Figure 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
![Numerical simulations predict localization of polyP accelerates thrombin production at low shear rates. Two-dimensional numerical simulations of the human blood coagulation cascade, comparing the generation of thrombin by polyP dispersed throughout a cylindrical channel versus polyP immobilized on the channel surface. The channel was 20 mm long with a radius of 2 mm. The overall number of polyP molecules was the same in all simulations (7.54 × 10 −9 moles). (A) Plots show [thrombin], which is the sum of concentrations of thrombin and meizothrombin, for a two-dimensional longitudinal cut of the cylinder at 500 s into the simulation. (B) The fold difference in the maximum [thrombin] generated in the channel when polyP was surface-immobilized (SI-polyP) versus dispersed (D-polyP) at varying shear rates.](publication/313582021/figure/fig1/AS:462520319254532@1487284845865/Numerical-simulations-predict-localization-of-polyP-accelerates-thrombin-production-at.png)
Numerical simulations predict localization of polyP accelerates thrombin production at low shear rates. Two-dimensional numerical simulations of the human blood coagulation cascade, comparing the generation of thrombin by polyP dispersed throughout a cylindrical channel versus polyP immobilized on the channel surface. The channel was 20 mm long with a radius of 2 mm. The overall number of polyP molecules was the same in all simulations (7.54 × 10 −9 moles). (A) Plots show [thrombin], which is the sum of concentrations of thrombin and meizothrombin, for a two-dimensional longitudinal cut of the cylinder at 500 s into the simulation. (B) The fold difference in the maximum [thrombin] generated in the channel when polyP was surface-immobilized (SI-polyP) versus dispersed (D-polyP) at varying shear rates.
Source publication
Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowi...
Similar publications
The study was carried out to determine the values of whole blood coagulation time (WBCT), haematocrit (HM), haemaglobin (HB) and total protein (TP) of one hundred and eighteen apparently healthy turkeys reared under an extensive management system in Zaria. The mean values for WBCT, HM, HB and TP were 1.12 ± 0.02 min, 31.31 ± 0.52%, 10.43 ± 0.17g% a...
Citations
... 11 However, concentration of short (platelet-sized) polymers through biochemical manipulation corrects this difference. [12][13][14] A similar, but natural, concentration phenomenon appears to occur inand on platelets, where polyphosphate exists in a crystalline state, complexed to divalent metal ions. 15 This helps to explain why platelet polyphosphate (despite its short polymer length) can drive pathological thrombus formation and blockade of polyphosphate is therapeutic in these models. ...
Medical device associated thrombosis is an important clinical problem. This type of thrombosis can result from Factor XII (FXII) binding to non-natural surface materials and subsequent activation of the contact pathway. This drives the development of new therapeutic strategies to block this pathway and information on the structural properties of FXII should catalyse this quest. Presently, there is no publicly available crystal structure of full-length FXII. However, the AlphaFold Protein Structure Database provides a model structure. We here explore this model in combination with previous structure-function studies to identify opportunities for selective pharmacological blockade of the contribution of FXII in medical device associated thrombosis. Previous studies demonstrated that FXII activation is dependent on molecular cleavage after R353. We subsequently proposed that protein conformation protects this cleavage site to ensure zymogen quiescence and prevent inappropriate FXII activation. The AlphaFold model shows that a small loop containing R353 indeed is buried in the globular molecule. This is the result of intra-molecular interactions between the (N-terminal) Fibronectin type II domain, (central) kringle and (C-terminal) protease domain, in a structure that resembles a three-point harness. Furthermore, this interaction pushes the intermediate domains, as well as the flexible proline-rich region (PRR), outward while encapsulating R353 in the molecule. The outward directed positively charged patches are likely to be involved in binding to anionic surfaces. The binding of FXII to surfaces (and several monoclonal antibodies) acccelerates its activation by inducing conformational changes. For prevention of medical device associated thrombosis, it is therefore important to target the surface binding sites of FXII without causing structural changes.
... However, there is limited experience with generating pure PolyP-NPs, moreover, developed particles are stable only for a few hours [10]. Our paper presents a further improved version of previously described method for preparing pure PolyP-NPs [10,11]. The generated particles were of a size similar to those secreted by platelets in vivo (100-200 nm [6]), retained the former introduced polyphosphate functions on clot forming and lysis and were stable for at least 5 days stored in BSA-TBS at 4˚C. ...
