation of heme biosynthesis, heme has also been shown to influence a number of gene functions that are not directly related to heme synthesis in various tissues.
The heme oxygenase (HO) system consists of two forms identified to date: the oxidative stress-inducible protein HO-1 (HSP32) and the constitutive isozyme HO-2. These proteins, which are different gene products, have little in common in primary structure, regulation, or tissue distribution. Both, however, catalyze oxidation of heme to biologically active molecules: iron, a gene regulator; biliverdin, an antioxidant; and carbon monoxide, a heme ligand. Finding the impressive heme-degrading activity of brain led to the suggestion that "HO in brain has functions aside from heme degradation" and to subsequent exploration of carbon monoxide as a promising and potentially significant messenger molecule. There is much parallelism between the biological actions and functions of the CO- and NO-generating systems; and their regulation is intimately linked. This review highlights the current information on molecular and biochemical properties of HO-1 and HO-2 and addresses the possible mechanisms for mutual regulatory interactions between the CO- and NO-generating systems.
The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4kappa monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass approximately 7.4 x 10(5) M-1 s-1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss < or = 1 x 10(-5) s-1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.
Introduction
Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein factor VIII. The activated form of the protein serves as an essential cofactor for factor IXa in the conversion of factor X to factor Xa. This surface-bound complex of enzyme and cofactor is referred to as the intrinsic factor Xase. Factor VIIIa dramatically increases the catalytic rate constant for substrate conversion by an unclear mechanism. The activity and stability of the factor Xase appears to be regulated by the integrity of the cofactor. Factor VIIIa possesses a labile structure, and subunit dissociation results in the decay of Xase activity. Furthermore, factor VIIIa is a substrate for proteolytic inactivation by several enzymes, including factor IXa, the enzyme for which it serves as a cofactor. Although interest in the structure, function, and metabolism of factor VIII is commensurate with its biochemical and clinical importance, the molecular basis for its role in coagulation and the regulation of function through complex intramolecular and intermolecular interactions remain poorly understood.
. ABO histo‐blood group is a major determinant of plasma levels of factor VIII (FVIII) and von Willebrand factor (vWF). Blood group O individuals have significantly (approximately 25%) lower plasma levels of both glycoproteins. This association is of clinical significance. Low plasma levels of either FVIII or vWF have long been established as causes of excess bleeding. Conversely, there is accumulating evidence that elevated FVIII–vWF levels may represent an important risk factor for ischaemic heart disease and venous thromboembolic disease. In spite of the well‐documented association between ABO blood group and FVIII–vWF levels, the underlying mechanism remains unknown. However, it has been established that the ABO effect is primarily mediated through a direct functional effect of the ABO locus on plasma vWF levels.
Theoretically, ABO blood group may alter the rate of vWF synthesis or secretion within endothelial cells. Alternatively, ABO group may affect vWF plasma clearance rates. ABH antigenic determinants have been identified on the N ‐linked oligosaccharide chains of circulating vWF and FVIII, according to the blood group of the individual. It remains unclear whether these carbohydrate structures are responsible for mediating the effect of ABO blood group on plasma vWF levels.
Excellular hemoglobin is an extremely active oxidant of low-density lipoproteins (LDL), a phenomenon explained so far by different mechanisms. In this study, we analyzed the mechanism of met-hemoglobin oxidability by comparing its mode of operation with other hemoproteins, met-myoglobin and horseradish peroxidase (HRP) or with free hemin. The kinetics of met-hemoglobin activity toward LDL lipids and protein differed from that of met-myoglobin and HRP, both quantitatively and qualitatively. Those differences were further clarified by analyzing heme transfer from the above-mentioned hemoproteins to LDL. It appeared that met-hemoglobin transferred most of its hemin to LDL, and the presence of H(2)O(2) accelerated the process. In contrast, met-myoglobin partially released hemin, but only in the presence of H(2)O(2), while HRP could not transfer heme at all. The minor amount of hemin transferred from met-myoglobin to LDL sufficed to trigger ApoB oxidation, forming covalent aggregates via inter-bityrosines. This indicated that heme bound to high affinity site(s) is responsible for oxidation. LDL components providing the sites were analyzed by binding heme-CO monomers to LDL. Soret spectra revealed that the high affinity site of monomeric hemin is located on the LDL protein, ApoB. The complex heme-CO-ApoB underwent instantaneous oxidation to hemin-ApoB, and the bound hemin then slowly disintegrated in conjunction with LDL oxidation. Hemopexin prevented LDL oxidation by trapping hemoprotein transferable heme. We concluded that met-hemoglobin exerts its oxidative activity on LDL via transfer of heme, which serves as a vehicle for iron insertion into the LDL protein, leading to formation of atherogenic LDL aggregates.
Severe hemolysis or myolysis occurring during pathological states, such as sickle cell disease, ischemia reperfusion, and malaria results in high levels of free heme, causing undesirable toxicity leading to organ, tissue, and cellular injury. Free heme catalyzes the oxidation, covalent cross-linking and aggregate formation of protein and its degradation to small peptides. It also catalyzes the formation of cytotoxic lipid peroxide via lipid peroxidation and damages DNA through oxidative stress. Heme being a lipophilic molecule intercalates in the membrane and impairs lipid bilayers and organelles, such as mitochondria and nuclei, and destabilizes the cytoskeleton. Heme is a potent hemolytic agent and alters the conformation of cytoskeletal protein in red cells. Free heme causes endothelial cell injury, leading to vascular inflammatory disorders and stimulates the expression of intracellular adhesion molecules. Heme acts as a pro-inflammatory molecule and heme-induced inflammation is involved in the pathology of diverse conditions; such as renal failure, arteriosclerosis, and complications after artificial blood transfusion, peritoneal endometriosis, and heart transplant failure. Heme offers severe toxic effects to kidney, liver, central nervous system and cardiac tissue. Although heme oxygenase is primarily responsible to detoxify free heme but other extra heme oxygenase systems also play a significant role to detoxify heme. A brief account of free heme toxicity and its detoxification systems along with mechanistic details are presented.
Heme and Hemoproteins Tetrapyrroles: Birth, Life and Death
- A Munro
- H Girvan
- K Mclean
- M Cheesman
- D Leys
Structure of human factor VIII
- Ga Vehar
- B Keyt
- Dh Eaton
- H Rodriguez
- O Brien
- Dp Rotblat
- F Oppermann
- H Keck
- R Wood
- Wi Harkins
- Rn Tuddenham
- Egd Lawn
- Rm Capon
Editorial: hematin and porphyria
- C J Watson
Watson CJ. Editorial: hematin and porphyria. N Engl J Med 1975;
293: 605-7.
The heme oxygenase system: a regulator of second messenger gases
- Maines
Maines MD. The heme oxygenase system: a regulator of second
messenger gases. Annu Rev Pharmacol Toxicol 1997; 37: 517-54.