Products Of Prothrombin Activation Research Articles

Activation of factor XI by products of prothrombin activation

AbstractThe prothrombinase complex converts prothrombin to α-thrombin through the intermediate meizothrombin (Mz-IIa). Both α-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains α-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. α-Thrombin can undergo additional proteolysis to β-thrombin and γ-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by β-thrombin or γ-thrombin, and by α-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, β-thrombin, γ-thrombin, and α-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than α-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than α-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factor-initiated coagulation and the proteases of the contact system.

Activation of Factor XI and the Contact Proteases by Products of Prothrombin Activation.

AbstractAbstract 1150During plasma coagulation the protease α-thrombin (αIIa) cleaves fibrinogen to form a fibrin clot. Conversion of prothrombin to αIIa is catalyzed by factor (f) Xa, and results in expression of two electropositive regions on αIIa designated anion binding exosites (ABE) I and II. ABE I is involved in fibrinogen binding. In the presence of fVa and phospholipid, fXa cleaves prothrombin preferentially after Arg320, generating the intermediate meizothrombin (MzIIa), which also expresses ABE I. MzIIa is rapidly converted to αIIa. αIIa can be converted to β-thrombin (βIIa) and γ-thrombin (γIIa), both of which are cleaved within ABE I, and have greatly reduced capacity to convert fibrinogen to fibrin. Physiologic functions for βIIa or γIIa are not established; however, both have been identified in clotting blood. αIIa up-regulates its own generation in plasma by converting fXI to the protease fXIa. Yun et al. (J Biol Chem 2003;278:48112) showed that amino acids in ABE I are required for optimal fXI activation in the presence of the polyanion dextran sulfate (DS). MzIIa also activates fXI, consistent with a role for ABE I in protease binding to fXI. Given the absence of ABE I in βIIa and γIIa, it seems reasonable to postulate these proteases would interact poorly with fXI.In a clotting assay in which thrombin is added to plasma anticoagulated with citrate (low calcium), βIIa (12.5 nM) and γIIa (50 nM) did not induce clot formation, consistent with their low capacity to cleave fibrinogen. However, when plasma was recalcified to allow thrombin to form from endogenous prothrombin, both βIIa and γIIa induced clot formation. Recalcified plasma in the absence of βIIa or γIIa did not clot (800 sec observation period), indicating fibrin formation was βIIa/γIIa-dependent. Addition of an antibody to fXI prolonged the clotting time with βIIa, and prevented clotting with γIIa, suggesting βIIa and γIIa were activating fXI. In addition, with γIIa, a fXIIa inhibitor modestly prolonged clotting time, indicating the plasma contact phase was activated.We studied fXI activation by thrombin using western blot. βIIa and γIIa activated fXI at approximately half the rate of αIIa, while MzIIa activated fXI ∼4 fold faster than αIIa. FXI activation by αIIa is greatly enhanced by DS. In the presence of DS, αIIa and βIIa activated fXI comparably, while results with γIIa were not informative because the protease does not interact well with DS. Importantly, fXI activation by αIIa was not affected by the ABE I blocking peptide hirugen, indicating ABE I is not required for fXI activation by thrombin.While βIIa and γIIa were less effective fXI activators than αIIa and MzIIa in solution, significantly different results were obtained in a plasma thrombin generation assay. Here coagulation is initiated in fXII deficient plasma with thrombin (10 nM), and subsequent thrombin generation from endogenous prothrombin is monitored. The system is fXI-dependent, as a fXI antibody blocks thrombin generation. Prior work with this system indicates fXI is probably converted to fXIa by the thrombin added to initiate the process. Initiation of coagulation with αIIa and MzIIa resulted in comparable thrombin generation (∼250 nM). βIIa and γIIa, as well as recombinant αIIa with mutations in amino acids in ABE I induced thrombin generation ∼2-fold greater than for αIIa and MzIIa. We hypothesized this was due to the inability of fibrinogen to compete with fXI for binding to thrombin species lacking ABE I. Consistent with this, hirugen peptide enhanced αIIa initiated thrombin generation ∼4-fold.Finally, we followed up on the observation that a fXIIa inhibitor prolonged time to γIIa-induced clot formation in recalcified plasma. In solution, γIIa, but not αIIa, βIIa, or MzIIa cleaves the contact factors fXII and PK. The cleaved proteases, in turn, are capable of cleaving chromogenic substrates, and have activity in a reciprocal fXII-PK activation assay.Our studies show that ABE I is not required for thrombin-mediated activation of fXI, that thrombin species not fully expressing ABE I may be better than αIIa and MzIIa as initiators of fXI-dependent thrombin generation in plasma, and that γIIa can activate the plasma contact proteases. Taken as a whole, the data indicate forms of thrombin other than αIIa may contribute directly to feedback activation of fXI, and may represent a previously unrecognized link between coagulation and the contact system. Disclosures:No relevant conflicts of interest to declare.

