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The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inlammation in a human model of endotoxemia
Abstract
The protein C pathway serves as a modulating system with both anti-inflammatory and anticoagulant properties and is intimately involved in the pathophysiology of inflammation and sepsis. Treatment with recombinant human activated protein C (rhAPC) can reduce the mortality of severe sepsis. We investigated whether an elevation of plasma protein C levels to supra-normal levels by infusion of a protein C zymogen concentrate has an effect on coagulation, protein C activation or inflammation in a human endotoxemia model. Eleven healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Ten minutes after infusion of 2ng/kg endotoxin each volunteer received either placebo or a plasma-derived protein C zymogen concentrate (Ceprotin, Baxter) (150 U/kg as a slow bolus infusion followed by 30 U/kg/h continuous infusion until 4 hours after LPS-infusion). Protein C antigen and activity increased 4- to 5-fold after infusion of the concentrate. APC was generated during endotoxin-induced inflammation in the placebo (1.6 fold increase) and the protein C period (4.0-fold increase). The increase of APC levels correlated with the TNF-alpha and IL-6 release in both periods (r = 0.65-0.68; p < 0.05) and paralleled the protein C antigen and activity levels in the period with supranormal protein C levels. Supra normal protein C levels resulted in slightly, although non-significant, lower tissue factor mRNA expression and thrombin generation (TAT, F1+2). Systemic inflammation (TNF-alpha, IL-6) was not influenced by protein C zymogen concentrate administration. Infusion of protein C zymogen was safe and no adverse effects occurred. The increase of protein C levels several fold above the normal range resulted in a proportional increase of the APC levels, but had no major anticoagulant, anti-inflammatory or profibrinolytic effects. Low grade endotoxemia itself induces significant protein C activation, which correlates with the TNF release.
... Unexpectedly, a supra-normal dose of protein C zymogen concentrate had no significant effect on LPS-induced biomarkers of coagulation, fibrinolysis, and inflammation while significant protein C activation and production of Tumor Necrosis Factor alpha (TNFα) were detected in human volunteers [48]. ...
Sepsis is one of the ten leading causes of death in developed and developing countries. In the United States, sepsis mortality approaches that of acute myocardial infarction and exceeds deaths from stroke. Neonates and the elderly are the most vulnerable patients, with these groups suffering from the highest sepsis mortality. In both groups, many survivors respectively display serious developmental disabilities and cognitive decline. The National Institute of Health National Heart Lung and Blood Institute Panel redefined sepsis as a "severe endothelial dysfunction syndrome in response to intravascular and extravascular infections causing reversible or irreversible injury to the microcirculation responsible for multiple organ failure. " Microvascular endothelial injury in sepsis due to microbial inflammation encompasses small blood vessels (< 100 μm in diameter). While the lungs remain the principal organ of interest due to sepsis-associated acute respiratory distress syndrome, "septic heart" or "septic cardiomyopathy" accelerates sepsis' transition to potentially lethal septic shock. This Review analyzes both new advances in understanding the septic mechanism and possible resolutions of sepsis. The concept of a "genomic storm", caused by microbes triggering florid production of inflammatory mediators, is based on septic reprogramming of the human genome. This "genomic storm" leads to microvascular endothelial injury, persistent hypotension, and organ failure. While very early control of sepsis-causing bacterial, fungal and viral infections remains crucial for the treatment of sepsis, supportive measures are likewise necessary to maintain blood pressure, respiration, and kidney function. New evidence indicates that preadmission β-blockers may reduce sepsis-associated mortality. The fundamental role of nuclear signaling in the progression and resolution of sepsis was established with a new class of cell-penetrating nuclear transport modifiers (NTMs). NTMs target the translocation of proinflammatory and metabolic transcription factors to the cell's nucleus while also enhancing bacterial clearance in experimental polymicrobial sepsis models. The result is a 700-fold reduction in the bacterial burden of the lungs and improvement of sepsis-associated thrombocytopaenia and blood markers of endothelial injury. When added to anti-microbial therapy, NTM has increased survival from 30% to 55%, when compared to antimicrobial therapy alone. Yet, the prevention of sepsis remains the most rational and beneficial path. Anti-pneumococcal vaccination has reduced the incidence of pneumonia and sepsis caused by increasingly antibiotic-resistant Streptococcus pneumoniae in all age groups. Similarly, the incidence of meningococcal sepsis known as "purpura fulminans" has been reduced by a recently approved vaccine thereby preventing hearing loss, neurologic damage, and limb amputations in young survivors of septic outbreaks. We urgently need further preventive, diagnostic, and therapeutic measures as the tide of sepsis rises in the United States and around the world.
... Additionally, zyPC, and especially zyPC-R15QS195A, would not have the same risk of bleeds as aPC. Interestingly, injections of recombinant human zyPC into septic, but otherwise healthy, volunteers at fold levels over baseline similar to those seen here following gene therapy caused no adverse effects 270 . It is possible that treatment of patients with cancers known to be metastatic, especially at the time of primary tumor resection, could have a disease progression benefit for patients. ...
