Platelet receptor-mediated factor X activation by factor IXa. High-affinity factor IXa receptors induced by factor VIII are deficient on platelets in Scott syndrome.

We have studied factor IXa binding and factor X activation with normal platelets and with platelets obtained from a patient with a bleeding disorder and an isolated deficiency of platelet procoagulant activity termed Scott syndrome. In the absence of factor VIIIa and factor X, normal, thrombin-treated platelets exposed 560 +/- 35 sites for factor IXa with a Kd of 2.75 +/- 0.27 mM, compared with 461 +/- 60 sites per patient platelet with Kd of 3.2 +/- 0.33 nM. The addition of factor VIIIa and factor X resulted in a decrease in the Kd for normal platelets to 0.68 nM but had no effect on the Kd for patient platelets. The concentrations of factor IXa required for half-maximal rates of factor X activation for normal (0.52 nM) and patient platelets (2.5 nM) were similar to those determined from equilibrium binding studies. Kinetic parameters for factor X activation by factor IXa showed that the Km and Kcat were identical for normal and patient platelets in the absence of factor VIIIa. In the presence of factor VIIIa, and kcat for patient platelets (163 min-1) was only 33% of that for normal platelets (491 min-1): This result can be explained by the difference in affinity for factor IXa between normal and patient platelets in the presence of factor VIIIa, suggesting impaired factor VIIIa binding to Scott syndrome platelets.     

Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa

Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane.     

Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex

Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.     

Endothelial cells and vascular hemostasis]

Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.     

Recombinant factor VIIa enhances platelet adhesion and activation under flow conditions at normal and reduced platelet count.

BACKGROUND: Recombinant factor VIIa (rFVIIa), which was developed for treatment of inhibitor-complicated hemophilia, appears suitable as prohemostatic agent in other clinical disorders including patients with thrombocytopenia. It is generally accepted that rFVIIa functions by enhancement of thrombin generation at the site of injury. It is, however, unknown if and how this affects platelet adhesion and aggregation. OBJECTIVES: To determine the effect of rFVIIa-mediated thrombin generation on platelet adhesion and aggregation under flow conditions at normal and reduced platelet counts. METHODS: Washed platelets and red cells were combined to obtain plasma-free blood with different platelet counts. The reconstituted blood was perfused over a collagen- or fibrinogen-coated surface in the absence or presence of a thrombin generating system consisting of purified coagulation factors rFVIIa, factor (F)X and prothrombin. RESULTS: Addition of coagulation factors rFVIIa, FX and prothrombin to washed platelets and red cells enhanced platelet adhesion and aggregation to collagen and adhesion and spreading to fibrinogen at normal platelet count and at platelet numbers as low as 10 000 microL(-1). rFVIIa-mediated thrombin generation enhanced the activation state of platelets as measured by intracellular calcium fluxes, and enhanced the exposure of procoagulant phospholipids as measured by annexin A5 binding. CONCLUSIONS: Taken together, increased platelet adhesion and aggregation by rFVIIa-mediated thrombin formation may explain the therapeutic effects of rFVIIa in thrombocytopenic conditions and in patients with a normal platelet count by (i) enhancement of primary hemostasis and (ii) enhancement of procoagulant surface leading to elevated fibrin formation.     

Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model.