Factor XI Contributes to Thrombin Generation in the Absence of Factor XIIa

Factor XI (fXI) is the zymogen of a plasma protease (fXIa) that contributes to coagulation by activating factor IX. The mechanism by which fXI is converted to fXIa in plasma has been a topic of recent debate. When plasma is exposed to a charged surface, factor XII (fXII) is converted to fXIIa, which then activates fXI. The importance of this reaction to hemostasis in vivo is questionable, as fXII deficiency does not cause abnormal bleeding. This suggests that fXI can be activated by other proteases, with α-thrombin receiving considerable attention in this regard. Results from several laboratories support a model in which α-thrombin activates fXI to propagate coagulation. This notion has been challenged by a recent study that foundno direct evidence of fXI activation by α-thrombin in plasma andthat fXI activation during plasma preparation can give the false impression that fXI is activated independent of fXIIa.We developed two plasma systems to examine thrombin generation (measured by calibrated automated thrombography) in the absence of fXII, with due consideration to the possibility that traces of fXIa can affect results. In the first system, fXI deficient plasma is initially treated with corn trypsin inhibitor to neutralize fXIIa, and then supplemented with fXI treated previously with DFP to neutralize contaminating fXIa. The second system uses fXII deficient plasma, and endogenous fXI is neutralized with an antibody if a fXI deficient state is required. Coagulation is initiated in both systems by addition of Ca2+ with or without tissue factor (TF - <10 pM), α-thrombin (5 nM), or factor Xa (6 pM). In both systems, significant thrombin generation was detected only in the presence of fXI, and required TF, α-thrombin, or factor Xa. Ca2+ alone did not stimulate thrombin generation. Thrombin generation was detected in fXI deficient plasma stimulated with as little as 3.0 pM fXIa. However, only 0.3 pM fXIa was required to induce thrombin generation if fXI was present, indicating additional fXIa is generated after addition of the fXIa trigger. The fXI deficient plasma system was not reconstituted by fXI variants defective in factor IX activation, nor by a fXI variant that is activated poorly by α-thrombin but normally by fXIIa. The results support a model in which fXI is activated in plasma by thrombin, with fXIa subsequently contributing to additional thrombin generation through factor IX activation. α-thrombin generated early in these reactions could promote subsequent thrombin generation through activation of factors V and VIII, as well as conversion of fibrinogen to fibrin. These reactions involve interactions with anion binding exosite I (ABE-I) on α-thrombin. When thrombin with a dysfunctional ABE-I (β-thrombin or α-thrombin with ABE-I mutations) were tested in the plasma systems, fXI-dependent thrombin generation was actually greater, and occurred earlier, than in the same system stimulated with α-thrombin. Studies with purified proteins and SDS-PAGE showed that β-thrombin and the ABE-I mutants convert fXI to fXIa similarly to α-thrombin. α-thrombin was also able to activate fXI in the presence of the ABE-I blocking peptide hirugen. β-thrombin and the exosite I mutants may promote fXI-dependent thrombin generation in plasma better than α-thrombin because there is no competition from fibrinogen. The different behavior of α-thrombin compared to β-thrombin and the ABE-I mutants supports the broader concept that thrombin activates fXI in plasma, and indicates that fXI activation by thrombin does not require ABE-I. Natural products of prothrombin activation lacking ABE-I, such as β-thrombin, therefore, may contribute to factor XI activation in plasma.

