Formation Of Prothrombinase Complex Research Articles

Microvesicles, blood cells, and endothelial cells mediate phosphatidylserine-related prothrombotic state in patients with periodontitis.

Phosphatidylserine (PS) is essential for inflammation-associated thrombogenesis, but the exact effect of PS on the prothrombotic state in periodontitis is uncertain. This study aimed to determine the PS-related procoagulant state in patients with periodontitis. A total of 138 patients with periodontitis were examined compared with 42 healthy controls. PS-exposing cells and microvesicles in blood samples were detected by confocal microscopy and flow cytometry. The clotting time assay and prothrombinase complex formation assay were used to measure the procoagulant activity of microvesicles, blood cells and endothelial cells. Periodontal clinical parameters and laboratory characteristics of patients with severe periodontitis were recorded and analyzed at baseline and 6 months after non-surgical periodontal therapy. Total PS-positive (PS+ ) microvesicles and the percentage of PS+ blood cells increased in patients with severe periodontitis compared with patients with moderate/mild periodontitis or healthy controls. Endothelial cells cultured in serum from patients with severe periodontitis expressed more PS compared with those cultured in serum from healthy controls. Specifically, PS exposure on blood cells and endothelial cells significantly decreased after inhibiting the effect of inflammatory cytokines. The elevated levels of PS+ cells and microvesicles in severe periodontitis shortened clotting time and led to increased prothrombinase complex formation. Non-surgical periodontal therapy significantly attenuated the release of microvesicles and the PS exposure of blood cells in severe periodontitis. The prothrombotic state of patients with periodontitis is mediated by PS+ cells and microvesicles stimulated by elevated levels of inflammatory cytokines.

Secondary hemostasis studies of crude venom and isolated proteins from the snake Crotalus durissus terrificus.

Among the activities triggered by Crotalus durissus terrificus snake venom, coagulation is intriguing and contradictory since the venom contains both coagulant and anticoagulant precursor proteins. This work describes the in vitro effects of crude venom and purified proteins from snake Crotalus durissus terrificus as they affect coagulation factors of clotting pathways. Coagulant and/or anticoagulant activities of crude venom, and purified proteins were all analyzed directly in human plasma. Clots formed by crude venom and Gyroxin presented as flexible hyaline masses in punctiform distribution. Clot formation time evaluation of isolated proteins with PT and APTT assays made it possible to infer that these proteins interfere in all coagulation pathways. However, regarding ophidism by C. d. terrificus, Gyroxin acts directly, breaking down fibrinogen to fibrin and increasing the amount plasminogen activator, which results in the formation of thrombi. Crotoxin complex, Crotoxin A and Crotoxin B proteins can act in prothrombinase complex formation; Crotoxin B can inhibit prothrombinase complex formation by direct interaction with Factor Xa. Crotamine interacts with negatively charged regions of differing coagulation factors in all coagulation pathways, and possesses a whole set of activities causing dysfunction, activation and/or inhibition of natural anticoagulants and disturbing hemostasis.

Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: Therapeutic implications

Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning − induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. Biological significanceEnvenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

Flow Regulates Thrombin Generation Kinetics

Shear stress plays important roles in platelet coagulation. However, in many studies, coagulation rate constants were determined under static conditions. In the present study, we aimed to determine how flow affects thrombin generation rates, when coagulation factors were immediately available. Washed platelets were exposed to various dynamic shear stress (physiological and pathological) in a cone and plate shearing device, with Factor Xa, prothrombin, and calcium. Thrombin generation was quantified using chromozym‐TH absorbance at 405 nm. Platelet activation was assessed by cell surface P‐selectin expression using flow cytometry. The results demonstrate that elevated dynamic shear stress induced a significant increase in P‐selectin expression, indicating platelet activation. However, thrombin generation rate was significantly reduced under elevated shear stress, compared to that under low or physiological shear stress. Such results were different from that observed with a prothrombinase assay following platelet exposure to dynamic shear stress. The immediate availability of coagulation factors likely contributed to the observed differences. These data suggest that dynamic shear stress affects thrombin generation kinetics, not only by affecting platelet activation state, but also by affecting prothrombinase complex formation on platelet surface. Thrombin generation rates could be reduced under accelerated flow and elevated shear stress, as reaction time for prothrombinase complex to form is significantly reduced.