To assess the contribution of Factor IX/IXa, to intravascular thrombosis, a canine coronary thrombosis model was studied. Thrombus formation was initiated by applying current to a needle in the circumflex coronary artery. When 50% occlusion of the vessel developed, the current was stopped and animals received an intravenous bolus of either saline, bovine glutamyl-glycyl-arginyl-Factor IXa (IXai), a competitive inhibitor of Factor IXa assembly into the intrinsic Factor X activation complex, bovine Factor IX, or heparin. Animals receiving saline or Factor IX developed coronary occlusion due to a fibrin/platelet thrombus in 70 +/- 11 min. In contrast, infusion of IXai prevented thrombus formation completely (greater than 180 min) at doses of 460 and 300 micrograms/kg, and partially blocked thrombus formation at 150 micrograms/kg. IXai attenuated the accumulation of 125I-fibrinogen/fibrin at the site of the thrombus by approximately 67% (P less than 0.001) and resulted in approximately 26% decrease in serotonin release from platelets in coronary sinus (P less than 0.05). Hemostatic variables in animals receiving IXai, remained within normal limits. Animals given heparin in a concentration sufficient to prevent occlusive thrombosis had markedly increased bleeding, whereas heparin levels that maintained extravascular hemostasis did not prevent intracoronary thrombosis. This suggests that Factor IX/IXa can contribute to thrombus formation, and that inhibition of IXa participation in the clotting mechanism blocks intravascular thrombosis without impairing extravascular hemostasis.     

Role of human factor VIII in factor X activation.

The cofactor function of human Factor VIII in Factor X activation was investigated by an initial-rate assay of 3H-Factor X activation in the presence of human factor IXa, Ca2+, and either phospholipid or fresh washed human platelets. Purified Factor VIII that has not been activated by thrombin or Factor Xa supports Factor X activation after a lag of several minutes. A specific inhibitor of Factor Xa, which had no inhibitory activity against Factor IXa, markedly prolonged this lag, whereas specific thrombin inhibitors did not prolong the lag. These data support the conclusion that unactivated Factor VIII has no ability to support Factor X activation in a purified system until it is activated by Factor Xa feedback during the lag period. When Factor VIII was optimally preactivated by thrombin, the lag was completely abolished, regardless of the order of addition of the other reactants or the phospholipid source. These data indicate that there is no slow, time-dependent ordering of the reactants at the phospholipid or activated platelet surface if Factor VIII has been preactivated. Unactivated platelets did not support Factor X activation by Factors IXa and VIII. The effect of activated Factor VIII on the kinetics of bovine Factor X activation was primarily to increase the Vmax (54-fold), whereas with human Factor X, Factor VIII both increased the Vmax 56-fold and decreased the Km sixfold to 0.14 microM, similar to the plasma concentration of Factor X. Therefore, a change in the plasma factor X concentration would be expected to have a major effect on the rate of Factor X activation in vivo.     

Disorders of platelet function.

Platelets provide for primary hemostasis by forming a hemostatic plug at sites of vascular damage. They also provide a surface for the assembly of the coagulation protein complexes that generate thrombin, serve as a nidus for fibrin clots, and secrete factors involved in wound repair. Normal platelet function can be divided into four phases: adhesion, aggregation, secretion, and expression of procoagulant activity. Platelet adhesion initiates plug formation as platelets adhere to the connective tissue at the edges of a wound within seconds after vascular damage. When damage occurs in regions of slow blood flow, platelets adhere to subendothelial collagen, fibronectin, and laminin. However, when damage occurs in regions of rapid flow, platelet adhesion requires the presence of subendothelial von Willebrand factor (vWf) and a specific platelet receptor, the glycoprotein Ib/IX (GPIb/IX) complex. Following initial adhesion, platelets aggregate to complete the formation of a hemostatic plug. Platelet aggregation requires active platelet metabolism, platelet stimulation by agonists such as ADP, thrombin, collagen, or epinephrine; the presence of calcium or magnesium ions and specific plasma proteins such as fibrinogen or vWf; and a platelet receptor, the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. Thus, platelet stimulation results in the generation of intracellular second messengers that transmit the stimulus back to the platelet surface, exposing protein binding sites on GPIIb/IIIa. Fibrinogen (or vWf) then binds to GPIIb/IIIa and crosslinks adjacent platelets to produce platelet aggregates. Platelet stimulation also results in platelet secretion and the elaboration of platelet procoagulant activity. During secretion, substances are released to propagate the aggregation response and to promote wound healing; the expression of procoagulant activity localizes thrombin generation to the site of vascular damage. Disorders of platelet function can be divided into those of congenital and those of acquired origin. Although congenital disorders are uncommon, acquired disorders are encountered frequently in clinical practice. Congenital absence of GPIb/IX and GPIIb/IIIa results in the Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT), respectively. Each is an autosomal recessive bleeding disorder in which absence of a protein complex renders the affected platelets incapable of undergoing either vWf-mediated adhesion (BSS) or fibrinogen-mediated aggregation (GT).(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of fibrinogen to human monocytes.

