Regulation of prothrombinase activity by protein S.

The independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (approximately 0.2 microM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd approximately 10(-8) M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and prothrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 x 10(8)/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.     

Platelet activation and blood coagulation

Platelet activation and blood coagulation are complementary, mutually dependent processes in haemostasis and thrombosis. Platelets interact with several coagulation factors, while the coagulation product thrombin is a potent platelet-activating agonist. Activated platelets come in a procoagulant state after a prolonged elevation in cytosolic [Ca2+]i. Such platelets, e.g. when adhering to collagen via glycoprotein VI, expose phosphatidylserine (PS) at their outer surface and produce (PS-exposing) membrane blebs and microvesicles. Inhibition of aminophospholipid translocase and activation of phospholipid scramblase mediate the exposure of PS, whereas calpain-mediated protein cleavage leads to membrane blebbing and vesiculation. Surface-exposed PS strongly propagates the coagulation process by facilitating the assembly and activation of tenase and prothrombinase complexes. Factor IXa and platelet-bound factor Va support these activities. In addition, platelets can support the initiation phase of coagulation by providing binding sites for prothrombin and factor XI. They thereby take over the initiating role of tissue factor and factor VIIa in coagulation activation.     

The influence of the surface charge of phospholipids on the activity of the prothrombin converting complex

During blood coagulation factor V is converted into its activated form, factor Va, by the action of thrombin. In the presence of Ca⁺⁺-ions, phospholipids, factor Xa and factor Va, a complex is formed, prothrombinase, which converts prothrombin into thrombin. The influence of the surface charge of various phospholipid micelles on the formation of this complex was studied. As phospholipids purified phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositin (PI), phosphatidylcholine (PC) and mixed micelles of some of these phospholipids were used.

The experiments revealed that after the addition of phospholipids to factors Va, Xa, II and Ca⁺⁺-ions two reaction phases could be distinguished. During the first phase a fall of factor Va activity occurred, which was faster as the negative charge of the micelles increased. The presence of factors II and Xa was not required for this reaction. This fall in activity suggests that Ca⁺⁺-ions are needed for the stability of factor Va. During the second phase maximal reduction rate of factor Va activity, accompanied by maximal thrombin generation from prothrombin, occurred only in the case of an optimal negative surface charge of the phospholipid micelles. The presence of factors II and Xa was essential for this reaction. The second reaction phase probably implies consumption of factor Va in favour of thrombin generation.     

Thrombin-induced platelet factor Va formation in patients with a gray platelet syndrome

The present study was initiated to establish the functional factor V concentration in platelets of patients with a mild bleeding disorder ascribed to a gray platelet syndrome. This inherited platelet disorder has been characterized by a specific deficiency of alpha-granules and subsequent deficiencies in the alpha-granule proteins. We found that the concentration of plasma factor V was slightly decreased (70% of normal values). In contrast, platelet factor Va formation was severely impaired. Besides a much lower factor V content than in control platelets (10-20% of normal), the dependency of platelet factor Va formation on thrombin concentration was altered. Increasing the thrombin concentration 4-fold compared to the concentration that results in maximal factor Va generation from normal platelets did not result in a maximal factor Va formation from gray platelets. When a suspension of washed gray platelets was incubated with a prostacyclin analogue prior to the stimulation with thrombin, a 10-fold lower factor Va activity was measured. Thus, thrombin-induced factor Va formation in a suspension of gray platelets is the result of a release reaction, followed by the thrombin-catalyzed activation of released factor V. Whereas the kinetics of the former reaction are apparently impaired, the kinetics of the latter one were found to be identical to those observed for normal platelet and plasma factor V activation.     

Evidence for appearance of “lysis independent” procoagulant activities in collagen or thrombin stimulated platelets

The availability of procoagulant activities in platelet suspensions lysed by sonication correlated directly with the degree of lysis (loss of LDH and ⁵¹Cr). Separate measurements of platelet factor 3 (PF3) and Factor V/Va activity revealed significant differences in appearance of these platelet activities in sonicated cell suspensions. Platelet factor 3 activity increased almost in parallel to the loss of ⁵¹Cr, whereas Factor V/Va activity became available at a significant slower rate. This might be explained by the localization of Factor V/Va activities inside the α-granules and therefore not immediately available on the surface of the partially lysed platelets.No increase in lactate dehydrogenase or ⁵¹Cr-loss was observed during 12 min incubation of platelet suspension with collagen or thrombin, whereas both PF3 and Factor V/Va activities increased. Thus, the high platelet factor 3 activity of 34% (lysed platelets 100%) measured in thrombin stimulated platelet suspensions probably represents “lysis-independent” activity.     