Factor Xa dimerization competes with prothrombinase complex formation on platelet-like membrane surfaces

Exposure of phosphatidylserine (PS) molecules on activated platelet membrane surface is a crucial event in blood coagulation. Binding of PS to specific sites on factor Xa (fXa) and factor Va (fVa) promotes their assembly into a complex that enhances proteolysis of prothrombin by approximately 10⁵. Recent studies demonstrate that both soluble PS and PS-containing model membranes promote formation of inactive fXa dimers at 5 mM Ca²⁺. In the present study, we show how competition between fXa dimerization and prothrombinase formation depends on Ca²⁺ and lipid membrane concentrations. We used homo-FRET measurements between fluorescein-E-G-R-chloromethylketone (CK)-Xa [fXa irreversibly inactivated by alkylation of the active site histidine residue with FEGR (FEGR-fXa)] and prothrombinase activity measurements to reveal the balance between fXa dimer formation and fXa-fVa complex formation. Changes in FEGR-fXa dimer homo-FRET with addition of fVa to model-membrane-bound FEGR-fXa unambiguously demonstrated that formation of the FEGR-fXa-fVa complex dissociated the dimer. Quantitative global analysis according to a model for protein interaction equilibria on a surface provided an estimate of a surface constant for fXa dimer dissociation (K(fXa×fXa)(d, σ)) approximately 10-fold lower than K(fXa×fVa)(d,σ) for fXa-fVa complex. Experiments performed using activated platelet-derived microparticles (MPs) showed that competition between fXa dimerization and fXa-fVa complex formation was even more prominent on MPs. In summary, at Ca²⁺ concentrations found in the maturing platelet plug (2-5 mM), fVa can compete fXa off of inactive fXa dimers to significantly amplify thrombin production, both because it releases dimer inhibition and because of its well-known cofactor activity. This suggests a hitherto unanticipated mechanism by which PS-exposing platelet membranes can regulate amplification and propagation of blood coagulation.

Platelets: Small in Size But Essential in the Regulation of Vascular Homeostasis - Translation From Basic Science to Clinical Medicine.

Platelets are small blood cells that adhere to the site of vessel injury where von Willebrand factor (VWF) is expressed. Platelets bind to VWF through interaction with a membrane protein, glycoprotein (GP) Ibα. Next, the accumulated platelets are activated to change their morphological and biochemical characteristics. Various vasoactive substances, such as immune-regulatory CD40 ligand, are released locally from activated platelet cells to maintain homeostasis of the vascular system. Major roles played by platelets in the regulation of hemostasis and thrombus formation include local activation of the coagulation cascade. Translocation of negatively charged phospholipids to the surface of activated platelets helps in the formation of prothrombinase complex, which efficiently produces thrombin. Thrombin produces fibrin around the activated platelets and further activates the platelets through thrombin receptor stimulation. Of the various platelet-stimulating receptors and activation signals, cyclo-oxygenase-1, P2Y12 adenosine 5'-diphosphate receptor, and thrombin receptor (protease activated receptor)-1 blockers are used clinically as antiplatelet agents. In the future, precise understanding of the quantitative contribution of platelet function in hemostasis and pathological thrombus formation should lead to the development of effective antithrombotic agents without increasing the risk of serious bleeding complications.

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

Protein S Regulates Factor IXa/VIIIa Activity Independent of Activated Protein C

AbstractAbstract 2196Protein S (PS) is a vitamin-K-dependent plasma protein that plays an important role in the feedback regulation of thrombin generation. PS is a well recognized anticoagulant protein, the absence of which causes venous and arterial thrombosis. While PS has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear. PS was initially identified as a cofactor for activated protein C (APC). Interestingly, recent reports suggest PS may function in an APC-independent manner as it has been suggested to inhibit prothrombinase complex formation and act as a stimulator of factor Xa-TFPI (tissue factor pathway inhibitor) complex.Based on these new data, we have begun to examine possible roles of PS outside of the PC pathway which contribute to the regulation of thrombin generation. We recently obtained exciting preliminary data which suggests that PS plays an important role in the inhibition of Xa generation by the intrinsic Xase complex. Our central hypothesis is that PS binds to the enzyme FIXa in the presence of Phosphatidyl serine (PhosSer) containing membranes and inhibits FXa generation in the presence and absence of VIIIa.We did clotting assays with FIX deficient plasma both with and without anti PS antibody. Clotting initiated with 0.1 nM FIXa shows a must faster clotting in the presence of anti PS antibody (57 seconds compared to 107 seconds without the incubation with anti PS antibody) (see Table 1). However, there is no difference in clotting times when the clotting is initiated with FVIIa in the presence and absence of anti PS antibody. Our results showing PS is only able to alter FIXa initiated clotting indicates that PS inhibits the FXa generation by FIXa/FVIIIa in plasma.Table 1: Clotting of FIX deficient plasma initiated with FIXa (A) & initiated with FVIIa (B)(A)Conc of FVIIa nMClotting time in seconds - AntiPS AntibodyClotting time in seconds + AntiPS Antibody0.1107571463153114.7103014.4(B)Conc of FVIIa nMClotting time in seconds - AntiPS AntibodyClotting time in seconds + AntiPS Antibody0.11131121565454142103635We determined the binding of PS to factor IXa by determining the change in anisotropy and fluorescence of DEGR(5 (dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl chloromethyl ketone)-IXa. The Kd was ~ 1 nM in both cases suggesting a tight binding of PS with factor IXa.We also determined the activation of FX by FIXa in the presence of FVIIIa and 50 μM Phosphatidylserine membrane in the presence of PS. We used physiological concentrations of FX, PS,0.1 nM FIXa, 5 nM FVIIIa and monitored the rate of FXa generation by using S2222 (chromogenic substrate). There was ~ 95 % inhibition in the rate of FXa generation by FIXa in the0.2 presence of FVIIIa. Our results clearly indicate that PS inhibits FXa generation by factors IXa/VIIIa.We believe that these new findings have major implications for better understanding how FXa generation is regulated during the initial phases of coagulation and may shed light on a major outstanding question in the field relating to how a key anticoagulant, PS contributes to the down regulation of the haemostatic response. Disclosures:No relevant conflicts of interest to declare.

Amino Acid Region 1000–1008 of Coagulation Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

AbstractAbstract 2221The intricate process of hemostasis is a highly regulated mechanism which implements the conversion of prothrombin to thrombin and the crucial formation of a fibrin clot. The direct progression of hemostasis is pivotal to the prevention of various clotting disorders associated to hypercoagulation and excess bleeding. Upon vascular injury, the proteolytic conversion of prothrombin to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of divalent metal ions. Coagulation factor V (fV) is synthesized as a multi-domain (A1-A2-B-A3-C1-C2) quiescent procofactor with nominal procoagulant activity. Following the three sequential catalytic cleavages by a-thrombin at Arg709, Arg1018 and Arg1545 amino acids 710–1545 of the B-domain are liberated to generate the noncovalently associated light and heavy chains of fVa. The cleavage at Arg1545 is crucial for full procoagulant activity. The heterodimer of fVa is composed of a heavy chain associated with the 2 A domains (residues 1–303 and 317–656) and a light chain composed of one A domain (1546-1877) and two C domains (residues 1878–2036 and 2037–2196). Since single chain fV does not bind fXa, the proper removal of the B-domain is vital to generate procoagulant activity. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Appropriate binding of fVa to fXa during prothrombinase function is essential to the proper activation of the substrate, prothrombin. Previous studies have determined the heavy and light chains of fVa to have fXa interactive sites. A highly basic region of amino acids in the B-domain suggests a potential sheathing of either the heavy or light chain fXa interface sites. To verify this hypothesis we investigated the role of amino acid region 1000–1008 that contains seven basic amino acid residues. To ascertain the role of this region we have constructed a recombinant mutant fV molecule with all activation cleavage sites (R709/R1018/R1545) mutated to glutamine (fV*T3Q), a mutant fV molecule with region 1000–1008 deleted (fVΔ1000-1008), and a mutant fV molecule containing the same deletion with all activation cleavage sites changed to glutamine (fVΔ1000-1008/*T3Q). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for their capability to bind fXa within prothrombinase prior (fV) and after incubation with thrombin (fVa). The data showed that fV*T3Q and fVa*T3Q were unable to interact with fXa. In contrast, the Kd values for fVΔ1000-1008 (0.9 nM), fVaΔ1000-1008 (0.4 nM), fVΔ1000-1008*T3Q (0.7 nM) and fVaΔ1000-1008*T3Q (0.5 nM), were similar to the affinity of fVaWT for fXa (0.22 nM). Two-stage clotting assays revealed that while fVa*T3Q was practically devoid of clotting activity, the mutant molecules fVaΔ1000-1008, and fVaD1000-1008*T3Q had clotting activities comparable to fVaWT. Thus, unactivated fVΔ1000-1008*T3Q has an affinity for fXa that is similar to the affinity of fVaWT for the enzyme. In addition, fVΔ1000-1008*T3Q that cannot be cleaved and activated by thrombin or activated during the course of the clotting assay, has similar clotting activity as fVaWT (∼3110 U/mg). The data presented in this study provide an important insight into one of the possible roles of the B domain of factor V, explicitly the fXa interactive sites on fVa are covered/inhibited by amino acids 1000–1008 of the fV B-domain. These data strongly suggest that amino acid region 1000–1008 of fV contains a regulatory sequence protecting the organisms from spontaneous binding of the procofactor to fXa and unnecessary prothrombinase complex formation which will result in catastrophic physiological consequences. Disclosures:No relevant conflicts of interest to declare.

Factor Xa Dimerization and Prothrombinase Complex Formation are Competitive Process on a Membrane Surface

Table of ContentsExposure of phosphatidylserine (PS) molecules on activated platelet membranes is a crucial event in blood coagulation. Binding of PS to a specific site on factor Xa (fXa) and factorVa (fVa) promotes their assembly into a complex that dramatically enhances proteolytic activity of fXa. Recent studies demonstrate that by both soluble PS and PS-containing membranespromote formation of inactive fXa dimer at 5mM Ca+2. The fluorescence anisotropy of active site labeled fXa, FEGR (Fluorescein-GLU-GLY-ARG-chloromethylketone)-Xa, is decreased in the presence of PS membrane on which it forms dimer. We report now the addition of fVa to membrane-bound FEGR-Xa produced fVa- FEGR-Xa complex formation with a Kd, surface approximately 60-fold lower than that characterizing FEGR-Xa surface dimerization, clearly indicating fVa strongly competed with fXa dimer formation in order to form active Xa-Va complex on the membrane surface. Analysis of FEGR-Xa fluorescence anisotropy yielded roughly constant Kd's for Xa-Va interaction with increasing Ca2+ concentration from 2 to 5 mM Ca2+ despite the fact that fXa dimer formation varied dramatically over this Ca2+ range. Experiments performed at varying membrane and fVa concentrations for both 23 nm and 120 nm confirmed that protein distribution between vesicles was sufficiently rapid as to overcome any possible effects of membrane discreteness. We conclude that PS-induced fXa dimerization on membrane strongly competes with fXa-fVa complex formation at high Ca2+ concentrations. Supported by USPHS grant HL072827 to BRL.

Factor Xa Dimerization and Prothrombinase Complex Formation Are Competitive Process On a Membrane Surface.

Abstract 2123Poster Board II-100Exposure of phosphatidylserine (PS) molecules on activated platelet membranes is a crucial event in blood coagulation. Binding of PS to a specific site on factors Xa and on Va promotes prothrombinase assembly and enhances proteolytic activity of factor Xa. PS binding produces significant structural changes in both proteins. Recent studies demonstrate formation of inactive Xa dimer by both soluble, short-chain PS and PS-containing membranes at high Ca+2 concentration (Chattopadhyay et al.,2009, Biophys. J, 974). Association of factor Xa with factor Va in the presence of soluble PS relieves this inactivation. From kinetic measurements, we found that apparent Kd's for the Xa-Va interaction in the presence of 50 mM PS/PC (25/75) membranes increased in with increasing factor Xa concentrations, suggesting that factor Va must compete with factor Xa dimer in order to form the Xa-Va complex on the membrane surface. The fluorescence anisotropy of active site labeled factor Xa, FEGR (Fluorescein-GLU-GLY-ARG-chloromethylketone)-Xa, is lowered in the presence of PS membrane on which it forms dimer (Koklic et al., October 2009, Biophys. J., V-97, Issue 8) We report now that addition of factor Va to FEGR-Xa/membrane samples produced a saturating increase in anisotropy, clearly indicating that factor Va competed with factor Xa dimer formation. This experiment was repeated for several Ca2+ concentrations and yielded increasing apparent Kd's for Xa-Va interaction with increasing Ca2+concentration. FEGR-Xa fluorescence anisotropy as well as DEGR-Xa (factor Xa labeled at the active site with Dansyl-GLU-GLY-ARG-chloromethylketone) fluorescence intensity at varying membrane and factor Va concentrations for both 23 nm and 120 nm vesicles were analyzed globally to obtain surface Kd's of dimerization and factor Va-factor Xa interaction. This showed that membrane discreteness (slow distribution of factor Xa between discrete membrane vesicles) had almost no effect on the competition between factor Xa dimer formation and Xa-Va complex formation. We conclude that prothrombinase complex formation can overcome PS-induced factor Xa inhibition through factor Xa dimer formation at Ca2+ concentrations just above plasma concentration. In this way, one can imagine that factor Va is required to overcome factor Xa dimerization and amplify thrombin production in the early platelet plug.Supported by USPHS grant HL072827 to BRL. Disclosures:No relevant conflicts of interest to declare.

Effects of TAK-442, a Direct FXa Inhibitor, and Fondaparinux on Platelet-Associated Prothrombinase in the Balloon Injured Artery of Rats

PCI patients routinely experience arterial injury during balloon angioplasty and stent deployment. At the injured arterial site, endothelial damage triggers platelet adhesion to the vascular wall and initiates platelet-associated prothrombinase complex (factor Xa [FXa]-factor Va-phospholipids-calcium) formation, resulting in the progression of thrombus formation. Recently, it was demonstrated that treatment with fondaparinux reduced the risk of death or recurrent myocardial infarction without an increase in the incidence of major bleeding compared to standard therapy in patients with STEMI. However, fondaparinux showed no superiority to placebo or heparin in patients undergoing PCI. On the other hand, small molecule direct FXa inhibitors, which are still in early stages of clinical development for the prevention of ACS, have been reported to strongly block reconstituted prothrombinase. Here, we compared the effects of TAK-442, a novel direct FXa inhibitor on platelet-associated prothrombinase activity, with fondaparinux. TAK- 442 and fondaparinux inhibited endogenous FXa activity in platelet-poor plasma from humans (IC50: 53 nM, TAK-442; 11 nM, fondaparinux) and rats (IC50: 32 nM, TAK-442; 19 nM, fondaparinux). When TAK-442 or fondaparinux were preincubated with free FXa, they each inhibited in vitro reconstituted prothrombinase activity, with IC50 values of 28 nM and 3.9 nM, respectively. In the absence of free FXa preincubation, TAK-442 retained this inhibitory effect (IC50, 51 nM); the inhibitory effect of fondaparinux, however, decreased dramatically (IC50, 1700 nM). In a rat model of balloon injury, thrombin activity on the surface of injured vessels increased to 3.3-, 20- and 5.7-fold that of intact aorta at 5 min, 1 hr, and 24 hrs after the injury, respectively. At 1 hr after the injury, 300 nM TAK-442 significantly (p<0.025) inhibited platelet-associated thrombin generation on the surface of injured aorta by 99% (IC50, 19 nM), whereas the same concentration of fondaparinux showed no significant (p=0.076) inhibition (IC50, >300 nM). These results highlight a limitation of fondaparinux in inhibiting platelet-associated prothombinase activity that leads to thrombus formation compared with TAK-442, and suggest that TAK-442 may be more effective in preventing arterial thrombosis in patients undergoing percutaneous coronary interventions.

INTERACTION OF SNAKE-VENOM PROTEINS WITH BLOOD COAGULATION FACTORS: MECHANISMS OF ANTICOAGULANT ACTIVITY

Snake venoms are rich in a large variety of proteins and peptides that can interfere with the hemostatic system. This review focuses on snake-venom proteins, with or without enzymatic activity, that bind to blood coagulation factors and exhibit anticoagulant effects. These proteins include (1) anticoagulant phospholipases A2, which bind to factor X and impair the formation of prothrombinase complex independently from their enzymatic activity; (2) factor IX- and X-binding proteins, which belong to the family of C-type lectin-like proteins and interact with the Gla domain of factor X/Xa and/or IX/IXa; (3) prothrombin- and thrombin-binding proteins, represented by bothrojaracin and salmorin, which are also C-type lectin-like molecules and either bind to prothrombin, impairing thrombin formation, or to thrombin, inhibiting its biological activities, such as clotting of fibrinogen, platelet activation, and so forth; and (4) L-amino oxidases that have recently been described to have anticoagulant activity. The general structural characteristics of these proteins and current knowledge regarding their mechanisms of action are discussed. These proteins have helped in our understanding of some molecular aspects of the hemostatic process. A more detailed analysis of the structure–function relationship of these molecules will certainly result in new medical and pharmacological applications. Furthermore, venom components offer attractive templates for the development of rationally designed therapeutic agents.

Murine macrophage P2X 7 receptors support rapid prothrombotic responses

Non-apoptotic externalization of phosphatidylserine (PS) can act as a reactive surface for the efficient assembly of the prothrombinase complex leading to thrombin generation and coagulation. Here we show that extracellular ATP, acting at the macrophage P2X 7 receptor, drives the rapid Ca 2+-dependent formation and release of PS-rich microvesicles that enhance the assembly of the prothrombinase complex and subsequent formation of thrombin. Incubation with P2X 7 receptor antagonists (KN-62 and Brilliant Blue G) attenuates ATP induced prothrombotic responses. Consistent with the hypothesis that exposed PS enhances prothrombinase activity; pre-incubation with annexin V blocks the increase in thrombin formation. The rapid translocation of PS and formation of pro-thrombotic microvesicles occurs in the absence of cell lysis. These data demonstrate that the pro-inflammatory P2X 7 receptor can also support and propagate rapid increases in thrombin formation.

Plasmodium falciparum-infected erythrocytes induce tissue factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes.

Plasmodium falciparum malaria infects 300-500 million people every year, causing 1-2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria. We have asked whether interaction of parasitized red blood cells (pRBC) with EC induces tissue factor (TF) expression in vitro and in vivo. The role of phosphatidylserine-containing pRBC to support the assembly of blood coagulation complexes was also investigated. We demonstrate that mature forms of pRBC induce functional expression of TF by EC in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late-stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, post-mortem brain sections obtained from P. falciparum-infected children who died from cerebral malaria and other causes display a consistent staining for TF in the EC. These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications.

Dipyridamole Decreases Protease-Activated Receptor and Annexin-V Binding on Platelets of Poststroke Patients with Aspirin Nonresponsiveness

Background: Although controversial, the phenomenon of aspirin resistance (AR) has been correlated in some small studies with poor clinical outcomes in patients with coronary artery disease. Even less is known regarding the role of AR in the poststroke population. The reason for and the underlying mechanism of AR is unknown. We hypothesized that excessive formation of thrombin on the platelet surface may contribute to this phenomenon and assessed how dipyridamole affects multiple platelet and thrombin generation biomarkers in AR patients after ischemic stroke. Methods: Whole blood samples from 20 poststroke AR patients were pretreated with dipyridamole, simulating the therapeutic range, and then incubated for 45 min at 37°C. Platelet characteristics were assessed by aggregometry, cartridge-based analyzer, and receptor expression by flow cytometry. Markers of thrombin generation were measured in the autologous plasma by ELISA. Results: Pretreatment of blood with dipyridamole resulted in 22–26% diminished expression of intact PAR-1 receptor (p = 0.021 and p = 0.024) and 28–31% decrease of annexin V binding (p = 0.031 and p = 0.02) after incubation with 2 µg/ml and 4 µg/ml of dipyridamole, respectively. Platelet aggregation and thrombin generation markers were not affected in vitro by dipyridamole. Conclusions: Dipyridamole may be capable of overcoming increased prothrombinase complex formation and be in part able to compensate for AR in patients with moderate carotid stenosis. This phenomenon may explain the clinical advantages of Aggrenox^®, known to reduce ischemic events in poststroke patients as proven in clinical trials, though an additional antithrombotic benefit beyond the platelet inhibition by aspirin alone.

Structure–function relationships and mechanism of anticoagulant phospholipase A 2 enzymes from snake venoms

Phospholipase A 2 (PLA 2) enzymes from snake venom are toxic and induce a wide spectrum of pharmacological effects, despite similarity in primary, secondary and tertiary structures and common catalytic properties. Thus, the structure–function relationships and the mechanism of this group of small proteins are subtle, complex and intriguing challenges. This review, taking the PLA 2 enzymes from spitting cobra ( Naja nigricollis) venom as examples, describes the mechanism of anticoagulant effects. The strongly anticoagulant CM-IV inhibits both the extrinsic tenase and prothrombinase complexes, whereas the weakly anticoagulant PLA 2 enzymes (CM-I and CM-II) inhibit only the extrinsic tenase complex. CM-IV binds to factor Xa and interferes in its interaction with factor Va and the formation of prothrombinase complex. In contrast, CM-I and CM-II do not affect the formation of prothrombinase complex. In addition, CM-IV inhibits the extrinsic tenase complex by a combination of enzymatic and nonenzymatic mechanisms, while CM-I and CM-II inhibit by only enzymatic mechanism. These functional differences explain the disparity in the anticoagulant potency of N. nigricollis PLA 2 enzymes. Similarly, human secretory enzyme binds to factor Xa and inhibits the prothrombinase complex. We predicted the anticoagulant region of PLA 2 enzymes using a systematic and direct comparison of amino acid sequences. This region between 54 and 77 residues is basic in the strongly anticoagulant PLA 2 enzymes and neutral or negatively charged in weakly and non-anticoagulant enzymes. The prediction is validated independently by us and others using both site directed mutagenesis and synthetic peptides. Thus, strongly anticoagulant CM-IV binds to factor Xa (its target protein) through the specific anticoagulant site on its surface. In contrast, weakly anticoagulant enzymes, which lack the anticoagulant region fail to bind specifically to the target protein, factor Xa in the coagulation cascade. Thus, these studies strongly support the target model which suggests that protein–protein interaction rather than protein–phospholipid interaction determines the pharmacological specificity of PLA 2 enzymes.

Role of protease‐activated and ADP receptor subtypes in thrombin generation on human platelets

The activated platelet surface serves as an integral part of the prothrombinase complex upon activation by potent platelet agonists such as thrombin and collagen. We determined the receptor specificity through which thrombin was enhancing collagen-induced thrombin generation. Whereas SFLLRN or AYPGKF alone produced minimal thrombin generation or phosphatidylserine exposure through protease activated receptor (PAR) stimulation, they caused a leftward shift in the collagen-induced thrombin generation dose-response curve. Although SFLLRN or AYPGKF potentiated collagen-induced thrombin generation, neither of them potentiated to the same extent as thrombin. However, SFLLRN and AYPGKF together potentiated collagen-induced thrombin generation to the same extent as thrombin. We conclude that thrombin mediates its procoagulant activity through activation of both PAR1 and PAR4 receptors. Similarly, neither PAR1 nor PAR4 stimulation alone mimicked the annexin V-binding response caused by thrombin stimulation. The combination of PAR activating peptides caused minimal increases in annexin V binding, but caused significant thrombin generation, suggesting that events other than phosphatidylserine exposure may play a role in platelet prothrombinase complex formation. We also investigated the ability of ADP to potentiate agonist-induced thrombin generation. Whereas P2Y(1) antagonism did not affect collagen or thrombin-induced thrombin generation, P2Y(12) antagonism did decrease both collagen- and thrombin-induced thrombin generation, suggesting that ADP potentiates thrombin generation primarily through the P2Y(12) receptor. Collectively, these results suggest that stimulation of both the PAR1 and PAR4 receptors are necessary for thrombin-induced procoagulant activity, and that the P2Y(12) receptor, but not the P2Y(1) receptor, is responsible for the potentiation of agonist-induced platelet procoagulant activity.

This month in J Lab Clin Med: Issue highlights for March 2002

This month in J Lab Clin Med: Issue highlights for March 2002

Pharmacological Characterization of a Novel Factor Xa Inhibitor, FXV673

FXV673 is a novel, potent, and selective factor Xa (FXa) inhibitor. FXV673 inhibited human, dog, and rabbit FXa with a K i of 0.52, 1.41, and 0.27 nM, respectively. FXV673 also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C (aPC), plasmin, and tissue-plasminogen activator (t-PA). FXV673 prolonged plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) in a dose-dependent fashion. In the APTT assays, the concentrations (μM) required for doubling coagulation time were 0.41 (human), 0.65 (monkey), 1.12 (dog), 0.25 (rabbit), and 0.80 (rat). The concentrations (μM) required in the PT assays were 1.1 (human), 1.32 (monkey), 2.31 (dog), 0.92 (rabbit), and 1.69 (rat). A coupled-enzyme assay was performed to measure thrombin activity following prothrombinase conversion of prothrombin to thrombin. FXV673 showed IC 50s of 1.38 and 2.55 nM, respectively, when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or fresh platelets were used as the phospholipid source for prothrombinase complex formation. It was demonstrated that FXV673 could inhibit further thrombin generation in the prothrombinase complex using PS/PC liposomes. FXV673 dose-dependently prolonged the time to vessel occlusion and inhibited thrombus formation in well-characterized canine models of thrombosis. Interspecies extrapolation (∼2.5-fold higher sensitivity for FXa inhibition in human than in dog) suggested that 100 ng/ml of FXV673 would be an effective plasma concentration for clinical studies. Currently FXV673 is undergoing clinical studies to be developed as an antithrombotic agent.