The interaction of fibrinogen with monocytes was studied. After stimulation with ADP (10 microM) or thrombin (1 U/ml), platelet-free suspensions of human monocytes bind 125I-fibrinogen with two different affinities in a specific and Ca2+-dependent reaction with saturation at 5.80-7.35 X 10(-7) M of added protein. The binding of fibrinogen to specific receptors on monocytes induces the procoagulant activity of these cells. Thrombasthenic cells or normal monocytes preincubated with a monoclonal antibody to the platelet glycoprotein IIb/IIIa complex (10E5) do not bind fibrinogen and have no procoagulant activity. Metabolic studies with [35S]methionine revealed that cultured monocytes actually synthesize a surface antigen precipitated by 10E5 antibody as a major band with 92,000 relative molecular weight. Our data indicate that monocytes express receptors for fibrinogen only in part related to the platelet glycoprotein IIb/IIIa complex. Furthermore, the binding of fibrinogen to monocytes enhances the cooperation of these cells in hemostasis.     

Binding studies of the enzyme (factor IXa) with the cofactor (factor VIIIa) in the assembly of factor-X activating complex on the activated platelet surface

Summary.  Activated platelet membranes expose binding sites for the enzyme factor (F)IXa, the substrate (FX) and the cofactor (FVIIIa) that colocalize to assemble the FX-activating complex and promote optimal rates of FX activation. To determine the stoichiometry and affinity of binding to activated platelets, coordinate, equilibrium binding studies with enzyme (¹²⁵I-FIXa) and cofactor (¹³¹I-FVIII or ¹³¹I-FVIIIa) were carried out in the presence of saturating concentrations of substrate (FX). Results of these studies indicate that in the presence of FX (1.5 µ m ), the enzyme (active-site-inhibited Glu-Gly-Arg-FIXa, EGR-FIXa) and procofactor (FVIII) bind to an equal number (approximately 700 sites/platelet) of receptors whereas the active cofactor (FVIIIa) binds an additional approximately 500 high-affinity FVIIIa binding sites per platelet (K_d approximately 0.8 n m ). With excess zymogen (FIX) to block shared FIX/FIXa-binding sites, the stoichiometry of ¹²⁵I-FIXa and ¹³¹I-FVIIIa binding was 1 : 4. These FIXa/FVIIIa binding studies together with previously reported evidence of the coordinate binding of FVIIIa and FX to equivalent numbers of binding sites on activated platelets provide strong evidence to support the conclusion that FVIIIa comprises the receptor that presents FX to FIXa for efficient catalysis on the activated platelet membrane.     

Recombinant human factor VIIa (rFVIIa) can activate factor FIX on activated platelets

The studies reported here show that factor (F)VIIa can activate factor (F)IX on activated platelets in the absence of tissue factor. Both FIX and FIXa bind to the activated platelet surface with a K(d) of 8 nM and 2 nM, respectively. With factor (F)VIIIa, FIXa binds more tightly to platelets (K(d) 0.6 nM). At rFVIIa concentrations < 100 nm, no direct binding to the activated platelet surface can be detected with electrophoretic light scattering. However, in the presence of FIX, rFVIIa binding to platelets at concentrations as low as 10 nm rFVIIa can be detected. This is reflected by a decrease in the FIX K(d) from 8 to 1.6 nM. When rFVIIa is added to activated platelets in the presence of both FIX and FVIIIa, the K(d) for FIX decreases to 0.6, suggesting that rFVIIa activates FIX on the surface of activated platelets in the absence of tissue factor. The activation of FIX by FVIIa on activated platelets can also be demonstrated by a functional assay for FIXa. These data show that pharmacological doses of rFVIIa result in the direct activation of FIX by rFVIIa to form additional tenase complexes ultimately resulting in improved thrombin generation. These results may explain, at least in part, the mechanism of action of rFVIIa in hemorrhagic conditions seen in otherwise normal patients who develop an acquired coagulopathy due to trauma, surgery or a variety of other events in which rFVIIa has been found to be effective.     