The role of platelet factor V in prothrombin conversion

The contribution of platelet factor V to prothrombin conversion was studied in a purified two-stage system designed to measure the ability of factor V to accelerate prothrombin conversion. When unstimulated gel-filtered platelets (GFP) were substituted for both factor V and phospholipid, thrombin evolution was linear following a long lag time. Gel filtration resulted in considerable phospholipid availability with minimal factor V release. Incubating platelets with collagen in increasing concentrations resulted in marked shortening of the lag time, an increase in the initial rate of thrombin formation, and release of platelet factor V. The inhibition of thrombin formation by preincubation of the platelets with metabolic inhibitors is consistent with previous observations that factor V is released from alpha-granules by collagen in a process requiring metabolic energy. Released platelet factor V added to metabolically inhibited platelets reproduces the acceleration of prothrombin conversion demonstrated in GFP incubated with collagen. Furthermore no acceleration of the clotting time at collagen concentrations used in this study was demonstrated in an assay designed to measure available platelet phospholipid in the presence of excess factor V. The rate of increased thrombin generation produced by collagen stimulation is primarily due to released platelet factor V in the system employed.     

Collagen derived octapeptide inhibits platelet procoagulant activity induced by the combined action of collagen and thrombin

Platelet prothrombin converting activity was measured in a system using washed human platelets and purified coagulation factors Xa, Va and prothrombin. Exposure of platelet prothrombin converting activity evoked by collagen or the combined action of collagen and thrombin was effectively inhibited when a collagen derived octapeptide was added prior to platelet activation. Half maximal inhibition of prothrombinase activity of platelets stimulated by collagen plus thrombin- or collagen alone was obtained at 0.9 mM and 0.5 mM octapeptide, respectively. This suggests a modifying effect of thrombin on the platelet-collagen interaction. Octapeptide either alone or in combination with thrombin was unable to enhance platelet procoagulant activity. The increased prothrombin converting activity seen upon treatment of platelets with ionophore A23187 was not affected by octapeptide, added either before or after treatment with ionophore. It is concluded that octapeptide specifically interferes with the platelet-collagen interaction required to generate a procoagulant surface which enhances the rate of thrombin formation.     

Phospholipid-bound tissue factor modulates both thrombin generation and APC-mediated factor Va inactivation

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.     

Effects of Ethanol on Platelet Responses Associated with Adhesion to Collagen

Adhesion of platelets to collagen in damaged blood vessels or ruptured atherosclerotic plaques is important in hemostasis and arterial thrombosis. Adhesion to collagen results in secretion of granule contents and formation of thromboxane A₂; thromboxane A₂ and released ADP synergistically promote aggregation around platelets adherent to collagen. Ethanol inhibits collagen-induced platelet aggregation, secretion, arachidonate mobilization, and thromboxane A₂ formation but does not inhibit platelet adhesion to de-endothelialized rabbit aortae. We investigated whether ethanol affects the initial signalling events and responses of platelets adherent to collagen, independent of the actions of secondary agonists. Suspensions of washed human platelets, labelled by incorporation of [³H]oleate into phospholipids, were used to measure platelet adhesion to collagen by a filtration method; studies were done in the presence of an ADP-removing system and blockers of receptors for thromboxane A₂, platelet-activating factor, serotonin, and fibrinogen. Ethanol (87 mM) did not affect the rate or extent of platelet adhesion to collagen or secretion of [¹⁴C]serotonin from prelabelled platelets adherent to collagen, but ethanol did inhibit thromboxane A₂ formation. Previous studies showed that ethanol does not affect platelet stimulation by arachidonate, leading to the suggestion that reduced mobilization of arachidonate, rather than inhibition of its conversion to thromboxane A₂, is responsible for inhibition by ethanol of thromboxane A₂ formation. Here, we show by a gel mobility shift assay and immunoblotting, that ethanol delays the collagen-induced increase in the phosphorylation of cytosolic phospholipase A₂, the enzyme responsible for arachidonate mobilization. However, ethanol has no effect on collagen-induced tyrosine phosphorylation of phospholipase Cγ2, determined by immunoprecipitation and immunoblotting. Thus, ethanol's effect on signal transduction in collagen-adherent platelets occurs distal to phosphorylation of phospholipase Cγ2 but proximal to phosphorylation of cytosolic phospholipase A₂.     

GPIIb-IIIa antagonist-induced reduction in platelet surface factor V/Va binding and phosphatidylserine expression in whole blood

In addition to inhibition of platelet aggregation, GPIIb-IIIa antagonists may reduce thrombotic events via other mechanisms. In a novel whole blood flow cytometric system, we investigated the effects of GPIIb-IIIa antagonists, in the presence or absence of thrombin inhibitors, on platelet surface-bound factor V/Va and platelet surface phospholipids. Diluted venous blood was incubated with either buffer or a GPIIb-IIIa antagonist (abciximab, tirofiban, or eptifibatide). Some samples were pre-incubated with clinically relevant concentrations of unfractionated heparin (UFH), a low molecular weight heparin, a direct thrombin inhibitor, or buffer only. Platelets were then activated and labeled with mAb V237 (factor V/Va-specific) or annexin V (binds phosphatidylserine), fixed, and analyzed by flow cytometry. In the absence of thrombin inhibitors, GPIIb-IIIa antagonists (especially abciximab) significantly reduced agonist-induced platelet procoagulant activity, as determined by reduced binding of V237 and annexin V. At high pharmacologic concentrations, unfractionated heparin and enoxaparin, but not hirudin, further reduced factor V/Va binding to the surface of activated platelets in the presence of GPIIb-IIa antagonists. Agonist-induced platelet procoagulant activity was reduced in a patient with Glanzmann's thrombasthenia. We conclude that GPIIb-IIIa antagonists reduce platelet procoagulant activity in whole blood and heparin and enoxaparin augment this reduction. Fibrinogen binding to GPIIb-IIIa is important in the generation of platelet procoagulant activity.     

Protein S and factor V in regulation of coagulation on platelet microparticles by activated protein C

Introduction

Platelets are the main source of microparticles in plasma and the concentration of microparticles is increased in many diseases. As microparticles expose negatively charged phospholipids, they can bind and assemble the procoagulant enzyme-cofactor complexes. Our aim was to elucidate possible regulation of these complexes on microparticles by the anticoagulant protein C system.

Materials and methods

Platelets were activated with thrombin ± collagen or the calcium ionophore A23187 ± thrombin to generate microparticles. The microparticles were analyzed using flow cytometry and functional coagulation assays to characterize parameters with importance for the activated protein C system.

Results

Activation with A23187 + thrombin was most efficient, fully converting the platelets to microparticle-like vesicles, characterized by high lactadherin and protein S binding capacity. Suppression of thrombin generation by activated protein C in plasma spiked with these microparticles was dependent on the presence of plasma protein S. Experiments with purified components showed that activated protein C inhibited both factor Va and factor VIIIa on the microparticle surface. Inhibition of factor Va was stimulated by, but not fully dependent on, the presence of protein S. In the factor VIIIa-degradation, activated protein C was dependent on the addition of protein S, and exogenous factor V further increased the efficiency.

Conclusions

Protein S is crucial for activated protein C-mediated inhibition of thrombin generation on platelet-derived microparticles in plasma. Moreover, protein S and factor V are synergistic cofactors in the inhibition of factor VIIIa. The results demonstrate that the activated protein C system has the capacity to counterbalance the procoagulant ability of microparticles.     

Thrombin stimulated platelet accumulation on protein coated glass capillaries: role of adhesive platelet alpha-granule proteins

The generation of trace amounts of thrombin at artificial surfaces in contact with blood is likely to be a contributing factor in thrombosis on biomaterials. Using an in vitro capillary perfusion system, platelet accumulation on glass surfaces, uncoated or precoated with purified bovine collagen or human plasma proteins, was determined in the presence or absence of preadsorbed purified human thrombin. Static adsorption for 15 min at 22 degrees C from solutions of thrombin 100 NIH units (33 micrograms)/ml gave surface concentrations in the range 0.019-0.101 micrograms/cm2. Protein coated capillaries, thrombin treated or untreated, were perfused for 2 min at 37 degrees C with suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer containing 40% washed red blood cells, under conditions of controlled, non pulsatile laminar flow (50 s-1 or 2,000 s-1). Platelet accumulation was increased in the presence of surface adsorbed thrombin on uncoated and albumin or fibrinogen coated glass but little affected on fibronectin or collagen coated glass. On von Willebrand factor (vWF) coated glass, thrombin enhancement was observed only at high shear forces. In experiments using antibodies against human platelet alpha-granule proteins, thrombin stimulated platelet deposition in uncoated glass capillaries was inhibited at 2,000 s-1 by anti-vWF and to a lesser extent by anti-fibrinogen but not by anti-thrombospondin antibodies.     

Human platelets contain forms of factor V in disulfide-linkage with multimerin

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage.The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.     

Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of alpha2beta1 but not alphaIIbbeta3 integrin

The role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in enhancing thrombin generation, in a way dependent on exposure of phosphatidylserine (PS) at the platelet surface. Soluble, non-fibrillar type-I collagen required pre-activation of integrin alpha2beta1 with Mn2+ for enhancement of thrombin generation. With all preparations, blocking of glycoprotein VI (GPVI) with 9O12 antibody abrogated the collagen-enhanced thrombin generation, regardless of the alpha2beta 1 activation state. Blockade of alpha2beta1 alone or antagonism of autocrine thromboxane A2 and ADP were less effective. Blockade of alphaIIbbeta3 with abciximab suppressed thrombin generation in platelet-rich plasma, but this did not abolish the enhancing effect of collagens. The high activity of type-I fibrillar collagens in stimulating GPVI-dependent procoagulant activity was confirmed in whole-blood flow studies, showing that these collagens induced relatively high expression of PS. Together, these results indicate that: i) fibrillar type-I collagen greatly enhances thrombin generation, ii) GPVI-induced platelet activation is principally responsible for the procoagulant activity of fibrillar and non-fibrillar collagens, iii) alpha2beta1 and signaling via autocrine mediators facilitate and amplify this GPVI activity, and iv) alphaIIbbeta3 is not directly involved in the collagen effect.     

The role of factor XI in thrombin generation induced by low concentrations of tissue factor.

Thrombin generation has been studied in the plasma of severely factor XI deficient patients under conditions in which contact activation did not play a role. In platelet-rich as well as platelet-poor plasma, thrombin generation was dependent upon the presence of factor XI at tissue factor concentrations of between 1 and 20 pg/ml i.e. approximately 0.01 to 0.20% of the concentration normally present in the thromboplastin time determination. The requirement for factor XI is low; significant thrombin generation was seen at 1% factor XI; at 10%, thrombin formation was nearly normalised. A suspension of normal platelets in severely factor XI deficient plasma did not increase thrombin generation. This implies that there is no significant factor XI activity carried by normal platelets, although the presence of factor XI and factor XI inhibitors in platelets cannot be ruled out.     

In vitro effects of ethanol on rabbit platelet aggregation, secretion of granule contents, and cyclic AMP levels in the presence of prostacyclin

Ethanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin-treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.     

Inhibition of thrombin- and collagen-induced phosphoinositides breakdown in rabbit platelets by a PAF antagonist--denudatin B, an isomer of kadsurenone

Denudatin B, an isomer of kadsurenone, was isolated from Magnolia fargesii. It inhibited the aggregation and ATP release of washed rabbit platelets caused by platelet-activating factor (PAF) in a concentration-dependent manner. The IC50 on PAF (2 ng/ml)-induced aggregation was about 10 micrograms/ml. High concentration of denudatin B (greater than 50 micrograms/ml) also inhibited the aggregation and ATP release of platelets caused by ADP, collagen, arachidonic acid and thrombin. However, shape change of platelets still existed. Prolongation of the incubation time with platelets could not cause further inhibition, and the aggregability of platelets could be restored after denudatin B was washed out from platelets. Thrombin-induced thromboxane B2 formation was almost completely suppressed. In the absence of extracellular calcium (EGTA 1 mM), ATP release caused by thrombin was inhibited. Thrombin-induced rise of the intracellular calcium concentration was suppressed by denudatin B, but not by BN52021 or kadsurenone. The generation of inositol phosphate in washed platelets caused by collagen, PAF and thrombin was also suppressed. The data indicate that PAF antagonist denudatin B has nonspecific antiplatelet action at high concentration by inhibiting phosphoinositides breakdown induced by collagen and thrombin.     

Importance of N-terminal residues in plasminogen activator inhibitor 1 on its antibody induced latency transition

Bothrojaracin (BJC) is a 27 kDa snake venom protein from Bothrops jararaca that has been characterized as a potent ligand (KD = 75 nM) of human prothrombin (Monteiro RQ, Bock PE, Bianconi ML, Zingali RB, Protein Sci 2001; 10: 1897-904). BJC binds to the partially exposed anion-binding exosite I (proexosite I) forming a stable 1:1, non-covalent complex with the zymogen whereas no interaction with fragment 1 or 2 domains is observed. In addition, BJC interacts with thrombin through exosites I and II (KD = 0.7 nM), and influences but does not block the proteinase catalytic site. In the present work we studied the effect of BJC on human prothrombin activation by factor Xa in the absence or in the presence of its cofactors, factor Va and phospholipids. In the absence of phospholipids, BJC strongly inhibited (80%) the zymogen activation by factor Xa in the presence but not in the absence of factor Va, suggesting a specific interference in the cofactor activity. In the presence of phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine), BJC also inhibited (35%) prothrombin activation by factor Xa in the presence but not in the absence of factor Va. BJC showed a higher inhibitory effect (70%) towards thrombin formation by prothrombinase complex assembled on phospholipid vesicles composed by 95% phosphatidylcholine, 5% phosphatidylserine. Activation of prothrombin by platelet-assembled prothrombinase complex (factor Xa, factor Va and thrombin-activated platelets) showed that hirudin (SO3-) and BJC efficiently inhibit the thrombin formation (43% and 84%, respectively). Taken together, our results suggest that proexosite I blockage decreases the productive recognition of prothrombin as substrate by factor Xa-factor Va complex and prothrombinase complex. Furthermore, data obtained with human platelets suggest that proexosite I may play an important role in the physiological activation of prothrombin.     

Roles of platelets and factor XI in the initiation of blood coagulation by thrombin

To account for the variable hemostatic defect in patients with factor XI (FXI) deficiency, with normal hemostasis in contact factor deficiencies, a coagulation paradigm is presented whereby trace quantities of thrombin, generated transiently by exposure of tissue factor at sites of vascular injury, activates FXI bound to the platelet surface in the presence of prothrombin or high Mr kininogen (HK). Tissue factor pathway inhibitor (TFPI) limits the flux of thrombin generated by the tissue factor pathway, and protease nexin II (PNII), released from activated platelets, inhibits solution phase FXIa and localizes FIX activation to the platelet surface where FXIa is protected from inactivation by PNII. Either prothrombin or HK binds to the Apple 1 (A1) domain of FXI, thereby exposing a platelet-binding site in the FXI A3 domain. Dimeric FXI binds to activated platelets directly through the A3 domain of one monomer. After proteolytic activation of platelet-bound FXI by thrombin (or FXIIa), a substrate binding site for FIX is exposed in the opposite monomer that promotes FIX activation on the platelet surface resulting in the local explosive generation of thrombin and the formation of hemostatic thrombi at sites of vascular injury.     

Factor Xa-driven thrombin generation in plasma: dependency on the aminophospholipid density of membranes and inhibition by phospholipid-binding proteins

Phosphatidylserine (PS) externalization of activated platelets plays a pivotal role in haemostasis and thrombosis. In the present study we have explored the relationship between the PS density of membranes and the rate of thrombin generation in plasma. Factor (F)Xa-initiated thrombin generation was measured in platelet-free plasma (PFP) containing either phospholipid vesicles of varying PS-content or non-stimulated platelets (reconstituted PRP). The duration of the initiation phase of FXa-driven thrombin generation decreased dramatically with increasing PS density. Concomitantly, the maximal rate of thrombin generation during the propagation phase (maxR) increased non-linearly, with the steepest incline between 5 and 10 mol% PS. Titration of FVa into plasma containing 2 mol% PS increased maxR proportionally and diminished the lag phase. In contrast, platelet-dependent thrombin generation was not influenced by addition of FVa. With increasing platelet concentration, the duration of the initiation phase drastically decreased, and maxR increased proportionally. At a physiologically relevant platelet concentration, maxR corresponded with the maxR found with 2 microM of 10 mol% PS. Annexin A5 (AnxA5) and lactadherin appeared to be powerful inhibitors of in-situ thrombin generation under all conditions examined, with AnxA5 being three- to four-fold more potent than lactadherin. In conclusion, maximal thrombin generation in plasma requires membranes with a density of 10-20 mol% PS. Our data further indicate that thrombin formed in situ induces externalization of PS to approx 10 mol% in a substantial platelet subpopulation.