Activation of bovine prothrombin as monitored by the clotting assay

The activation of bovine prothrombin was studied with highly purified clotting factors and using a coagulation assay developed to look at the initial rate of prothrombin conversion as well as the conversion rate over a time course of 75 min. Activation of prothrombin by factor Xa alone was slow. The rate of prothrombin conversion increased markedly with the addition of each of the accessory components Ca2+, phospholipid and bovine factor V, respectively. With the complete prothrombinase complex comprising factor Xa, Ca2+, phospholipid and factor V, the rate increase was about 22,000-fold higher compared to the action of factor Xa and Ca2+ on prothrombin alone. The rates of thrombin formation obtained with activated factor X1 were only about 70% the values obtained with factor X2. The rate of prothrombin activation and the difference between the activities of the activated factors X1 and X2 are discussed.     

Studies on the formation of the prothrombin-converting complex

1. It has been shown that factor V accelerates the rate of conversion of prothrombin into thrombin by activated factor X, and that this activity does not depend on the presence of phospholipid. 2. Although the mechanism by which factor V increases the rate of reaction is not clear, it seems unlikely that it is converted into an enzyme. 3. The rate of conversion of prothrombin by purified activated factor X and factor V was increased threefold in the presence of phospholipid, and it is suggested that the lipid acts by adsorbing the proteins on its surface and hence increasing the local concentration of the reactants.     

Activation of human factor V by factor Xa and thrombin

The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by either autoradiography of 125I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of Mr 220,000 and 105,000. Although thrombin cleaved the Mr 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the Mr 220,000 peptide. The factor Xa dependent functional assessment of 125I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the Mr 220,000 peptide. This observation facilitated the study of the kinetics of factor V activation by allowing the activation of factor V to be monitored by the appearance of the Mr 220,000 peptide (factor Xa activation) or the Mr 105,000 peptide (thrombin activation). Factor Xa catalyzed activation of factor V obeyed Michaelis-Menten kinetics and was characterized by a Km of 10.4 nM, a kcat of 2.6 min-1, and a catalytic efficiency (kcat/Km) of 4.14 X 10(6) M-1 s-1. The thrombin-catalyzed activation of factor V was characterized by a Km of 71.7 nM, a kcat of 14.0 min-1, and a catalytic efficiency of 3.26 X 10(6) M-1 s-1. This indicates that factor Xa is as efficient an enzyme toward factor V as thrombin.     

The control of prothrombin conversion. Kinetic control by mechanisms inherent in two activation pathways.

It is known that the activation of prothrombin to thrombin can proceed via two pathways: one initiated by the prothrombin-converting complex (factor Xa, factor V, phospholipid, and CA2+ ions) and the other initiated by the product, thrombin. A kinetic study has shown that the pathways do not proceed with equal ease under all conditions. At high levels of the converting complex, both go to completion: some prothrombin is always cleaved by thrombin, but the resulting intermediate is then activated to give quantitative conversion to thrombin. At slower rates of activation, the product-initiated pathway occurs to a relatively greater extent. Moreover, the intermediate then is not cleaved further but accumulates, so that the generation of thrombin is curtailed. The reason the intermediate is productive only at higher levels of activator may be partly that it is a poorer substrate for the converting complex than prothrombin. More importantly, the activity of the complex is also modulated by thrombin, which rapidly destroys the activity of factor V and factor Xa in a feedback reaction. These concerted controls ensure that prothrombin activation damps itself. Thus thrombin production occurs as a burst, the size of which is regulated by the amounts of factor Xa and factor V initially available.     

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.     

Inhibition of factor X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombin-mediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contact-activated plasma. Heparin (approximately 0.1 microM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were approximately 50 microM and approximately 5 microM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of approximately 5 microM pentasaccharide and approximately 0.05 microM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.     

Thrombin-mediated proteolysis of factor V resulting in gradual B-domain release and exposure of the factor Xa-binding site

To investigate the relationship between the individual thrombin cleavages in factor V (FV) and the generation of activated factor X (FXa) cofactor activity, recombinant FV mutants having the cleavage sites eliminated separately or in combination were used. After thrombin incubation, the ability of the FV variants to bind FXa and support prothrombin activation was tested. The interaction between FVa and FXa on the surface of phospholipid was investigated with a direct binding assay as well as in a functional prothrombin activation assay. FV mutated at all cleavage sites functioned poorly as FXa cofactor in prothrombin activation, the apparent K(d) for FXa being approximately 10 nm. Fully activated wild type FVa, yielded an apparent K(d) of around 0.2 nm. The Arg(709) and Arg(1018) cleavages occurred at low thrombin concentrations and decreased the K(d) for FXa binding 5- and 3-fold, respectively. The Arg(1545) cleavage, being less sensitive to thrombin, decreased the K(d) for FXa binding approximately 20-fold. The K(m) for prothrombin was the same for all FV variants, demonstrating B-domain dissociation to result in exposure of binding site for FXa but not for prothrombin. In conclusion, we demonstrate FV activation to be associated with the stepwise release of the B-domain, which results in a gradual exposure of the FXa-binding site.     

Identification and characterization of novel salivary thrombin inhibitors from the ixodidae tick, Haemaphysalis longicornis.

Novel antithrombin molecules were identified from the ixodidae tick, Haemaphysalis longicornis. These molecules, named madanin 1 and 2, are 7-kDa proteins and show no significant similarities to any previously identified proteins. Assays using human plasma showed that madanin 1 and 2 dose-dependently prolonged both activated partial thromboplastin time and prothrombin time, indicating that they inhibit both the intrinsic and extrinsic pathways. Direct binding assay by surface plasmon resonance measurement demonstrated that madanin 1 and 2 specifically interacted with thrombin. Furthermore, it was clearly shown that madanin 1 and 2 inhibited conversion of fibrinogen into fibrin by thrombin, thrombin-catalyzed activation of factor V and factor VIII, and thrombin-induced aggregation of platelets without affecting thrombin amidolytic activity. These results suggest that madanin 1 and 2 bind to the anion-binding exosite 1 on the thrombin molecule, but not to the active cleft, and interfere with the association of fibrinogen, factor V, factor VIII and thrombin receptor on platelets with an anion-binding exosite 1. They appear to be exosite 1-directed competitive inhibitors.     

Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.     

Inhibition of factor X, factor V and prothrombin activation by the bis(lactobionic acid amide) LW10082.

The minimum concentrations of heparin, dermatan sulfate, hirudin, and D-Phe-Pro-ArgCH2Cl required to delay the onset of prothrombin activation in contact-activated plasma also prolong the lag phases associated with both factor X and factor V activation. Heparin and dermatan sulfate prolong the lag phases associated with the activation of the three proteins by catalyzing the inhibition of endogenously generated thrombin. Thrombin usually activates factor V and factor VIII during coagulation. The smallest fragment of heparin able to catalyze thrombin inhibition by antithrombin III is an octadecasaccharide with high affinity for antithrombin III. In contrast, a dermatan sulfate hexasaccharide with high affinity for heparin cofactor II can catalyze thrombin inhibition by heparin cofactor II. A highly sulfated bis(lactobionic acid amide), LW10082 (Mr 2288), which catalyzes thrombin inhibition by heparin cofactor II and has both antithrombotic and anticoagulant activities, has been synthesized. In this study, we determined how the minimum concentration of LW10082 required to delay the onset of intrinsic prothrombin activation achieved this effect. We demonstrate that, like heparin and dermatan sulfate, LW10082 delays the onset of intrinsic prothrombin activation by prolonging the lag phase associated with both factor X and factor V activation. In addition, LW10082 is approximately 25% as effective as heparin and 10 times as effective as dermatan sulfate in its ability to delay the onset of prothrombin activation. The strong anticoagulant action of LW10082 is consistent with previous reports which show that the degree of sulfation is an important parameter for the catalytic effectiveness of sulfated polysaccharides on thrombin inhibition.     

Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.     

Kinetics of thrombin-induced release and activation of platelet factor V

The kinetics of thrombin-induced platelet factor V activation were studied in suspension of washed human platelets. The effect of thrombin in stimulating the release reaction could be separated from its effect on factor V activation by use of a potent inhibitor of the release reaction, the prostacyclin analogue ZK 36374. When platelets were incubated with ZK 36374 prior to stimulation with thrombin, the amount of ZK 36374 required to inhibit 50% of factor Va formation was 15 pM. ZK 36374 at a final concentration of 1 nM was found to block instantaneously and completely the release of factor Va, whereas it has no effect neither on platelet factor V activation nor on the factor Va assay. By varying the time interval between the addition of thrombin (0.5 nM) and ZK 36374 to suspensions of 4.6 X 10(6) platelets/ml the rate of factor V release was found to be 12 pM factor V/min. In the absence of ZK 36374 the total amount of factor V released was 8 pM, whereas Triton X-100-treated platelets gave 13 pM factor V. It appeared that the amount of factor V that could be released was dependent on the thrombin concentration. Maximum release was obtained at 1 nM thrombin. The rate of factor V release increased in proportion to the thrombin concentration. The rate of factor V activation was found to be proportional to the thrombin concentration as well as to the amount of released factor V. When 4.6 X 10(6) platelets/ml were activated by 0.5 nM thrombin, the rates of factor V activation were found to be 0.3 pM and 1.2 pM factor Va/min at 20% and 90% completion of the release reaction. Therefore, the rate of factor V release was at least one order of magnitude faster than the rate of factor V activation. The kinetics of thrombin-induced platelet factor V activation were compared to those of plasma factor V activation in platelet-rich and platelet-free plasma. The results clearly demonstrate that platelets have no effect on the rate of factor V activation and that the kinetics of plasma factor V activation are identical to those of platelet factor V activation.     

The factor V activation paradox

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.     

Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor X(a) in the absence of factor V(a) and in the absence and presence of membranes.

Activation of prothrombin by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X(a) of human prothrombin was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-1) s(-1), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X(a), (2) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X(a) for rapid conversion to thrombin.     

The in situ inhibition of prothrombinase-formed human alpha-thrombin and meizothrombin(des F1) by antithrombin III and heparin

The inhibition of thrombin by antithrombin III (AT III) and heparin has been studied in pure systems to determine the kinetics of inhibition during human prothrombin activation. The present study shows that prothrombinase-catalyzed prothrombin activation resulted in the generation of thrombin and meizothrombin(des F1). In the absence of heparin the second-order rate constants of the inactivation of both thrombin and meizothrombin(des F1) formed in the reaction mixture appeared to be identical, k = 3.7 X 10(5) M-1 min-1. The rate constant of inhibition of purified thrombin was 6.5 X 10(5) M-1 min-1. In the presence of heparin the decay of the amidolytic activity was biexponential and could be modeled by a four-parameter equation to determine the pseudo first-order rate constants of inhibition as well as the composition of the reaction with respect to the levels of thrombin and meizothrombin(des F1). The ratio of thrombin over meizothrombin(des F1) varied with the initial prothrombin concentration. Heparin catalyzed the AT III inhibition of thrombin but not meizothrombin(des F1) formed during the prothrombin activation. Thrombin, generated by (Xa-Va-phospholipid-Ca2+) was inhibited by AT III/heparin more slowly than purified thrombin, and the saturation kinetics of the inhibition with respect to AT III differed from those found with purified thrombin.     

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.     

Thrombin generation and inactivation in the presence of antithrombin III and heparin

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.     

Fibroblasts, glial, and neuronal cells are involved in extravascular prothrombin activation.

A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.     

Exposure of platelet membrane phosphatidylserine regulates blood coagulation

This article addresses the role of platelet membrane phosphatidylserine (PS) in regulating the production of thrombin, the central regulatory molecule of blood coagulation. PS is normally located on the cytoplasmic face of the resting platelet membrane but appears on the plasma-oriented surface of discrete membrane vesicles that derive from activated platelets. Thrombin, the central molecule of coagulation, is produced from prothrombin by a complex ("prothrombinase") between factor Xa and its protein cofactor (factor V(a)) that forms on platelet-derived membranes. This complex enhances the rate of activation of prothrombin to thrombin by roughly 150,000 fold relative to factor X(a) in solution. It is widely accepted that the negatively charged surface of PS-containing platelet-derived membranes is at least partly responsible for this rate enhancement, although there is not universal agreement on mechanism by which this occurs. Our efforts have led to an alternative view, namely that PS molecules bind to discrete regulatory sites on both factors X(a) and V(a) and allosterically alter their proteolytic and cofactor activities. In this view, exposure of PS on the surface of activated platelet vesicles is a key regulatory event in blood coagulation, and PS serves as a second messenger in this regulatory process. This article reviews our knowledge of the prothrombinase reaction and summarizes recent evidence leading to this alternative viewpoint. This viewpoint suggests a key role for PS both in normal hemostasis and in thrombotic disease.