Assembly of the prothrombin activator complex on rabbit alveolar macrophage high-affinity factor Xa receptors. A kinetic study

Efficient prothrombin activation occurs after assembly of factors Va, Xa, and phospholipid surface cofactor as a multimolecular complex. These components are provided by platelets and plasma within the vascular space, but molecules and membranes for prothrombin activator assembly in extravascular spaces have not been identified. In the present study, purified alveolar macrophages were found to produce high-affinity factor Xa receptors that mediate formation of enzymatic prothrombinase complexes and rapid prothrombin to thrombin conversion in the absence of exogenous factor V/Va or platelets. Thus, in reaction mixtures with alveolar macrophages cultured for 20 h in serum-free medium, the thrombin formation rate was 152 nM/min/0.66 X 10(6) cells, after adding prothrombin (1.5 microM), Ca2+ (5 mM), and factor Xa (3.7 nM). The observed Kd of factor Xa interaction with macrophage receptors is 2.1 +/- 0.94 X 10(-10) M. Kinetic analysis and inhibition studies using isolated factor V and anti-factor V antibody show that macrophage Xa receptors are functionally and antigenically similar to plasma factor V. By contrast, freshly isolated cells lacked receptors promoting prothrombin conversion at high rates. Inhibitors of protein synthesis and glycosylation, puromycin and monensin, respectively, abrogated production of Xa receptors in culture. Additionally, subcellular fractionation and enzyme-marker studies (alkaline phosphodiesterase I) indicate that internal and external membranes of alveolar macrophages have phospholipid surface cofactor activity required for prothrombinase complexes. Pulmonary surfactant is also shown to express this cofactor activity. Alveolar macrophages and surfactant comprise an efficient prothrombin activator system that is independent of plasma factor V. This system may facilitate rapid extravascular alveolar thrombin formation even at very low concentrations of factor Xa during lung defense reactions to inflammation or edema.     

Nucleotide receptor signaling in platelets

Upon injury to a vessel wall the exposure of subendothelial collagen results in the activation of platelets. Platelet activation culminates in shape change, aggregation, release of granule contents and generation of lipid mediators. These secreted and generated mediators trigger a positive feedback mechanism potentiating the platelet activation induced by physiological agonists such as collagen and thrombin. Adenine nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), released from damaged cells and that are secreted from platelet-dense granules, contribute to the positive feedback mechanism by acting through nucleotide receptors on the platelet surface. ADP acts through two G protein-coupled receptors, the Gq-coupled P2Y1 receptor, and the Gi-coupled P2Y12 receptor. ATP, on the other hand, acts through the ligand-gated channel P2X1. Stimulation of platelets by ADP leads to shape change, aggregation and thromboxane A2 generation. ADP-induced dense granule release depends on generated thromboxane A2. Furthermore, costimulation of both P2Y1 and P2Y12 receptors is required for ADP-induced platelet aggregation. ATP stimulation of P2X1 is involved in platelet shape change and helps to amplify platelet responses mediated by agonists such as collagen. Activation of each of these nucleotide receptors results in unique signal transduction pathways that are important in the regulation of thrombosis and hemostasis.     

A factor X-activating cysteine protease from malignant tissue.