Introduction
Beyond the three-dimensional fibrin network, the mechanical and lytic stability of thrombi is supported by the matrix of neutrophil extracellular traps (NETs) composed of polyanionic DNA meshwork with attached proteins including polycationic histones. Polyphosphates represent another type of polyanions, which in their linear form are known to enhance the fibrin stabilizing effects of DNA and histones. However, in vivo polyphosphates are also present in the form of nanoparticles (PolyP-NP), the interference of which with the fibrin/NET matrix is poorly characterized.
Aims
To compare the effects of linear and nanoparticulate polyphosphates, and their combinations with relevant NET components (DNA, histone H3) on fibrin formation, structure, and lysis in in vitro assays focusing on histone-polyphosphate interactions.
Methods
Transmission electron microscopy and dynamic light scattering for stability of the PolyP-NP preparations. Turbidimetry for kinetics of fibrinogen clotting by thrombin and fibrin dissolution by tissue-type plasminogen activator/plasminogen. Scanning electron microscopy for fibrin structure. Surface plasmon resonance for strength of histone-PolyP interactions.
Results
Both linear PolyP and PolyP-NP accelerated the fibrin formation and slowed down its dissolution and these effects were strongly dependent on the number of individual PolyP particles and not on their size. Addition of DNA did not modify significantly the PolyP-NP effects on fibrin formation and lysis. Both linear and nanoparticulate PolyP counteracted the effect of histone in the acceleration of fibrinogen clotting by thrombin. PolyP-NP, but not linear PolyP enhanced the prolongation of lysis time in fibrin containing histone and caused more pronounced thickening of the fibrin fibers than the linear form. Finally, PolyP-NP bound weaker to histone than the linear form.
Conclusions
The interaction of PolyP with histone was a stronger modulator of fibrin formation and lysis than its interaction with DNA. In addition, the PolyP nanoparticles enhanced the thrombus stabilizing effects of histone more effectively than linear PolyP.
... 73 Due to the important role of platelet-derived PolyP in modulating coagulation kinetics and clot structure, some approaches are also exploring PolyP delivery using nanoparticle platforms. 74,75 In another recent approach, we have explored the exposure of anionic phospholipids F I G U R E 3 Various design approaches for platelet-inspired hemostatic nanomedicine constructs (e.g. phosphatidylserine, PS) on the surface of SynthoPlate nanoparticles in an injury site-selective manner, inspired by the platelet procoagulant function. ...
Platelets are anucleate cell‐fragments derived predominantly from megakaryocytes in the bone marrow and released in the blood circulation, with a normal count of 150,000‐400,00 per μL and a lifespan of approximately 10 days in humans. A primary role of platelets is to aid in vascular injury site‐specific clot formation to stanch bleeding, termed hemostasis. Platelets render hemostasis by a complex concert of mechanisms involving platelet adhesion, activation and aggregation, coagulation amplification, and clot retraction. Additionally, platelet secretome can influence coagulation kinetics and clot morphology. Therefore, platelet defects and dysfunctions result in bleeding complications. Current treatment for such complications involve prophylactic or emergency transfusion of platelets. However, platelet transfusion logistics constantly suffer from limited donor availability, challenges in portability and storage, high bacterial contamination risks, and very short shelf life (~ 5 days). To address these issues, an exciting area of research is focusing on the development of microparticle‐ and nanoparticle‐based platelet surrogate technologies that can mimic various hemostatic mechanisms of platelets. On the other hand, aberrant occurrence of the platelet mechanisms lead to the pathological manifestation of thrombosis and thromboinflammation. The treatments for this are focused on inhibiting the mechanisms or resolving the formed clots. Here, platelet‐inspired technologies can provide unique platforms for disease‐targeted drug delivery to achieve high therapeutic efficacy while avoiding systemic side‐effects. This review will provide brief mechanistic insight into the role of platelets in hemostasis, thrombosis and thromboinflammation, and present the current state‐of‐art in the design of platelet‐inspired nanomedicine for applications in these areas.
... PolyP concentrations in most mammalian cells and tissues are considerably lower than those in in yeast (Ͻ100 M versus Ͼ200 mM, respectively) (27, 28), although the dense granules of platelets are an exception (29). Here, local polyP concentrations can reach ϳ100 mM, and the release of polyP from these structures following injury plays important roles in blood coagulation (29)(30)(31)(32)(33)(34)(35)(36). PolyP also impacts cell cycle and growth control (37-39), mitochondrial functions (40-42), and neuronal signaling (43-45). ...
During stress, bacterial cells activate a conserved pathway called the stringent response that promotes survival. Polyphosphates are long chains of inorganic phosphates that modulate this response in diverse bacterial species. In this issue (pgs. XXX-XXX) (1), Michael Gray provides an important correction to the model of how polyphosphate accumulation is regulated during the stringent response in E. coli. With other recent publications, this study provides a revised framework for understanding how bacterial polyphosphate dynamics might be exploited in infection control and industrial applications.
... 57 Consistently, localization onto particles increases procoagulant activity of short-chain polyP even under flow conditions. 58 The procoagulant properties of insoluble polyP packed in nanoparticles largely differ from those of molecularly dissolved soluble molecules. FXII-activating properties of polymers in nanoparticle form largely exceed that of dispersed polyP in solution. ...
Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. The factor XII-driven-contact system starts coagulation and inflammatory mechanisms via the intrinsic pathway of coagulation and the bradykinin-producing kallikrein-kinin system, respectively. The biochemistry of the contact systemin vitrois well understood, however itsin vivofunctions are just beginning to emerge. Challenging the concept of the coagulation balance, targeting factor XII or its activator polyphosphate provides protection from thromboembolic diseases without interfering with hemostasis. This suggests that the polyphosphate/factor XII axis contributes to thrombus formation while being dispensable for hemostatic processes. In contrast to deficiency in factor XII providing safe thromboprotection, excessive FXII activity is associated with the life-threatening inflammatory disorder hereditary angioedema. The current review summarizes recent finding of the polyphosphate/factor XII-driven contact system at the intersection of procoagulant and proinflammatory disease states. Elucidating the contact system offers the exciting opportunity to develop strategies for safe interference with both thrombotic and inflammatory disorders.
Polyphosphate (PolyP) is a polymer comprised of linear phosphate units connected by phosphate anhydride bonds. PolyP exists in a diverse range of eukaryotes and prokaryotes with varied chain lengths ranging from six to thousands of phosphate units. Upon activation, human platelets and neutrophils release short-chain PolyP, along with other components, to initiate the coagulation pathway. Long-chain PolyP derived from cellular or bacterial organelles exhibits higher proinflammatory and procoagulant effects compared to short-chain PolyP. Notably, PolyP has been identified as a low-hemorrhagic antithrombotic target since neutralizing plasma PolyP suppresses the thrombotic process without impairing the hemostatic functions. As an inorganic polymer without uniform steric configuration, PolyP is typically targeted by cationic polymers or recombinant polyphosphatases rather than conventional antibodies, small-molecule compounds, or peptides. Additionally, because of its procoagulant property, PolyP has been incorporated in wound-dressing materials to facilitate blood hemostasis. This review summarizes current studies on PolyP as a low-hemorrhagic antithrombotic target and the development of hemostatic materials based on PolyP.
Coronary artery thrombosis is the major risk associated with Kawasaki disease (KD). Long-term management of KD patients with persistent aneurysms requires a thrombotic risk assessment and clinical decisions regarding the administration of anticoagulation therapy. Computational fluid dynamics has demonstrated that abnormal KD coronary artery hemodynamics can be associated with thrombosis. However, the underlying mechanisms of clot formation are not yet fully understood. Here we present a new model incorporating data from patient-specific simulated velocity fields to track platelet activation and accumulation. We use a system of Reaction-Advection-Diffusion equations solved with a stabilized finite element method to describe the evolution of non-activated platelets and activated platelet concentrations [AP], local concentrations of adenosine diphosphate (ADP) and poly-phosphate (PolyP). The activation of platelets is modeled as a function of shear-rate exposure and local concentration of agonists. We compared the distribution of activated platelets in a healthy coronary case and six cases with coronary artery aneurysms caused by KD, including three with confirmed thrombosis. Results show spatial correlation between regions of higher concentration of activated platelets and the reported location of the clot, suggesting predictive capabilities of this model towards identifying regions at high risk for thrombosis. Also, the concentration levels of ADP and PolyP in cases with confirmed thrombosis are higher than the reported critical values associated with platelet aggregation (ADP) and activation of the intrinsic coagulation pathway (PolyP). These findings suggest the potential initiation of a coagulation pathway even in the absence of an extrinsic factor. Finally, computational simulations show that in regions of flow stagnation, biochemical activation, as a result of local agonist concentration, is dominant. Identifying the leading factors to a pro-coagulant environment in each case—mechanical or biochemical—could help define improved strategies for thrombosis prevention tailored for each patient.
In this review, we highlight well-described and emerging polyanions, and the way these molecules can be targeted in the design of potential therapeutics (synthetic and biologics) with applications in thrombosis and hemostasis. It is important to strike a balance between bleeding and clotting. In thrombosis, unwanted blood clots are formed in the lumen of a blood vessel, obstructing the flow of blood through the circulatory system. Over many years of research, several polyanionic biopolymers that can either impede (anticoagulant) or promote (procoagulant) blood clotting have been identified. Mediators impeding blood clotting including polyanionic polysaccharides such as heparins and heparin mimics are widely used as antithrombotics although they impart adverse complications such as bleeding. Emerging synthetic polycations and well-described cationic proteins that are specifically designed to neutralize the biological activity of heparins to prevent bleeding complications are discussed. On the other hand, there is growing evidence that several polyanions bear a procoagulant nature in blood; polyphosphate (polyP), neutrophil extracellular traps (NETs), extracellular RNA and cell free DNA are shown to promote blood clotting. Recent research highlights the use of polycations and enzymes that either inhibit or cleave these procoagulant polyanions and demonstrates the proof-of-concept design of new antithrombotics without bleeding side effects. Additional studies have shown that some of these procoagulant polyanions can be used as a hemostat to prevent bleeding in emergency. There are significant opportunities for chemists in the design of new inhibitors and agents with improved selectivity towards these biological polyanions furthering the development of novel therapeutics.
Inorganic polyphosphate (PolyP) is a potential hemostatic material. However, the effect of PolyP chain length on the immune response and hemostatic function remains to be established. We have developed PolyP-conjugated hyaluronans (HA-PolyPs) with three different short-chain PolyPs (n = 13, 40, and 100 phosphate units). All short-chain PolyP showed biocompatibility in the cell viability and inflammatory cytokine secretion test in vitro and in vivo, wherein shorter PolyPs showed milder response in some cases. We then produced HA-PolyP hydrogels (HAX-PolyPs) with three different short-chain PolyPs as hemostats. Interestingly, in vivo biocompatibility and hemostatic activity of HAX-PolyP was not significantly affected by the length of conjugated PolyPs. HAX-PolyP with all chain length significantly decreased the amount of bleeding in a novel mouse liver bleeding model. These results indicated that the shortest PolyP (n = 13) induced milder acute inflammation and had an efficient hemostatic effect when conjugated to hyaluronic acid. The present study provides key insights into the design of PolyP-based biomaterials and bioconjugates, which are expected to grow in importance for various medical applications.
This illustrated review focuses on polyphosphate as a potent modulator of the plasma clotting cascade, with possible roles in hemostasis, thrombosis, and inflammation. Polyphosphates are highly anionic, linear polymers of inorganic phosphates that are widespread throughout biology. Infectious microorganisms accumulate polyphosphates with widely varying polymer lengths (from a few phosphates to over a thousand phosphates long), while activated human platelets secrete polyphosphate with a very narrow size distribution (about 60‐100 phosphates long). Work from our lab and others has shown that long‐chain polyphosphate is a potent trigger of clotting via the contact pathway, while polyphosphate of the size secreted by platelets accelerates factor V activation, blocks the anticoagulant activity of tissue factor pathway inhibitor, promotes factor XI activation by thrombin, and makes fibrin fibrils thicker and more resistant to fibrinolysis. Polyphosphate also modulates inflammation by triggering bradykinin release, inhibiting the complement system, and modulating endothelial function. Polyphosphate and nucleic acids have similar physical properties and both will trigger the contact pathway—although polyphosphate is orders of magnitude more procoagulant than either DNA or RNA. Important caveats in these studies include observations that nucleic acids and polyphosphate may co‐purify, and that these preparations can be contaminated with highly procoagulant microparticles if silica‐based purification methods are employed. Polyphosphate has received attention as a possible therapeutic, with some recent studies exploring the use of polyphosphate in a variety of formulations to control bleeding. Other studies are investigating treatments that block polyphosphate function as novel antithrombotics with the possibility of reduced bleeding side effects.