Treatment of hemophilia, which involves infusion of the missing clotting factor, is often hindered by the development of neutralizing antibodies to the replaced clotting factor. We utilized liver-directed AAV gene therapy to tolerize outbred hemophiliac dogs with pre-existing anti-factor VIII and IX antibodies and to treat their underlying hemophilia. Additionally, we sought to shed light on the immunologic mechanisms responsible for this tolerization. Staining for CD4+CD25+FoxP3+ T cells and cytokine profiles of treated dogs suggest that induced Tregs are at least partially responsible for inducing and maintaining tolerance.
The second part of the dissertation attempts to determine the underlying mechanism of the known anti-metastatic effects of activated protein C (aPC). We again utilized liver-directed AAV gene therapy to tease apart the anticoagulation and cytoprotective effects of aPC, as these were the most likely candidates based on available literature, in the B16F10 murine model of metastatic melanoma. Upon finding, however, that neither of these functions were involved in aPC's cancer protection and that zymogen PC is even more protective, we endeavored to find the novel mechanism through which the protein C pathway can modulate tumor progression. While the mechanism has not been found, a number of potential candidates and receptors have been eliminated, including involvement of PAR-4, EPCR, and a potential integrin binding site. We also demonstrate that it is not tumor growth, but rather a step in the metastatic pathway that is inhibited.
... Thrombin generated on the surface of injured endothelium activates an physiologic anticoagulant mechanism that is vested in the thrombomodulin-protein C axis [48]. Surprisingly, in humans, a supra-normal dose of protein C zymogen concentrate had no significant effect on LPS-induced biomarkers of coagulation, fibrinolysis and inflammation, whereas significant protein C activation and production of tumor necrosis factor alpha (TNFa) were detected [70]. Likewise, heparin administration remains controversial, awaiting a well-designed prospective study. ...
Sepsis, also known as septicemia, is one of the ten leading causes of death world-wide. The rising tide of sepsis due to bacterial, fungal, and viral infections cannot be stemmed by current antimicrobial therapies and supportive measures. New paradigms for sepsis mechanism, resolution, and consequences for sepsis survivors are emerging. Consistent with Benjamin Franklin's dictum "an ounce of prevention is worth of a pound of cure", sepsis can be prevented by vaccinations against pneumococci and meningococci. Recently, the NIH NHLBI Panel redefined sepsis as "severe endothelial dysfunction syndrome in response to intravascular and extravascular infections causing reversible or irreversible injury to microcirculation responsible for multiple organ failure". Microvascular endothelial injury underlies sepsis-associated hypotension, edema, Disseminated Intravascular Coagulation, Acute Respiratory Distress Syndrome, and Acute Kidney Injury. Microbial genome products trigger "genome wars" in sepsis that reprogram the human genome and culminate in a "genomic storm" in blood and vascular cells. Sepsis can be averted experimentally by endothelial cytoprotection through targeting nuclear signaling that mediates inflammation and deranged metabolism. Endothelial "rheostats", e.g. inhibitors of NF-κB, A20 protein, CRADD/RAIDD protein, and microRNAs regulate endothelial signaling. Physiologic "extinguishers", e.g. Suppressor of Cytokine Signaling 3, can be replenished through intracellular protein therapy. Lipid mediators, e.g. resolvin D1, hasten sepsis resolution. As sepsis cases rose from 387,330 in 1996 to 1.1 million in 2011, and are estimated to reach 2 million by 2020 in the US, sepsis mortality approaches that of heart attacks and exceeds stroke deaths. More preventive vaccines and therapeutic measures are urgently needed. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... In two rat studies high-dose APC modified plasma cytokine response to high-dose LPS exposure [17,18]. However, in human endotoxemia neither APC nor protein C zymogen had any effects on plasma cytokines [19][20][21]. Two of the studies showed increased activated partial thromboplastin time, but none of the three models of low-dose endotoxemia found any profibrinolytic, antithrombotic, or anti-inflammatory effects of APC. ...
To assess the anti-inflammatory effects of recombinant human activated protein C (rhAPC) in a porcine model of acute endotoxemia.
Animal randomized controlled study at the Laboratory of Clinical Institute, Aarhus University Hospital.
Eighteen female landrace pigs (30 kg).
By pairwise randomization, pigs were given either LPS or LPS and rhAPC. Both groups received a stepwise increasing LPS infusion for 30[Symbol: see text]min; whereafter the infusion continued at a lower rate (300 min LPS in both groups). The LPS+rhAPC group received rhAPC (100 microg/kg per hour) 15 min before the LPS infusion began and throughout the trial period.
While rhAPC showed no modifying effects on peak plasma levels of pro- or anti-inflammatory cytokines (TNF-alpha, IL-6, IL-8, IL-10), TNF-alpha and IL-10 peaked significantly later in the rhAPC-treated animals. The profibrinolytic effects of rhAPC were confirmed by decreased plasminogen activator inhibitor 1 levels, while no differences were found in other coagulation markers, hemodynamic, metabolic, or leukocyte data between the two groups.