A proteolytic procoagulant has been identified in extracts of human and animal tumors and in cultured malignant cells. It directly activated Factor X but its similarity to other Factor S-activating serine proteases was not clear. This study describes work done to determine whether this enzyme, cancer procoagulant, is a serine or cysteine protease. Purified cancer procoagulant from rabbit V2 carcinoma was bound to a p-chloromercurialbenzoate-agarose affinity column and was eluted with dithiothreitol. The initiation of recalcified, citrated plasma coagulation activity by cancer procoagulant was inhibited by 5 mM diisopropylfluorophosphate, 1 mM phenylmethylsulfonylfluoride, 0.1 mM HgCl2, and 1 mM iodoacetamide. Activity was restored in the diisopropylfluorophosphate-, phenylmethylsulfonylfluoride-, and HgCl2-inhibited samples by 5 mM dithiothreitol; iodoacetamide inhibition was irreversible. Russell's viper venom, a control Factor X-activating serine protease, was not inhibited by either 0.1 mM HgCl2 or 1 mM iodoacetamide. The direct activation of Factor X by cancer procoagulant in a two-stage assay was inhibited by diisopropylfluorophosphate and iodoacetamide. Diisopropylfluorophosphate inhibits serine proteases, and an undefined impurity in most commercial preparations inhibits cysteine proteases. Hydrolysis of diisopropylfluorophosphate with CuSO4 and imidazole virtually eliminated inhibition of thrombin, but cancer procoagulant inhibition remained complete, suggesting that cancer procoagulant was inhibited by the undefined impurity. These results suggest that cancer procoagulant is a cysteine endopeptidase, which distinguishes it from other coagulation factors including tissue factor. This and other data suggest that neoplastic cells produce this unique cysteine protease which may initiate blood coagulation.     

Molecular models of the procoagulant Factor VIIIa–Factor IXa complex

BACKGROUND: Formation of the intrinsic tenase complex is an essential event in the procoagulant reactions that lead to clot formation. The tenase complex is formed when the activated serine protease, Factor IXa (FIXa), and its cofactor Factor VIIIa (FVIIIa) assemble on a phospholipid surface to proteolytically convert the zymogen Factor X (FX) into its active form FXa. The physiological relevance of the tenase complex is evident in hemophilia A or B patients who present with bleeding disorders. OBJECTIVES: The purpose of this study was to establish three-dimensional (3D) models of the FVIIIa-FIXa complex. METHODS: First, we built two new theoretical models of FVIIIa via homology modeling, inter-domain docking and loop simulation algorithms as well as a model for FIXa. This was followed by pseudo-Brownian protein-protein docking in internal coordinates with the ICM (Internal Coordinates Mechanics) program between the two FVIIIa and the FIXa structures. RESULTS: Ten representative models of this complex are presented based on agreements with known experimental data and according to structural criteria. CONCLUSIONS: These novel 3D models will help guide future site directed mutagenesis aimed at improving the functionality of FVIIIa and/or FIXa and will contribute to a better understanding of the role of this macromolecular complex in the blood coagulation cascade.     

Procoagulant platelets: Generation,characteristics, and therapeutic target

Platelets play a pivotal role in hemostasis. Activated platelets are classified into two groups, according to their agonist response: aggregating and procoagulant platelets. Aggregating platelets consist of activated integrin αIIbβ3 and stretch out pseudopods to further attract platelets to the site of injury by connecting with fibrinogen. They mainly gather in the core of the thrombus and perform a secretory function, such as releasing adenosine diphosphate (ADP). Procoagulant platelets promote the formation of thrombin and fibrin by interacting with coagulation factors and can thus be considered as the connector between primary and secondary hemostasis. In addition to their functions in blood coagulation, procoagulant platelets play a proinflammatory role by releasing platelet microparticles and inorganic polyphosphate. Considering these important functions of procoagulant platelets, this subpopulation warrants detailed study to analyze their potential in preventing human diseases. This review summarizes the generation and important characteristics of procoagulant platelets, as well as their potential for preventing the adverse effects associated with current antiplatelet therapies.     

