Factor Xa-catalyzed Prothrombin Activation Research Articles

Autocatalytic peptide bond cleavages in prothrombin and meizothrombin.

During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg51-Thr52/Arg54-Asp55 in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s-1), Arg155-Ser156 in prothrombin (k = 2 x 10(4) M-1 s-1), and Arg284-Thr285 in prethrombin 1 (k = 0.02 x 10(4) M-1 s-1). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg51-Thr52/Arg54-Asp55) was not detectable, whereas cleavage at Arg155-Ser156 (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg284-Thr285 (formation of prethrombin 2 des-1-13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t1/2 = approximately 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 microM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s-1 in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg284-Thr285 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des-1-13 by a combination of intramolecular (k = 0.8 x 10(-4) s-1) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s-1). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s-1), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s-1) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) M-1 s-1, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.

Human Coagulation Factor V Is Activated to the Functional Cofactor by Elastase and Cathepsin G Expressed at the Monocyte Surface

The ability of intact peripheral blood monocytes to modulate factor V procoagulant activity was studied using electrophoretic and autoradiographic techniques coupled to functional assessment of cofactor activity. Incubation of plasma concentrations of factor V with monocytes (5 x 10(6)/ml) resulted in the time-dependent cleavage of the 330-kDa protein. Activation occurred via several high molecular mass intermediates (> or = 200 kDa) to yield peptides of 150, 140, 120, 94, 91, 82, and 80 kDa, which paralleled the expression of cofactor activity. The cleavage pattern observed differed from that obtained with either thrombin or factor Xa as an activator. The incubation time required to achieve full cofactor activity was dependent on the monocyte donor and ranged from 10 min to 1 h and was consistently slightly lower than that obtained with thrombin-activated factor Va. Cofactor activity was not diminished by additional incubation. The cofactor activity generated bound to the monocyte such that a competent prothrombinase complex was formed at the monocyte membrane surface. Furthermore, within 5 min of factor V addition to monocytes, near maximal cofactor activity (approximately 70%) was bound and expressed on the monocyte membrane. The proteolytic activity toward factor V was associated primarily with the monocyte membrane, as little proteolytic activity was released into the cell-free supernatant. Proteolytic activity was inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. However, the inhibitor profile obtained with alpha 1-antiproteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin suggested membrane-bound forms of elastase and cathepsin G were mediating, in large part, the proteolysis observed. These data were confirmed using purified preparations of both proteases and a specific anti-human leukocyte elastase antibody. Thus, expression of these proteases at the monocyte surface may contribute to thrombin generation at extravascular tissue sites by catalyzing the activation of the essential cofactor, factor Va, which binds to the monocyte surface and supports the factor Xa-catalyzed activation of prothrombin.

Blood Coagulation Factor Xa Interacts with a Linear Sequence of the Kringle 2 Domain of Prothrombin

Prothrombin is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca2+, and phospholipids. During the activation, prothrombin is cleaved into three fragments: fragment 1, containing a domain rich in gamma-carboxyglutamic acid residues and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human prothrombin during the activation. The isolated fragment 2 inhibited the activation of prothrombin by either prothrombinase complex or factor Xa alone in a dose-dependent manner, whereas fragment 1 and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to fragment 2 immobilized to microwell plates with a Kd of 9.0 x 10(-8) M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with Kds of 2.0 x 10(-7) and 1.5 x 10(-7) M, respectively, suggesting that factor Xa interacts with the kringle 2 domain in these molecules. The binding of factor Xa to immobilized fragment 2 was Ca(2+)-dependent with an optimal concentration at 6 mM. In the presence of Ca2+, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His205 to Arg220. Factor Xa bound to a synthetic peptide corresponding to the residues His205 to Arg220 immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of prothrombin, but a peptide with the reversed sequence of His205 to Arg220 did not. These findings indicate that factor Xa interacts with at least a linear sequence, His205 to Arg220, in the kringle 2 domain of prothrombin during its activation into thrombin.

Functional properties of human factor Va lacking the Asp683-Arg709 domain of the heavy chain.

A protease purified from the venom of the elapid snake Naja naja oxiana converts human blood coagulation factor Va into a molecule (factor VaNO) with greatly reduced cofactor activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the venom protease cleaved a small peptide from the heavy chain of factor Va and reduced the apparent M(r) from 105,000 to 101,000. This peptide was isolated by high performance liquid chromatography on a reversed-phase column. Amino acid sequence analysis of the peptide indicated that the venom enzyme cleaved the peptide bond between His682 and Asp683, thus removing 27 amino acids from the carboxyl-terminal part of the heavy chain. The cofactor activities of factors Va and VaNO were compared by measuring their abilities to support factor Xa-catalyzed prothrombin activation in the presence of phospholipids and calcium ions. Both factor Va molecules stimulated the binding of factor Xa to negatively charged phospholipids. However, the amounts of factor Va required for half-maximal incorporation of factor Xa into the membrane-bound factor Xa-Va complex were much lower for native factor Va (0.25 nM) than for factor VaNO (2.01 nM). At saturating concentrations of factor Va or factor VaNO the kcat values for prothrombin activation were 114 s-1 for factor Va and 128 s-1 for factor VaNO. The Km values for prothrombin determined under these conditions were 0.24 and 0.83 microM for prothrombinase complexes with native factor Va and factor VaNO, respectively. Direct binding studies revealed that factors Va and VaNO bind with equal affinity to phospholipids. These data indicate that factor VaNO is impaired in its ability to interact with factor Xa and prothrombin. Together with the structural data this implies that the carboxyl-terminal Asp683-Arg709 domain of the heavy chain is required for optimal interaction of factor Va with factor Xa and prothrombin.

Characterization of two forms of human factor Va with different cofactor activities.

Factor Va is an essential cofactor in factor Xa-catalyzed prothrombin activation. Purified human factor Va appears to consist of a heavy chain (M(r) approximately 105,000) and a light chain doublet with M(r) approximately 74,000 and approximately 71,000. We separated factor Va by chromatography on a Mono-S column into two fractions, designated factors Va1 and Va2. Factor Va1 contains the light chain with M(r) approximately 74,000, and factor Va2 exclusively contains the light chain with M(r) approximately 71,000. The two forms of factor Va express different cofactor activities when prothrombin is activated at low phospholipid concentrations or on membranes containing low amounts of phosphatidylserine in phosphatidylcholine. Compared with factor Va2, much higher amounts of factor Va1 are required for factor Xa. Va complex formation at the membrane surface. Once incorporated into the prothrombinase complex, factors Va1 and Va2 are equally active in prothrombin activation. This indicates that the two forms of factor Va do not differ in their ability to promote the catalytic activity of factor Xa or to interact with prothrombin. Direct binding experiments show that the different cofactor activities are explained by a greatly impaired ability of factor Va1 to bind to negatively charged membranes. Factor V is also separated into two protein peaks after chromatography on a Mono-S column. Upon incubation with thrombin, the first peak yields factor Va1 and the second peak factor Va2. The same two forms of factor Va were generated when freshly prepared plasma samples or platelet suspensions were treated with thrombin. This shows that the heterogeneity of the light chain domain is an intrinsic property of both plasma and platelet factor V. It is hypothesized that the heterogeneity is caused by small differences in the carboxylterminal C2 domain of factor V that are introduced as the result of post-ribosomal processing.

Factor Xa interacts with two sites on monocytes with different functional activities

Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (alpha-HFV1). At the monocyte surface, the factor Va/Xa- catalyzed activation of prothrombin was maximally inhibited with 0.25 mumol/L alpha-HFV1, whereas 1.0 mumol/L alpha-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did not cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.

Factor Xa Interacts With Two Sites on Monocytes With Different Functional Activities

AbstractStudies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IXα through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (α-HFV1). At the monocyte surface, the factor Va/Xa-catalyzed activation of prothrombin was maximally inhibited with 0.25 µmol/L α-HFV1, whereas 1.0 µmol/L α-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did hot cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.

Sphingosine inhibits factor Xa-catalyzed prothrombin activation on the surface of cultured calf pulmonary artery endothelium perturbed by hydrogen peroxide

The effects of hydrogen peroxide on factor Xa-catalyzed prothrombin activation on the surface of vascular endothelial cells, and the inhibitory effect of sphingosine on prothrombin activation were studied in cultured calf pulmonary artery endothelial cells (CPAEC). Hydrogen peroxide enhanced factor Xa-catalyzed prothrombin activation on the cell surface in a time and dose-dependent manner. Sphingosine, a potent inhibitor of protein kinase C and the extrinsic pathway of blood coagulation, showed a partial inhibition of prothrombin activation on the surface of CPAEC. When cells were perturbed by hydrogen peroxide, sphingosine inhibited prothrombin activation and displaced factor Xa bound to the cell surface.

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma.

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.

Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation on endothelial cells. The influence of thrombin on the reaction mechanism

Incubation of prothrombin on cultured human umbilical vein endothelial cells with factor Xa and calcium ions induced the activation of prothrombin. The mechanism of prothrombin activation was analyzed on sodium dodecyl sulfate gels using immuno- and amido-blotting techniques. It was demonstrated that meizothrombin was formed as an intermediate in prothrombin activation on the endothelial cell surface. In addition, considerable amounts of meizothrombin des-fragment-1 accumulated during prothrombin activation and were not further converted to thrombin. Although preincubation of the endothelial cells with thrombin did not influence the formation of meizothrombin, addition of hirudin to the prothrombin activation mixture inhibited the formation of meizothrombin and meizothrombin des-fragment-1 almost completely. This indicated that the activity of endogenously formed thrombin influenced the formation of meizothrombin via a feedback mechanism. The increased formation of meizothrombin and accumulation of meizothrombin des-fragment-1 in a latter phase of prothrombin activation points to a regulatory mechanism in hemostasis which subdues the formation of the procoagulant alpha-thrombin.

Prothrombin activation on membranes with anionic lipids containing phosphate, sulfate and/or carboxyl groups

Factor Xa catalyzed prothrombin activation is strongly stimulated by the presence of negatively charged membranes plus calcium ions. Here we report experiments in which we determined the prothrombin-converting activity of phosphatidylcholine (PC) membranes that contain varying amounts of different anionic lipids, viz., phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylmethanol (MePA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidyl-beta-lactate (PLac), sulfatides (SF), sodium dodecyl sulfate (SDS), and oleic acid. All anionic lipids tested were able to accelerate factor Xa catalyzed prothrombin activation, in both the absence and presence of the protein cofactor Va. This shows that the prothrombin-converting activity of negatively charged membranes is not strictly dependent on the presence of a phosphate group but that lipids which contain a carboxyl or sulfate moiety are also able to promote the formation of a functionally active prothrombinase complex. In the absence of factor Va, the prothrombin-converting activity of membranes with MePA, PG, PE, PLac, SF, or SDS was strongly inhibited at high ionic strength, while the activity of PS- and PA-containing membranes was hardly affected by ionic strength variation. This suggests that in the case of the ionic strength sensitive lipids electrostatic forces play an important role in the formation of the membrane-bound prothrombinase complex. For PS and to a lesser extent for PA we propose that the formation of a coordinated complex (chelate complex) with Ca2+ as central ion and ligands provided by the gamma-carboxyglutamic acid residues of prothrombin and factor Xa and the polar head group of phospholipids is the major driving force in protein-membrane association. Our data indicate that the anionic lipids used in this study can be useful tools for further investigation of the molecular interactions that play a role in the assembly of a membrane-bound prothrombinase complex. Membranes that were solely composed of PC can also considerably enhance prothrombin activation in the presence of factor Va. This activity of PC is only observed on membranes which are composed of PC that contains unsaturated hydrocarbon side chains. Membranes prepared from phosphocholine-containing lipids with saturated hydrocarbon side chains such as dimyristoyl-PC, dipalmitoyl-PC, distearoyl-PC, and dioctadecylglycerophosphocholine hardly accelerated prothrombin activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid.

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.

Formation of factor Va by atherosclerotic rabbit aorta mediates factor Xa-catalyzed prothrombin activation.

Vascular cell procoagulant activity may be important in the pathogenesis of atherosclerosis. In previous studies, we described the ability of the atherogenic metabolite homocysteine to activate endothelial cell Factor V, a key coagulation cofactor for thrombin generation. The present study was designed to investigate Factor V activity and Factor Xa-catalyzed prothrombin activation by control and atherosclerotic aorta from normal and hypercholesterolemic rabbits. Factor Xa generated ninefold more thrombin on atherosclerotic aortic segments than on control segments. Atherosclerotic segments activated 125I-prothrombin with Factor Xa in the presence of the thrombin inhibitor dansyl arginine-4-ethylpiperidine amide and cleaved 125I-Factor V. This suggests that increases in vessel-wall Factor V activity and Factor Xa-catalyzed prothrombin activation result from activation of vessel-wall Factor V. 125I-Factor Va peptides generated by atherosclerotic aorta were very similar in molecular weight to those generated by homocysteine-treated cells. When vascular endothelium was mechanically removed by brushing, atherosclerotic vessels still generated four- to fivefold more thrombin than control vessels. These data and results from immunocytochemical studies suggest that Factor V in atherosclerotic vessels is associated with both endothelium and other cells of the lesion. In contrast, Factor V in control vessels is associated primarily with endothelium. The increases in Factor V activity and thrombin formation in the blood vessel wall of hypercholesterolemic rabbits may contribute to the development of atherosclerosis and its complications.

Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation.

The conversion of prothrombin into thrombin by Factor Xa requires the cleavage of two peptide bonds in prothrombin. Dependent on the order of cleavage, prethrombin 2 or meizothrombin occurs as intermediate. Since prethrombin 2 has as yet been the only observed intermediate, prothrombin activation is generally considered to proceed via prethrombin 2. In this paper we present new methods that allow differentiation between meizothrombin and thrombin formed during the initial phase of prothrombin activation. These methods, which make use of the different reactivities of meizothrombin and thrombin toward fibrinogen and antithrombin III plus heparin, enabled us to show the generation of considerable amounts of meizothrombin during Factor Xa-catalyzed prothrombin activation. Both meizothrombin and thrombin incorporated the active site-directed fluorescent chloromethyl ketone 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-CH2Cl. Gel electrophoretic analysis of chloromethyl ketone-treated aliquots of prothrombin activation mixtures confirmed meizothrombin formation. These observations demonstrate that prothrombin may also be converted into thrombin via meizothrombin.

The effects of bovine prothrombin fragment 1 and fragment 1.2 on prothrombin activation

In this paper we describe the effects of the activation peptides prothrombin fragment 1 and fragment 1.2 on factor Xa-catalyzed prothrombin activation. Prothrombin activation in free solution by either factor Xa or factor Xa together with factor Va is unaffected by the activation fragments. When negatively charged phospholipids are present we observed considerable inhibition of prothrombin activation by both fragment 1 and fragment 1.2. For the activation of 0.25 μM prothrombin by factor Xa in the presence of 50 μM phospholipid (phosphatidylserine/phosphatidylcholine, 25/75; mol/mol) and 5 mM CaCl 2 50% inhibition was obtained at 0.28 μM fragment 1 or fragment 1.2. Much higher fragment concentrations were required for 50% inhibition of a prothrombinase complex consisting of factor Xa, factor Va, Ca 2+ and phospholipid. This shows that factor Va protects prothrombin activation against inhibition by its own activation peptides. Less inhibition by activation fragments was also observed at higher phospholipid and prothrombin concentrations or when the mole fraction phosphatidyl-serine in the phospholipid vesicles was decreased. The effects of fragment 1 and fragment 1.2 on prothrombin activation were identical throughout all experiments, indicating that the inhibition is due to the γ-carboxyglutamic acid containing region of the activation peptides. Our observations suggest that the activation fragments inhibit prothrombin activation by competing with prothrombin and factor Xa for binding sites at the phospholipid surface. In such a model factor Va will protect against the inhibition since it is known to promote the assembly of the prothrombinase complex through interactions with factor Xa and prothrombin that are independent of the gla-residues. The kinetic properties of fragment inhibition also suggest that in vivo prothrombin activation will not be affected by the generation of activation peptides.

The contribution of bovine Factor V and Factor Va to the activity of prothrombinase.

The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.

Effect of oral contraceptive use on platelet prothrombin converting (platelet factor 3) activity

Platelets obtained from women using estrogen-progesterone oral contraceptives are evaluated for activity as cofactor for Factor Xa-catalyzed prothrombin activation. Donor platelet-rich plasma is diluted into a standard platelet-poor plasma to minimize variations in plasma Factor X and prothrombin concentrations. Activation is initiated with Russell's viper venom, arrested at intervals with EDTA, and residual prothrombin is measured. Compared with controls, platelets from women using oral contraceptives promote an increase in both rate and extent of prothrombin activation, with control and test groups falling into non-overlapping populations. The increased prothrombin activation correlates with an increase in antithrombin III consumption. Enhanced antithrombin III consumption explains the decreased serum antithrombin III levels among women using oral contraceptives.