We found no significant effect of rhAPC on plasma levels of either pro- or anti-inflammatory cytokines in this porcine model of acute endotoxemia. However, TNF-alpha and IL-10 peaked significantly later in the rhAPC-treated animals.
Despite the success of the anti-coagulant protease protein C (PC) in treating septic shock in humans, the signaling pathways used are still unclear. To explore the effects of treatment with PC zymogen and its activated form aPC in a setting of sepsis, we employed a piglet model of endotoxic shock. In the aPC group, we observed a 65%-90% reduction in plasma TNF-alpha levels and a concomitant clinical improvement. Unexpectedly, administration of aPC also resulted in stabilization of the plasma pH above 7.2. Moreover, phosphorylated p38 mitogen-activated protein kinase (p38MAPK) was virtually absent in the livers of those piglets receiving aPC. In cultured human umbilical vein endothelial cells, we observed that nanomolar concentrations of PC and aPC inhibited the phosphorylation of p38MAPK. Furthermore, we showed that the regulation of the pro-apoptotic cell cycle regulator p53 by PC and aPC is dependent on the reduction of p38MAPK activation. The transduction of these effects involves all three receptors associated with protein C signaling, namely endothelial protein C receptor, protease-activated receptor 1, and sphingosine 1-phosphate receptor 1. Ultimately, this study elucidates novel signaling pathways regulated by protein C and emphasises the pivotal importance of its multiple modes of action beyond anticoagulation. APC's clinical success may, in part, be due to p38MAPK inhibition.
The protein C system plays an active role in modulating severe systemic inflammatory processes such as sepsis, trauma and acute respiratory distress syndrome via its anticoagulant and anti-inflammatory properties. Plasma levels of aPC are lower than normal in acute inflammation in humans, except early after severe trauma where high plasma levels of aPC may play a mechanistic role in the development of posttraumatic coagulopathy. Thus, following positive results of preclinical studies, a clinical trial (PROWESS) with high continuous doses of recombinant human aPC given for 4 days demonstrated a survival benefit in patients with severe sepsis. This result was not confirmed by subsequent clinical trials, including the recently published PROWESS-SHOCK trial in patients with septic shock and a phase II trial with patients with non-septic ARDS. A possible explanation for the major difference in outcome between PROWESS and PROWESS-SHOCK trials is that lung protective ventilation was used for the patients included in the recent PROWESS-SHOCK, but not in the original PROWESS trial. Since up to 75% of sepsis originates from the lung, aPC treatment may not have added enough to the beneficial effect of lung protective ventilation to show lower mortality. Thus, whether aPC will continue to be used to modulate the acute inflammatory response in humans remains uncertain. Because recombinant human aPC has been withdrawn from the market, a better understanding of the complex interactions between coagulation and inflammation is needed before considering the development of new drugs that modulate both coagulation and acute inflammation in humans.
Some case reports and case series suggest that protein C concentrates may improve the outcome in patients with congenital or acquired protein C deficiency (not only in those with sepsis induced purpura fulminans). We reviewed the published literature on the use of protein C concentrates in adult septic patients and found that it is limited to less than 70 patients reported in observational studies with a 70% survival, and added our personal experience (two adult patients with sepsis and contraindications to recombinant activated protein C).
Endothelial dysfunction is considered a major factor in the pathogenesis of a wide array of diseases and often leads to increased production of, or increased responsiveness to endogenous vasoconstrictive mediators, such as endothelin-1 (ET-1) or adrenergic agonists. Based on previous studies in animals and in-vitro, we hypothesized that ET-1 and alpha adrenergic stimulation by phenylephrine (PE) may alter plasma levels of von Willebrand factor-Ag (vWF) and protein S in humans. Ten healthy men were studied in a prospective randomized double blind trial: we investigated the effects of infusions of ET-1 and PE on plasma levels of vWF-Ag and protein S. Aditionally, we examined the effects of these vasoconstrictors on thrombomodulin, tissue plasminogen activator and on protein C. ET-1 increased plasma levels of vWF-Ag by 19% (CI: 9-36%; p=0.008) and increased plasma levels of protein-C by 7% (CI: 0.2 -12 %; p=0.028), even at doses which produced no increase in blood pressure. Plasma levels of protein S and TPA were not significantly affected by ET-1. After PE-infusion we observed a relative increase in plasma levels of protein S by 20% (CI: 5-28%; p=0.036) and of TPA by 14 % (CI: 2-39%; p=0.036). Also, platelet counts increased by 36% (CI: 12-53%; p=0.028) which correlated (r(2)=0.86; p=0.014) with an increase in leukocytes by 49 % (CI: 18-84%; p=0.028). Plasma levels of vWF and protein C were not affected by PE and neither drug had a significant effect on plasma levels of thrombomodulin. In conclusion, our results support the study hypothesis, that ET-1 and PE increase the plasma levels of vWF and protein S, respectively. ET-1 may induce a procoagulant status in various disease states by increasing vWF-Ag levels.
Some case reports and case series suggest that protein C concentrates may improve the outcome in patients with congenital or acquired protein C deficiency (not only in those with sepsis induced purpura fulminans). We reviewed the published literature on the use of protein C concentrates in adult septic patients and found that it is limited to less than 70 patients reported in observational studies with a 70% survival, and added our personal experience (two adult patients with sepsis and contraindications to recombinant activated protein C).
Recognition of the link between coagulation activation and inflammation has led to the hypothesis that anticoagulants may be effective in the treatment of septic patients by altering the inflammatory response. However, only limited methodologies exist that can be used in human volunteers to mimic the physiologic alterations observed in critically ill patients. The human endotoxemia model represents a model of inflammation-induced tissue factor triggered coagulation activation. As it permits elucidation of a key player in this proinflammatory and procoagulant response, it serves as a useful tool to investigate novel therapeutics in a standardized setting. The aim of this review is to focus on coagulation interventions in the human endotoxemia model.
BIBT986 is a dual inhibitor of factors Xa and IIa. The aim of this study was to compare with placebo the effect of three doses of BIBT986 on coagulation, platelet activation, and inflammation. This was a prospective, randomized, double-blind, placebo-controlled, parallel-group dose escalation trial in 48 healthy male volunteers. Participants received one of three doses of BIBT986 or placebo intravenously together with a bolus infusion of 2 ng/kg lipopolysaccharide (LPS). BIBT986 dose-dependently changed global coagulation parameters and in vivo markers of thrombin generation and action: BIBT986 doses, which prolonged activated partial thromboplastin time by 100%, completely suppressed the LPS-induced increases in prothrombin fragment, thrombin-antithrombin complexes, and D-dimer, which were 6.1-, 14.5, and 3.5-fold in the placebo group, respectively. BIBT986 did not influence inflammation, fibrinolysis, or platelet activation. Therefore, BIBT986 is a potent anticoagulant in the human endotoxemia model.
The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with alpha-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.
Drotrecogin alfa (activated), or recombinant human activated protein C, has antithrombotic, antiinflammatory, and profibrinolytic properties. In a previous study, drotrecogin alfa activated produced dose-dependent reductions in the levels of markers of coagulation and inflammation in patients with severe sepsis. In this phase 3 trial, we assessed whether treatment with drotrecogin alfa activated reduced the rate of death from any cause among patients with severe sepsis.
We conducted a randomized, double-blind, placebo-controlled, multicenter trial. Patients with systemic inflammation and organ failure due to acute infection were enrolled and assigned to receive an intravenous infusion of either placebo or drotrecogin alfa activated (24 microg per kilogram of body weight per hour) for a total duration of 96 hours. The prospectively defined primary end point was death from any cause and was assessed 28 days after the start of the infusion. Patients were monitored for adverse events; changes in vital signs, laboratory variables, and the results of microbiologic cultures; and the development of neutralizing antibodies against activated protein C.
A total of 1690 randomized patients were treated (840 in the placebo group and 850 in the drotrecogin alfa activated group). The mortality rate was 30.8 percent in the placebo group and 24.7 percent in the drotrecogin alfa activated group. On the basis of the prospectively defined primary analysis, treatment with drotrecogin alfa activated was associated with a reduction in the relative risk of death of 19.4 percent (95 percent confidence interval, 6.6 to 30.5) and an absolute reduction in the risk of death of 6.1 percent (P=0.005). The incidence of serious bleeding was higher in the drotrecogin alfa activated group than in the placebo group (3.5 percent vs. 2.0 percent, P=0.06).
Treatment with drotrecogin alfa activated significantly reduces mortality in patients with severe sepsis and may be associated with an increased risk of bleeding.
Impairment of the protein C anticoagulation pathway is critical to the thrombosis associated with sepsis and to the development of purpura fulminans in meningococcemia. We studied the expression of thrombomodulin and the endothelial protein C receptor in the dermal microvasculature of children with severe meningococcemia and purpuric or petechial lesions.
We assessed the integrity of the endothelium and the expression of thrombomodulin and the endothelial protein C receptor in biopsy specimens of purpuric lesions from 21 children with meningococcal sepsis (median age, 41 months), as compared with control skin-biopsy specimens.
The expression of endothelial thrombomodulin and of the endothelial protein C receptor was lower in the patients with meningococcal sepsis than in the controls, both in vessels with thrombosis and in vessels without thrombosis. On electron microscopical examination, the endothelial cells were generally intact in both thrombosed and nonthrombosed vessels. Plasma thrombomodulin levels in the children with meningococcal sepsis (median, 6.4 ng per liter) were higher than those in the controls (median, 3.6 ng per liter; P=0.002). Plasma levels, protein C antigen, protein S antigen, and antithrombin antigen were lower than those in the controls. In two patients treated with unactivated protein C concentrate, activated protein C was undetectable at the time of admission, and plasma levels remained low.
In severe meningococcal sepsis, protein C activation is impaired, a finding consistent with down-regulation of the endothelial thrombomodulin-endothelial protein C receptor pathway.
Inflammatory and procoagulant host responses are closely related in sepsis. The protein C pathway serves as a regulatory pathway with anti-inflammatory and anticoagulant properties. Recently, recombinant human activated protein C (rhAPC) was shown to reduce mortality in severe sepsis. Nevertheless, the effects of rhAPC in humans are still ill defined. The infusion of low endotoxin doses into humans provides a standardized model to study inflammatory and hemostatic mechanisms. Thus, we investigated whether rhAPC acts as an anticoagulant or anti-inflammatory drug in human endotoxemia. There were 24 volunteers randomized to receive either 24 microg/kg per hour rhAPC or placebo intravenously for 8 hours. Lipopolysaccharide (LPS, 2 ng/kg) was administered 2 hours after starting the infusions. rhAPC decreased basal tissue factor (TF)-mRNA expression, and thrombin formation and action. In contrast, rhAPC did not significantly blunt LPS-induced thrombin generation. Consistently, rhAPC did not reduce LPS-induced levels of TF-mRNA or D-dimer and had no effect on fibrinolytic activity or inflammation. Finally, endogenous APC formation was enhanced during endotoxemia and appeared to be associated with inflammation rather than thrombin formation. In conclusion, even low-grade endotoxemia induces significant protein C activation. Infusion of rhAPC decreases "spontaneous" activation of coagulation but does not blunt LPS-induced, TF-mediated coagulation in healthy volunteers, which is in contrast to a number of anticoagulants.
We studied, in 40 children (mean age: 52 months) with severe infectious purpura, the relationships between protein C (PC) and protein S (PS) levels, and shock, disseminated intravascular coagulation (DIC) and outcome. We determined, on admission, PC antigen (ELISA) and activity (chromogenic test), and total PS (ELISA). Results were expressed as % of normal adult values. Statistical analysis was performed with SAS. Thirty children were in shock, 20 had DIC. All children with DIC, and 10 without DIC were in shock. Of 20 children who were in shock and had DIC, 7 died and 3 had an amputation. PC antigen was significantly decreased in shock children (p less than 0.05), in children with DIC (p less than 0.0005), and in non-survivors (p less than 0.05). PC activity was significantly decreased in shock children (p less than 0.05), in children with DIC (p less than 0.0005), and in non-survivors (p less than 0.005). Total PS was not decreased in shock children, but was significantly decreased in children with DIC (p less than 0.005), and in non-survivors (p less than 0.005). We conclude that PC and PS levels were decreased in our children, and that PC levels were significantly decreased in the presence of shock, DIC, and fatal outcome. PC and antithrombin III (AT III) supplementation, should be evaluated in children with severe infectious purpura with shock and DIC.
Cathepsin G was used in vitro to digest human factor VII and factor IX. Clotting assays indicated that the proteinase affected a rapid loss in coagulant activity while in the presence of calcium ions the activity was almost totally protected. SDS-polyacrylamide gel electrophoresis indicated the removal of a peptide from each zymogen, VII-L from factor VII and IX-L from factor IX. This lead to the formation of VII-H and IX-H respectively. N-terminal analysis of the VII-H and IX-H products and COOH-terminal analysis of the VII-L and IX-L products confirmed that cathepsin G had cleaved position Phe40:Trp41 in factor VII and factor IX. The cleavage site is the same as that when cathepsin G is reacted with factor II, factor X and protein C. The unique action of cathepsin G may be part of a regulatory system for controlling the coagulant activity of vitamin K dependent clotting in vivo.
Although protein C (PC) and activated protein C (APC) have been postulated to be useful for treating patients with thrombosis, their critical effect remains to be studied in human subjects. To examine whether purified PC or APC are useful for treating patients with thrombosis without showing any adverse effect, wc studied effects on coagulation and fibrinolysis in normal human subjects.
When highly purified human PC was administered intravenously to healthy subjects, plasma levels of immunoreactive PC decreased with a half-life of 10.9 h. Intravenously administered APC decreased with a half-life of 23 min as measured by prolongation of activated partial thromboplastin time (APTT). However, 1.7 h was obtained for the plasma half-life of APC when it was measured immunologically. These findings suggested that a significant fraction of the administered APC was rapidly inhibited by plasma inhibitor. Upon administration of APC, APTT was prolonged and plasma levels of clotting factor VIII (FVIII) decreased transiently as measured by clotting assay. However, when determined by a chromogenic assay method in which 120-fold diluted plasma samples were used, plasma levels of FVIII remained unchanged. Plasma levels of F-V did not decrease after APC administration. These findings suggested that prolongation of APTT and apparent decrease in plasma F-VIII clotting activity might be due to the in vitro-effect of APC present in plasma samples used. Diurnal fluctuation of plasminogen activator inhibitor in normal subjects was not affected by administration of APC.
Thus, PC or APC seems to function selectively at the site of thrombin-formation without lowering plasma levels of coagulation factors.
Protein C replacement therapy with a monoclonal antibody purified, virus inactivated protein C concentrate was carried out in nine infants (three male, six female) with severe congenital protein C deficiency and life-threatening purpura fulminans and/or thrombosis associated with disseminated intravascular coagulation (DIC). Eight infants were homozygous for protein C deficiency; one was a compound heterozygote. The treatment period varied from 22 days to three years. The half-life of protein C was found to be as short as two to three hours during activation of the coagulation system, increasing to approximately ten hours after stabilization. During the acute phase, protein C levels of 0.10 to 0.25 IU/mL were associated with elevated markers of coagulation activation indicating DIC, while protein C levels greater than 0.25 were associated with normalization of coagulation markers. No product-related side effects were reported. Episodes of bleeding or purpura recurred in all patients who were switched to oral anticoagulant therapy, necessitating reinstatement of protein C replacement therapy, either as needed to control symptoms, or on a long-term prophylactic schedule, alone or in addition to oral anticoagulation. Home treatment with protein C concentrate allowed a near-normal life-style for patients who otherwise would be hospitalized for long periods of time.
To evaluate the clinical and laboratory effects of the substitution of protein C (PC) as an adjunct to conventional therapy in the treatment of purpura fulminans associated with meningococcal sepsis.
case series.
Medical and medical-surgical intensive care units of two university hospitals.
Three patients with purpura fulminans and multiple organ failure caused by Neisseria meningitidis.
Intravenous administration of PC concentrate (100 IU/kg every 6 to 8 hrs).
The administration of PC resulted in normal or above normal levels of the plasma PC activity in all patients. The laboratory and clinical parameters reflecting the severity of coagulopathy improved during the treatment, as did peripheral ischemia and the clinical manifestations of multiple organ failure. No adverse events were noted. One patient died of cerebral edema.
The administration of PC had a beneficial effect on coagulopathy and peripheral gangrene formation associated with meningococcal disease and showed no adverse effects.
We report the effects of substitution with a virus-inactivated protein C (PC) concentrate in disseminated intravascular coagulation (DIC) in infants and children with meningococcal sepsis associated with purpura fulminans. It was a prospective open-label study. Eight pediatric and adolescent patients age 0.2 to 18.25 years with DIC associated with severe acquired PC deficiency (range 0.02 to 0.48 IU/mL; median, 0.22 IU/mL) in meningococcal septic shock and purpura fulminans were studied. Replacement therapy was initiated with a virus-inactivated PC concentrate with an initial intravenous bolus of 80 to 120 IU/kg followed by 50 IU/kg up to six times per day as an adjunctive therapeutic regimen to otherwise optimal intensive care treatment. After initial PC administration, plasma PC levels rose to normal ranges and were maintained under PC replacement therapy. Improving or even normalizing global hemostatic parameters were assessed in all patients. Markedly elevated plasminogen activator inhibitor type 1 (PAI-1) levels prior to treatment, reflecting a reduced fibrinolytic potential, decreased rapidly under PC substitution. Concomitantly improving signs of purpura fulminans reflected by decreasing size of skin lesions, demonstrated a restoring microcirculation. Six of the eight patients survived. One patient required limb amputation; two patients died because of multiorgan failure. Both presented with a severely low plasma PC activity of 0.02 IU/mL on admission to the hospital. No adverse effects were observed with the PC concentrate administration. It can be concluded that the administration of PC concentrate had a marked benefit on the deranged coagulation status of patients with purpura fulminans and meningococcal septicemia. Normalization or even partial correction of hemostasis as well as improvement of microcirculation accompanied by improving signs of purpura fulminans were demonstrated in all patients.
Meningococcal septicaemia is a devastating disease with the potential to develop severe vascular complications. The incidence in Northern Ireland has risen from 27 cases notified in 1992 to 56 notified in 1997. We describe the first use of protein C concentrate in addition to antithrombin III infusion in the management of a life-threatening case of meningococcal septicaemia in the Regional Intensive Care Unit, Royal Group of Hospitals, Belfast, UK. The rationale and the evidence to support the use of protein C concentrate are discussed. Despite the apparent efficacy and safety of this treatment, subsequent cases of meningococcal septicaemia have not received protein C concentrate due to a lack of availability.
Severe meningococcemia, which is associated with hemodynamic instability, purpura fulminans and disseminated intravascular
coagulation, still has a high mortality rate, and patients who survive are often left invalids because of amputations and
organ failure. Clinical studies have shown that levels of protein C are markedly decreased in patients with severe meningococcemia
and that the extent of the decrease correlates with a negative clinical outcome. There is a growing body of data demonstrating
that activated protein C, in addition to being an anticoagulant, is also a physiologically relevant modulator of the inflammatory
response. The dual function of protein C may be relevant to the treatment of individuals with severe meningococcal sepsis.
In the present review we give a basic overview of the protein C pathway and its anticoagulant activity, and we summarize experimental
data showing that activated protein C replacement therapy clearly reduces the mortality rate for fulminant meningococcemia.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Coumarin derivatives are still widely used for prophylaxis of thromboembolic events and therefore represent important comparator substances for new anticoagulants. Measurement of the efficacy of such novel compounds in a human coagulation model with adequate biomarkers could be useful for early-phase clinical drug development. To evaluate the applicability of a well-established model of tissue factor-dependent coagulation for defining anticoagulant potency, we investigated the effects of acenocoumarol in experimental human endotoxemia.
In a randomized, controlled, 2-by-2 factorial design, healthy volunteers received an infusion of 2 ng/kg endotoxin or placebo after 18 days of pretreatment with acenocoumarol or placebo. Prothrombin fragment 1+2 (F(1+2)), soluble fibrin, and D-dimer were used as markers of thrombin and fibrin formation.
As expected, pretreatment with acenocoumarol decreased vitamin K-dependent coagulation factors, but it also decreased spontaneous thrombin formation. Acenocoumarol inhibited endotoxin-induced thrombin generation as measured by F(1+2) levels: endotoxin infusion increased F(1+2) levels 8-fold-from 0.5 to 4.1 nmol/L-in the placebo group, whereas peak F(1+2) levels reached only 1.0 nmol/L in subjects after acenocoumarol pretreatment. This inhibition was also reflected in decreased formation of soluble fibrin and decreased D-dimer levels, showing that depletion of endogenous coagulation factors limits the propagation of nonovert disseminated intravascular coagulation.
Human endotoxemia is a suitable tool for measurement of the efficacy of oral anticoagulants and therefore may become a valuable addition for expeditious pharmacodynamic characterization of lead compounds with anticoagulant potency.
Inhibition of the tissue factor-factor VIIa pathway attenuated the activation of coagulation and prevented death in a gram-negative bacteremia primate model of sepsis. This lethal animal model suggested that tissue factor also influences inflammatory cascades.
This trial examined the pharmacodynamic effects of active site-inhibited factor VIIa (FFR-recombinant factor VIIa [rFVIIa]; ASIS) on endotoxin-induced procoagulant, fibrinolytic, and inflammatory responses in healthy humans. A double-blind, randomized, placebo-controlled, parallel-group study was conducted in 12 healthy male volunteers. Subjects received a bolus infusion of 2-ng/kg endotoxin, followed by a bolus infusion of ASIS (400 microg/kg) or placebo 10 minutes later.
Endotoxin injection induced inflammation, activation of coagulation, and activation and subsequent inhibition of fibrinolysis. ASIS infusion completely blocked thrombin and fibrin generation, as measured by plasma levels of prothrombin fragment (no increase in the ASIS group, as compared with a 13-fold increase in the placebo group at 4 hours; P <.01), soluble fibrin, and fibrin split product D-dimer. ASIS did not alter endotoxin-induced changes in the fibrinolytic system, cytokine levels, or markers of endothelial (E-selectin, thrombomodulin) or platelet (P-selectin) activation.
In summary, ASIS effectively and selectively attenuates tissue factor-induced thrombin generation. Because ASIS was well tolerated, this study provides seminal data to further characterize its anticoagulant and putative anti-inflammatory effects in critically ill patients.
We aimed to characterize the pharmacokinetics and pharmacodynamics of drotrecogin alfa (activated) (recombinant human activated protein C) in patients with severe sepsis.
Patients (N = 1690) in a randomized, double-blind, placebo-controlled phase 3 trial received a 96-hour infusion of placebo (n = 840) or drotrecogin alfa (activated) (n = 850), 24 microg x kg(-1) x h(-1). Plasma samples from 680 patients were collected for pharmacokinetic assessment. Pharmacodynamic effects on activated partial thromboplastin time, D-dimer, protein C, and interleukin 6 were analyzed by drotrecogin alfa (activated) steady-state plasma concentration (C(ss)) quartile.
Transient endogenous activated protein C concentrations above 10 ng/mL were observed in 11 placebo-treated patients (3.3%). In drotrecogin alfa (activated)-treated patients, the median C(ss) was 44.9 ng/mL and the median plasma clearance (CL(p)) was 40.1 L/h. C(ss) was reached within 2 hours after the infusion was started. Plasma concentrations were below the assay quantitation limit of 10 ng/mL within 2 hours after the infusion was stopped in 92% of patients. CL(p) increased with increasing body weight, so infusion rates should be based on predose body weight. Mean CL(p) associated with age, sex, or baseline hepatic or renal function differed by less than 30% from the mean CL(p) in all patients and resided within the interquartile range of CL(p) in all patients. Dose adjustment is not required on the basis of these factors alone or in combination. No correlation was detected between C(ss) quartile and bleeding risk or the magnitudes of effect on biomarkers of coagulopathy (D-dimers and protein C) and inflammation (interleukin 6).
Plasma concentrations of drotrecogin alfa (activated) attain steady state rapidly after the infusion is started and decline rapidly after the infusion is stopped. The infusion rate should be based on predose body weight and not on any other demographic or baseline clinical covariate.
To document in patients with meningococcal purpura fulminans (PF), the effects of a combined supplementation with antithrombin (AT) and protein C (PC) plasma concentrates and to estimate the pharmacokinetics and dose requirements of each inhibitor.
Retrospective study of 15 patients. SETTING. One paediatric and one adult ICU in a university hospital.
In addition to standard intensive care, all patients received a 100 IU/kg loading dose of AT and PC concentrates, followed by a continuous infusion (AT: 100-150 IU.kg.day; PC: 100 IU.kg.day in adults, and 400 IU/kg in infants).
Clinical data, coagulation, and fibrinolysis parameters, AT and PC activities, and free protein S (PS) levels were sequentially measured. Restitution ratio, median increment after supplementation, and half-life of clearance from plasma were calculated for the two plasma substitutes. RESULTS. At admission, all patients had a severe decrease in AT, PC, and PS levels. The supplementation regimen induced a substantial increase in AT and PC activities, peaking at H18 and H48, respectively. The supplementation procedure did not modify free PS levels. The median values of AT and PC restitution ratio, increment in plasma activity observed after 100 IU/kg concentrate, and apparent half-life of clearance from plasma were 0.85 U.ml.U.kg and 0.59 U.ml.U.kg, 23% and 21%, 16 h and 6 h, respectively.
If AT and PC concentrates are to be given in fulminant meningococcemia, the doses of supplementation should be at least 150 IU/kg AT and 250 IU/kg PC as loading dose and 150 IU/kg AT and 200 IU/kg PC as daily maintenance therapy. Taking into account the individual variability in inhibitor deficiency and restitution ratio, repeated measurements of plasma levels are mandatory to obtain a patient-based adjustment of the supplementation.
Meningococcal septic shock in children results in high mortality and morbidity, and decreased protein C levels in these patients are associated with a poor outcome. We carried out a randomized, double-blinded, placebo-controlled study by supplying protein C concentrate. This phase 2 study was designed to assess the activation process of protein C and to study the dosing regimen of protein C concentrate in children with purpura fulminans and meningococcal septic shock in the perspective of a possible phase 3 trial.
Forty children were randomized to receive placebo or protein C concentrate (200 IU/kg, 400 IU/kg, or 600 IU/kg), for a maximum of 7 days. Clinical and laboratory data, including plasma levels of protein C and activated protein C (APC), were collected at various time points. All patients received standard therapy for septic shock, including antibiotics, inotropic/vasoactive drugs, and blood products.
Increased APC levels relative to baseline were observed for the 27 of 28 patients treated with protein C concentrate, and the areas under the curve of protein C and APC were correlated with the dosage of protein C concentrate administered. Activation of coagulation, as evidenced by d-dimer levels, as well as the ratio of thrombin vs. APC normalized significantly faster with increasing dosages of protein C concentrate. No adverse reactions related to protein C concentrate were observed. Nine of the 40 (23%) patients died, and five survivors required amputations, with no differences in these rates among the randomized groups. Baseline APC levels were positively correlated with sequential organ failure assessment and pediatric risk of mortality scores and with d-dimers, tumor necrosis factor-alpha, interleukin-1, interleukin-6, interleukin-8, plasminogen activator inhibitor-1, TAT complexes, and PAP complexes.
Treatment with protein C concentrate is safe in children with purpura fulminans and meningococcal septic shock and leads to dose-related increases of plasma APC and resolution of coagulation imbalances.
The clinical spectrum of sepsis, severe sepsis, and septic shock is responsible for a growing number of deaths and excessive health care expenditures. Until recently, despite multiple clinical trials, no intervention provided a beneficial outcome in septic patients. Within the last 2 years, studies that involved drotrecogin alfa (activated), corticosteroid therapy, and early goal-directed therapy showed efficacy in those with severe sepsis and septic shock. These results have provided optimism for reducing sepsis-related mortality.
Disseminated intravascular coagulation (DIC) is a common phenomenon in patients with sepsis, but the clinical implications of this condition are not clear. Clinical trials with coagulation inhibitors have failed to show a significant benefit concerning survival. DIC is primarily a laboratory diagnosis, based on the combination of elevated fibrin-related markers (FRM), with decreased procoagulant factors and platelets. Non-overt DIC is observed in most patients with sepsis, whereas overt DIC is less frequent. Patients with overt DIC may display consumption coagulopathy and purpura fulminans. Consumption coagulopathy is a bleeding disorder caused by low levels of platelets and procoagulant factors associated with massive coagulation activation. Purpura fulminans is caused by widespread microvascular thrombosis, resulting in tissue necrosis. Treatment with drotrecogin alfa (activated) improves survival and other outcome parameters in severe sepsis, including a subgroup of patients fulfilling the laboratory criteria of overt DIC. No randomized trials demonstrating effective therapies in consumption coagulopathy have been published. Bleeding patients with consumption coagulopathy are most frequently treated with platelet transfusions and various plasma products including fresh frozen plasma and coagulation factor concentrates. Based on case reports, treatment with drotrecogin alfa (activated) or substitution of protein C have been recommended for adjuvant treatment of sepsis-related purpura fulminans.