Platelet polyphosphates: the nexus of primary and secondary hemostasis

For decades it has been known that activated platelets promote plasma clotting and that the fibrin forming activity of activated platelets is dependent on blood coagulation factor XII. However, because factor XII deficiency is not associated with any bleeding disorder, platelet-driven factor XII activation was believed to be an in vitro artefact with no importance for coagulation in vivo. Using arterial and venous thrombosis models in factor XII deficient mice we recently demonstrated that factor XII is essential for thrombosis in vivo. However it was not known how factor XII is activated by procoagulant platelets within the growing thrombus. Here, we review our studies of the last 5 years that led to the identification of the endogenous factor XII activator. We found that platelets release polyphosphates, linear inorganic polymers of 60-100 phosphate residues that directly bound to and activated factor XII. Platelet polyphosphates potently initiate fibrin formation via the factor XII-driven intrinsic pathway. Inhibition of factor XII or polyphosphates protected mice from lethal thrombotic disease and strongly reduced clot stability in patients. Our data identify polyphosphates as the endogenous factor XII activator in vivo linking platelet activation (primary hemostasis) and fibrin production (secondary hemostasis). Targeting polyphosphate-mediated factor XII activation offers the exciting possibility for a safe anticoagulation with minimal therapy-associated bleeding.     

Platelet polyphosphates: The nexus of primary and secondary hemostasis

Abstract

For decades it has been known that activated platelets promote plasma clotting and that the fibrin forming activity of activated platelets is dependent on blood coagulation factor XII. However, because factor XII deficiency is not associated with any bleeding disorder, platelet-driven factor XII activation was believed to be an in vitro artefact with no importance for coagulation in vivo. Using arterial and venous thrombosis models in factor XII deficient mice we recently demonstrated that factor XII is essential for thrombosis in vivo. However it was not known how factor XII is activated by procoagulant platelets within the growing thrombus. Here, we review our studies of the last 5 years that led to the identification of the endogenous factor XII activator. We found that platelets release polyphosphates, linear inorganic polymers of 60–100 phosphate residues that directly bound to and activated factor XII. Platelet polyphosphates potently initiate fibrin formation via the factor XII-driven intrinsic pathway. Inhibition of factor XII or polyphosphates protected mice from lethal thrombotic disease and strongly reduced clot stability in patients. Our data identify polyphosphates as the endogenous factor XII activator in vivo linking platelet activation (primary hemostasis) and fibrin production (secondary hemostasis). Targeting polyphosphate-mediated factor XII activation offers the exciting possibility for a safe anticoagulation with minimal therapy-associated bleeding.     

血小板相关组织因子及其意义的研究

目的探讨正常人血小板是否含有组织因子(TF),血小板相关TF是否具有促凝活性。方法用Sepharose2B凝胶柱分离纯化血小板,ELISA法测定洗涤血小板破碎液TF含量;一期血浆凝固时间测定血小板相关TF促凝活性;RT-PCR法检测正常人血小板相关TF基因的表达。结果正常人血小板破碎液中TF含量为(16.37±6.39)ng/L;胶原活化的血小板可将TF释放到血浆中引起血浆TF水平明显升高(P<0.05);静息血小板无TF的促凝活性,胶原活化的血小板促凝活性显著增高(P<0.01),TF单抗及乏凝血因子Ⅶ(FⅦ)血浆能明显阻断该效应(P<0.01);RT-PCR法在血小板上未能检测到TF mRNA的存在;冠心病患者血小板破碎液TF水平为(20.71±8.78)ng/L,与正常对照相比明显增高(P<0.05),体外未经胶原活化的血小板促凝活性也较正常对照明显增高,并能被TF单抗抑制。结论血小板内含有TF;活化血小板可表现出TF促凝活性;血小板所含的TF可能并非自身合成;冠心病患者血小板相关TF的含量及活性较正常对照明显增高,提示血小板可能可以通过释放TF而参与凝血过程及血栓形成。     

Impact of procoagulant concentration on rate,peak and total thrombin generation in a model system

Summary. Using a cell‐based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0–200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV‐deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell‐based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin.