Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation

Tissue factor is the major initiator of coagulation. Both factor IX and factor X are activated by the complex of factor VIIa and tissue factor (VIIa/TF). The goal of this study was to determine the specific roles of factors IXa and Xa in initiating coagulation. We used a model system of in vitro coagulation initiated by VIIa/TF and that included unactivated platelets and plasma concentrations of factors II, V, VIII, IX, and X, tissue factor pathway inhibitor, and antithrombin III. In some cases, factor IX and/or factor X were activated by tissue factor- bearing monocytes, but in some experiments, picomolar concentrations of preactivated factor IX or factor X were used to initiate the reactions. Timed samples were assayed for both platelet activation and thrombin activity. Factor Xa was 10 times more potent than factor IXa in initiating platelet activation, but factor IXa was much more effective in promoting thrombin generation than was factor Xa. In the presence of VIIa/TF, factor X was required for both platelet activation and thrombin generation, while factor IX was only required for thrombin generation. We conclude that VIIa/TF-activated factors IXa and Xa have distinct physiologic roles. The main role of factor Xa that is initially activated by VIIa/TF is to activate platelets by generating an initial, small amount of thrombin in the vicinity of platelets. Factor IXa, on the other hand, enhances thrombin generation by providing factor Xa on the platelet surface, leading to prothrombinase formation. Only tiny amounts of factors IX and X need to be activated by VIIa/TF to perform these distinct functions. Our experiments show that initiation of coagulation is highly dependent on activation of small amounts of factors IXa and Xa in proximity to platelet surfaces and that these factors play distinct roles in subsequent events, leading to an explosion of thrombin generation. Furthermore, the specific roles of factors IXa and Xa generated by VIIa/TF are not necessarily reflected by the kinetics of factor IXa and Xa generation.     

Newer concepts of blood coagulation

Summary. In this report we describe an in vitro model of blood coagulation reactions that mimics as closely as possible the in vivo condition. Our model indicates that the tissue factor—factor VIIa complex initiates coagulation by activating small amounts of both factor IX and factor X in the environment of the tissue factor bearing cell. Factor Xa and factor IXa formed in the initial reaction then play very distinct roles in the subsequent interactions of the clotting mechanism leading to a burst of thrombin generation on the platelet surface. Our results also indicate that factor XI can be activated by thrombin in the absence of factor XII and that the function of factor XI is simply to enhance conversion of factor IX to factor IXa resulting in enhanced thrombin generation on the platelet surface.     

Platelet procoagulant complex assembly in a tissue factor-initiated system

Summary. The aim of this study was to examine the assembly of the factor IXa/VIIIa (Xase) and factor Xa/Va (IIase) complexes on the platelet surface in a system designed to mimic tissue factor-initiated coagulation. The experimental system contained tissue factor-bearing monocytes, unactivated platelets, and plasma concentrations of factors V, VIII, IX, X, prothrombin, tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and small amounts of factor VIIa. The time courses of platelet activation, coagulation factor binding and thrombin generation were compared. In this system, thrombin generation by the combination of monocytes and platelets was synergistic compared to each cell type alone. Platelet activation and thrombin generation were minimal in the absence of prothrombin or factor X. After a lag period, platelet activation began, followed by progressive binding of factors Va and VIIIa. This was followed by factor IXa and Xa binding and the onset of thrombin generation. Unexpectedly, a transient early increase in platelet-associated factor IX and X was also seen, that was due to release from platelets. The amount of factor IX bound to isolated activated platelets was increased by addition of factor VIIIa, or by activation of factor IX to IXa. In contrast, factor VIIIa binding was not altered by the presence of factor IX or IXa. We conclude that in a tissue factor-initiated system, assembly of the procoagulant complexes on the platelet surface begins after platelet activation occurs. Platelet activation requires thrombin generation in the vicinity of the tissue factor bearing cells. The cofactors Va and VIIIa bind to the platelets and facilitate subsequent binding of factors IXa and Xa to form functional procoagulant complexes.     

On the coagulation of platelet-rich plasma. Physiological mechanism and pharmacological consequences.

Thrombin formation and blood platelet reactions are intimately linked in haemostasis and in thrombosis. In vivo, procoagulant phospholipids required for the coagulation mechanism are mainly provided by activated platelets, and thrombin is the most potent platelet activator. To study these interactions, an ancient tool of coagulation physiology, the thrombin generation test, was revived and the results obtained were reviewed. The amount of thrombin activity that develops, expressed as the endogenous thrombin potential (the area under the thrombin generation curve), is influenced by the clotting factors (except XII and XIII), the activated protein C system and natural inhibitors on the one hand and by platelet activity on the other. The platelet reactions that we found to be involved are induced by thrombin via glycoprotein (GP) IIb/IIIa activation and by fibrin via interaction with GPIb. von Willebrand factor is crucial in both reactions and therefore an obligatory factor for normal thrombin generation in the presence of platelets. All antithrombotics, be it anticoagulants (e.g. OAC, all heparins or hirudin) or antiplatelet drugs (aspirin, GPIIb/IIIa blockers) diminish thrombin generation.     

Feedback activation of factor XI by thrombin does not occur in plasma

In this study, we tested the hypothesis that factor XI (FXI) activation occurs in plasma following activation of the extrinsic pathway by thrombin-mediated feedback activation. We used two different assays: (i) a direct measurement of activated FXI by ELISA and (ii) a functional assay that follows the activation of the coagulation cascade in the presence or absence of a FXI inhibiting antibody by monitoring thrombin activity. We failed to detect any FXI activation or functional contribution to the activation of the coagulation cascade in platelet poor or platelet-rich plasma, when activation was initiated by thrombin or tissue factor. Additionally, we found that, in the absence of a contact system inhibitor during blood draw, contact activation of FXI can mistakenly appear as thrombin- or tissue-factor-dependent activation. Thus, activation of FXI by thrombin in solution or on the surface of activated platelets does not appear to play a significant role in a plasma environment. These results call for reevaluation of the physiological role of the contact activation system in blood coagulation.     

A cell-based model of thrombin generation

We have developed a cell-based model of thrombin generation using activated monocytes as a source of tissue factor (TF) and platelets serving as a surface for thrombin generation. Monocytes are activated by lipopolysaccharide and express cell-bound TF. To these are added physiologic (plasma) concentrations of all the plasma procoagulants as well as TF pathway inhibitor, antithrombin, and C1-esterase inhibitor. Coagulation takes place in microtiter wells and is initiated by factor VIIa (FVIIa) and calcium. At time intervals, aliquots are removed, platelet activation is measured by the expression of P-selectin, and thrombin generation is measured by chromogenic assay. In addition, one can measure the activation of FIX, FX, FVIII, FV, and FXI. Initial results reveal that the FVIIa-TF interaction results in the activation of FX to FXa and FIX to FIXa. FXa stays in the vicinity of the TF-bearing cell and, in the presence of FVa, converts a small amount of prothrombin to thrombin on the surface of the TF cell. This small amount of thrombin is not sufficient to clot fibrinogen, but is sufficient to activate platelets and FVIII, FV, and FXI. Following platelet activation, FVIIIa, FVa, and FXa occupy sites on the activated platelet surface. FIXa, activated by TF-FVIIa, does not remain on the TF cell, but converts FX to FXa on the platelet surface. FXIa acts to boost FIXa generation on the activated platelet, increasing FXa and subsequent thrombin generation. We have also shown that activated protein C does not inactivate Va on the platelet surface but rather on endothelial cell surfaces.     

Role of blood coagulation factor XI in downregulation of fibrinolysis

Factor XI is a component of the intrinsic pathway of coagulation. A deficiency of factor XI is associated with a mild to moderate bleeding disorder especially from tissues with a high local fibrinolytic activity. In contrast, high levels of factor XI are a risk factor for venous thrombosis. The recent finding that factor XI can be activated by thrombin led to a revised model of coagulation. In this model the primary thrombin generation that results in fibrin formation takes place via the extrinsic pathway. Additional thrombin generation takes place inside the fibrin clot via the intrinsic pathway after the activation of factor XI by thrombin. High concentrations of thrombin are formed that are necessary for the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Activated TAFI protects the fibrin clot against lysis. The role of factor XI in hemostasis can therefore be seen as a combination of procoagulant and antifibrinolytic actions. The new insights in the role of factor XI in coagulation and fibrinolysis may lead to new strategies for the treatment of thrombotic disorders.     

Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.     

Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation

Platelets shed microparticles, which support haemostasis via adherence to the damaged vasculature and by promoting blood coagulation. We investigated mechanisms through which storage-induced microparticles might support blood coagulation. Flow cytometry was used to determine microparticle number, cellular origin and surface expression of tissue factor (TF), procoagulant phosphatidylserine (PtdSer) and glycoprotein (GP) Ib-alpha. The influence of microparticles on initiation and propagation of coagulation were examined in activated factor X (factor Xa; FXa) and thrombin generation assays and compared with that of synthetic phospholipids. About 75% of microparticles were platelet derived and their number significantly increased during storage of platelet concentrates. About 10% of the microparticles expressed functionally active TF, as measured in a FXa generation assay. However, TF-driven thrombin generation was only found in plasma in which tissue factor pathway inhibitor (TFPI) was neutralised, suggesting that microparticle-associated TF in platelet concentrates is of minor importance. Furthermore, 60% of all microparticles expressed PtdSer. In comparison with synthetic procoagulant phospholipids, the maximal rate of thrombin formation in TF-activated plasma was 15-fold higher when platelet-free plasma was titrated with microparticles. This difference could be attributed to the ability of microparticles to propagate thrombin generation by thrombin-activated FXI. Collectively, our findings indicate a role of microparticles in supporting haemostasis by enhancement of the propagation phase of blood coagulation.     

Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX- deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.     

Evidence that Collagen Releases Human Platelet Constituents by Two Different Mechanisms

SUMMARY
Collagen is believed to be involved in the initial events in haemostasis and has been shown by others to cause platelet aggregation and release, and also to initiate the intrinsic pathway of coagulation. The present experiments provide evidence which suggests how these many effects of collagen may be involved in haemostasis.
It is shown here that collagen releases platelet constituents by two different pathways. Collagen causes platelets washed free of loosely adsorbed coagulation factors to release constituents. This activity is, therefore, independent of the intrinsic pathway of coagulation, and is not inhibited by heparin or hirudin. Collagen also releases platelet constituents by an alternative pathway which is inhibited by heparin and hirudin and is independent of factor XII, but is dependent on factor XI, subsequent factors in the intrinsic pathway of coagulation and calcium. These results suggest that collagen-induced release of platelet constituents is in part due to a direct effect on the platelet, and, in part, to an indirect effect involving coagulation factors and mediated by thrombin. The present results suggest that irreversible aggregation by collagen is also mediated by thrombin.
The possible significance of this dual action of collagen in the haemostatic process is shown in Fig 7.     

Activated protein C cleaves factor Va more efficiently on endothelium than on platelet surfaces

The protein C/protein S system is known to regulate thrombin generation in vivo by cleaving factors Va and VIIIa. We have examined the activity of activated protein C in several tissue factor-initiated models of coagulation. We used 4 models: monocytes as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as both the tissue factor source and the thrombin-generating surface; and relipidated tissue factor with lipid vesicles providing the surface for thrombin generation. With the lipid surface, activated protein C dose-dependently reduced thrombin generation. Similarly, when endothelial cells provided the only surface for thrombin generation, activated protein C dose-dependently decreased thrombin generation significantly. By contrast, whenever platelets were present, activated protein C only minimally affected the amount of thrombin generated. When endothelial cells were the tissue factor source with platelets providing the surface for thrombin generation, activated protein C did increase the time until the burst of thrombin generation but had minimal effects on the total amount of thrombin generated. Activated protein C had essentially no effect on thrombin generation when monocytes were the tissue factor source with platelets providing the surface for thrombin generation. From the studies reported here, we conclude that in vivo, despite the important role of the protein C system in regulating thrombosis, activated protein C does not serve as a primary regulator of platelet-dependent thrombin generation.     

Activation of factor XI in plasma is dependent on factor XII [see comments]

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.     

Enzymatic Basis for Platelet Aggregation and Release: The Significance of the ''Platelet Atmosphere'' and the Relationship between Platelet Function and Blood Coagulation

Summary . Blood coagulation and platelet reactions such as aggregation and release are both essential to haemostasis and, although relationships between these two processes have been demonstrated, they have been regarded as different phenomena. The results reported here show that the two are inseparable, and that platelet aggregation and release are dependent on the ‘plasmatic atmosphere’ of coagulation factors surrounding platelets. Test systems were developed for studying platelets in plasma (Han & Ardlie, 1973) and in artificial media. When coagulation factors were removed from platelets by washing, only slight reversible aggregation occurred upon the addition of ADP and fibrinogen. Trace amounts of thrombin restored aggregation and TAMe completely inhibited aggregation. Irreversible aggregation and the release reaction of platelets were shown to be dependent on the generation of thrombin by the interaction of clotting factors on the platelet surface. Polymerizing fibrin caused irreversible aggregation and, conversely, inhibitors of fibrin polymerization prevented irreversible aggregation. Irreversible aggregates were insoluble in 5 M urea and 1% monochloroacetic acid suggesting involvement of factor XIII in this aspect of aggregation. It is proposed that aggregation of platelets by ADP is mediated initially by thrombin-fibrinogen complexes between adjacent adhering platelets. It is also proposed that the nature of the bond between platelets changes when the enzymatic phase of the thrombin—fibrinogen reaction is followed by the polymerization phase, and aggregation then becomes irreversible under the influence of factor XIII, activated by thrombin. Thrombin action also results in the release of platelet constituents. A scheme for haemostasis which emerges from this unification of platelet reactions and blood coagulation is presented in Fig 15, and possible ways in which various other platelet and plasma factors may be involved in haemostasis are briefly discussed.     

The Effects of Collagen and Kaolin on the Intrinsic Coagulant Activity of Platelets. EVIDENCE FOR AN ALTERNATIVE PATHWAY IN INTRINSIC COAGULATION NOT REQUIRING FACTOR XII

Collagen and kaolin have been shown by other workers to initiate intrinsic coagulation by activating factor XII in plasma and to have complex effects on platelets. Because of the presence of collagen at sites of vascular injury there is good reason to believe that collagen has physiological importance in haemostasis. The present experiments were done to determine the effects of collagen and kaolin on platelets and to distinguish the platelet effects from the activity which these surface-active agents produce in plasma.
Using an albumin-density-gradient separation (ADGS) method for washing platelets free of loosely adsorbed coagulation factors, it is shown here that collagen can induce a coagulant activity in platelets which initiates intrinsic coagulation. This activity is independent of factor XII, provided factor XI is present. It is postulated that this collagen-induced coagulant activity of platelets provides an alternative pathway, by-passing factor-XII activation, for initiating intrinsic coagulation. The existence of this alternative pathway may provide an explanation for the absence of a haemostatic defect in Hageman trait. The effects of kaolin were similar to those of collagen, but kaolin had greater capacity to activate plasma factor XII and platelet factor 3 and relatively less capacity to activate platelet-associated factor XI.     

Influence of coagulation factors on intrinsic thrombin generation.

The intrinsic coagulation activity assay (INCA) is a new thrombin-generation test that imitates the intrinsic pathway of blood coagulation. The aim of the present study was to investigate the influence of the main coagulation factors on the INCA. The INCA was performed with citrated plasma samples supplemented with different amounts of fibrinogen. The INCA and activated partial thromboplastin time determination were performed with factor-depleted plasmas and with mixtures of depleted plasmas with normal plasma. Supplemented purified fibrinogen resulted in a decrease of intrinsic thrombin generation (50% inhibitory concentration = 0.8 g/l). The INCA depends on the intrinsic factors (factors VIII, IX, XI and XII) and on the factors of the common pathway (factors II, V and X): for normal thrombin generation, at least about 50% of normal factor II is necessary. For the majority of factors, the sensitivity of the INCA appears to be approximately one order of magnitude better than that of the activated partial thromboplastin time. The INCA allows one to diagnose defects in the intrinsic coagulation system and might be a useful test to support development and characterization of new drugs targeted at the intrinsic generation of thrombin.     

Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow

The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.     

Influence of coagulation factors on extrinsic thrombin generation.

The extrinsic coagulation activity assay (EXCA) is a new thrombin generation test for the tissue factor pathway of coagulation. The EXCA was performed with 10 parts citrated plasma of different contents of fibrinogen. One part tissue factor, 250 mmol/l CaCl(2), generating about 1 IU/ml thrombin within 1 min (37 degrees C). After 0-30 min 2.5 mol/l arginine (pH 8.6) Generated thrombin was detected by addition of CHG-Ala-Arg-pNA and measurement of triangle upA/t. The EXCA is dependent on factors 10% of the factor VII norm in the sample achieves 70-80% of the thrombin generation norm. The EXCA is not dependent on factors VIII, IX, XI and XII. Even in antithrombin III-deficient plasma, a phase of thrombin inhibition appears after the thrombin peak. Supplemented purified fibrinogen resulted in decreased thrombin generation in the important. Fibrinogen seems to act as antithrombin I; thrombin might be entrapped in the nascent fibrin. The EXCA is suitable to diagnose the level of extrinsic factors in patient plasma.     

Venous thromboembolism in heparin-induced thrombocytopenia

Deep-vein thrombosis (DVT) and pulmonary embolism are among the most common complications of heparin-induced thrombocytopenia (HIT), an antibody-mediated adverse effect of heparin that leads paradoxically to in vivo activation of platelets and the coagulation system. Inappropriate treatment of HIT-associated DVT with warfarin can cause the DVT to progress to limb gangrene: this results from impaired ability of the protein C natural anticoagulant pathway to down-regulate thrombin generation, thus leading to microvascular thrombosis and tissue necrosis. Appreciation of the importance of coagulation system activation in HIT provides a rationale for treatments that reduce thrombin generation, either via inhibiting factor Xa (danaparoid) or via inhibiting thrombin directly (lepirudin). Clinicians should know how to distinguish HIT from other thrombocytopenic disorders: for example, thrombocytopenia associated with pulmonary embolism can mimic HIT (pseudo-HIT), and acute dyspnea that can mimic acute pulmonary embolism can result from acute in vivo platelet activation in a patient with HIT antibodies who receives heparin bolus therapy (pseudo-pulmonary embolism).     

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.