Drug Monitoring of Argatroban Using the Ecarin Chromogenic Assay

The ecarin chromogenic assay (HaemoSys ECA; JenAffin GmbH, Jena, Germany) was developed for quantitative determination of direct thrombin inhibitors. As a further development of the ecarin clotting time (ECT), the ECA is based on the same principle of measurement: the activation of prothrombin by ecarin, a snake venom from Echis carinatus. In the ECA, the prothrombin activation products meizothrombin and meizothrombin-des-F1 cleave a chromogenic substrate. The activity of meizothrombin/meizothrombin-des-F1 is inhibited in a concentration-dependent manner by direct thrombin inhibitors. The ECA-T is an assay for quantitative determination of active site directed synthetic thrombin inhibitors. For argatroban ECA-T, there is a very precise and sensitive method of quantitative drug monitoring. Only very low interindividual variations were found compared with activated partial thromboplastin time and even ECT. ECA-T is independent of variations in prothrombin levels of the plasma samples. Heparin does not influence the measuring result. ECA-T is performed on manual coagulation analyzers with an option for optical measurement but can also be applied to automated laboratory systems. By the use of ECA-T as a drug-monitoring method, the drug safety of argatroban, most of all in critically ill patients, can be increased.

Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg306 and Arg506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg306 and Arg506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 microm prothrombin, whereas half-maximal inhibition was obtained at 0.7 microm prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.

Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom

Bleeding at the site of bite and/or systemic hemorrhage are symptoms frequently observed in envenomation by Bothrops jararaca snakes. In this study, we purified and characterized a prothrombin activator from B. jararaca that is probably involved in these clinical manifestations. The enzyme was isolated by a combination of gel filtration and ion exchange chromatographies and named bothrojaractivase. It has a single polypeptide chain with a molecular weight of 22,829 Da as measured by mass spectroscopy. Bothrojaractivase generates active thrombin from prothrombin, independently of cofactors. SDS-PAGE analysis of the prothrombin activation products shows that bothrojaractivase converts prothrombin into meizothrombin producing similar fragments to those generated by group A prothrombin's activators. In addition, bothrojaractivase degraded fibrinogen and fibrin. Chelating agents completely inhibited the enzymatic activity, whereas inhibitors of serine and cysteine proteinases had no effect. Amino acid sequence of four peptides demonstrated high similarity of bothrojaractivase with P-I class of snake venom metalloproteinases. Thus, our results indicate that bothrojaractivase is a new metalloproteinase that acts on different protein factors of the clotting cascade especially displaying a key and most relevant functional action in the generation of thrombin through prothrombin activation in a similar mode of action as that of group A activators.

The Interaction of Prothrombin and Its Activation Products with Platelets.

The interaction of human 125I-prothrombin and its activation products with unactivated and thrombin-stimulated human platelets was studied. 125I-prothrombin binding to unactivated platelets was found to be a reversible and calcium-dependent process (n=48,000 sites/platelet, Kd=3.0μM). Thrombin stimulation of platelets resulted in increased prothrombin binding both in the presence and absence of calcium (n=84,000 sites/platelet, Kd=6.0μM and n=53,000 sites/platelet, Kd=8.0μM, respectively). Thrombin stimulation of platelets also increased the calcium-independent binding of prethrombin-1 compared with unstimulated cells. Using thrombin-stimulated platelets as a membrane surface for prothrombinase, 125I-prothrombin was converted exclusively to 125I-Fragment 1.2 and 125I-thrombin when the reactions were carried out in the presence of the reversible thrombin inhibitor, 5-dimethylaminonapthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). 125I-thrombin was the major prothrombin activation product that remained bound to the thrombin-stimulated platelets (50,000 molecules/platelet). Platelet-bound 125I-thrombin either added directly or generated from 125I-prothrombin by the action of prothrombinase was found to be inacessible to human antithrombin (AT)/heparin-induced inactivation. Utilization of phosphatidylcholine/phosphatidylserine (PCPS) vesicles as a membrane surface for prothrombinase in the presence of DAPA demonstrated that 125I-prothrombin activation proceeded initially through a meizothrombin intermediate that was replaced at later times by Fragment 1.2 and the A and B chains of α-thrombin. 125I-Fragment 1.2 was the main prothrombin activation product that remained bound to the PCPS vesicles after Sephadex G-150 gel filtration chromatography. The above results indicate that human prothrombin binds specifically to unstimulated human platelets via its Fragment 1 domain only in the presence of calcium and thrombin-stimulation results in the exposure of additional calcium-dependent and -independent prothrombin binding sites. In addition, thrombin was the major prothrombin activation product that remains associated with the thrombin-stimulated human platelets and this platelet-bound thrombin is protected from AT/heparin- induced inactivation. During prothrombin activation on platelets, large amounts of thrombin remain bound and Fragment 1.2 is released from the platelet surface. In contrast, Fragment 1.2 was the major prothrombin activation product that binds PCPS vesicles. Therefore, although both thrombin-stimulated human platelets and PCPS vesicles may each serve as an effective membrane component of prothrombinase, they appear to differ both in their prothrombin activation pathway as well as their ability to bind the products of prothrombin activation.

Ecarin chromogenic assay: an innovative test for quantitative determination of direct thrombin inhibitors in plasma

The ecarin chromogenic assay (ECA) was developed for quantitative determination of direct thrombin inhibitors. As a further development of the ecarin clotting time (ECT), the ECA is based on the same principle, the activation of prothrombin by ecarin a snake venom from Echis carinatus. In the ECA the prothrombin activation products meizothrombin and meizothrombin-desF1 cleave a chromogenic substrate, whereas in the clotting assay ECT plasma fibrinogen is converted to fibrin. The activity of meizothrombin/meizothrombin-desF1 is inhibited in a concentration-dependent fashion by direct thrombin inhibitors. The ECA can be used as ECA-H for quantitative determination of hirudin and as ECA-T for determination of synthetic thrombin inhibitors. As shown for hirudin, argatroban and melagatran, the ECA turned out as a very precise and sensitive method, which combines the advantages of ECT with those of chromogenic assays. In ECA very low interindividual variations were found compared to aPTT and even ECT. The ECA is independent of the variations of the coagulation variables prothrombin and fibrinogen.

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.

Factor Xa is highly protected from antithrombin–fondaparinux and antithrombin–enoxaparin when incorporated into the prothrombinase complex

Antithrombin and its cofactor, heparin, target both the product of prothrombin activation by prothrombinase, thrombin, as well as the enzyme responsible for the reaction, factor (F)Xa. These studies were carried out to quantify the effects of each of the prothrombinase components on the half-life of FXa in the presence of antithrombin and the low-molecular-weight heparins (enoxaparin, Aventis, Laval, Quebec, Canada) or the heparin pentasaccharide (fondaparinux, Organon Sanofi-Synthelabo, Cypress, TX, USA). Experiments were carried out using a recombinant form of prothrombin in which the active site serine has been mutated to cysteine and subsequently labeled with fluorescein. This mutant allowed calculation of the second order rate constant for inhibition of FXa by antithrombin in such a way that competition for antithrombin by thrombin is eliminated and competition for FXa by prothrombin is accounted for. Intrinsic rate constants for the inhibition of FXa by antithrombin-enoxaparin and antithrombin-fondaparinux, in the presence of the various prothrombinase components, were calculated. Addition of phospholipid had no significant effect on the second order rate constant for inhibition of FXa by antithrombin, while addition of FVa appeared to be mildly protective. Further addition of prothrombin however, caused profound protection of FXa, increasing its half-life from 1.1 to 353 s in the case of fondaparinux, and from 0.4 to 42 s in the case of enoxaparin. Similar results were reported for unfractionated heparin previously [1]. Therefore, in the presence of unfractionated heparin, fondaparinux, or enoxaparin, prothrombinase is profoundly protected from antithrombin.

Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than alpha-thrombin.

The conversion of protein C into activated protein C (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells initiates an essential negative feedback reaction on blood coagulation. APC, together with its non-enzymic cofactor protein S, inactivates factors Va and VIIIa, the non-enzymic protein cofactors of the prothrombinase and intrinsic tenase complex, by proteolytic degradation. In this study we report that prothrombin activation products, generated by the prothrombinase complex on the surface of quiescent endothelial cells, are able to activate protein C. Subsequent inactivation of factor Va by the APC that was formed decreased the rate of prothrombin activation, thus demonstrating in vitro the negative feedback loop on coagulation factor activation. The anticoagulant feedback reaction of APC on the prothrombinase complex was stimulated 3-4-fold by the addition of protein S but not by thrombin-cleaved protein S or by protein S complexed with C4b-binding protein. Stimulation of endothelial cells with 50 pM tumour necrosis factor (TNF) or 500 pM interleukin 1 (IL-1) resulted in a 70% decrease in activation of protein C by exogenously added alpha-thrombin, which seemed to be due to down-regulation of thrombomodulin activity on the surface of endothelial cells. However, when prothrombin activation products generated in situ were allowed to activate protein C, stimulation of endothelial cells with TNF and IL-1 resulted in only a 25% decrease in activation of protein C. Stimulation with TNF or IL-1 did not affect the ability of endothelial cells to support prothrombinase activity. We investigated whether the differences in extent of protein C activation by exogenously added alpha-thrombin and by prothrombin activation products generated in situ were due to meizothrombin formed during prothrombin activation. Previous reports from our groups revealed that meizothrombin is generated as a transient intermediate during prothrombin activation on phospholipid vesicles and endothelial cells. Here we show that meizothrombin is at least a 6-fold better activator of protein C on the surface of endothelial cells than is alpha-thrombin. These results demonstrate that meizothrombin, formed during the initial phase of prothrombin activation, efficiently down-regulates both its own formation and that of thrombin.

Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis

In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation. The detection limit of the so-called "amidoblot" is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting. It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

Structural and fuctional characterization of a prothrombin activator from the venom of Bothrops neuwiedi

A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn 2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a K m of 38 μM and a V max of 120 μmol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca 2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents, such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl 2. These results indicated that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrobmin activator from B. Neuwiedi are similar to those reported for the venom activator from Echis carinatus.

Neutralization of heparin by prothrombin activation products

The neutralization of heparin by active site blocked meizothrombin and thrombin, prothrombin fragment 1.2, fragment 1 and fragment 2 was probed by the heparin-dependent factor Xa inactivation by antithrombin III (AT III). Meizothrombin had no effect on the inactivation of factor Xa, whereas thrombin had an inhibitory effect (IC 50 = 700 nM). After factor Xa catalyzed cleavage of meizothrombin, the resulting products, prothrombin fragment 1.2 plus thrombin, did not show any heparin neutralizing properties. However, after isolation of the reaction products, both thrombin and prothrombin fragment 1.2 exhibited heparin neutralizing properties in the factor Xa inactivation reaction. The IC 50-values were 700 nM and 100 nM, respectively. Prothrombin fragment I, when present at 125 nM, caused a 50% reduction of the heparin-dependent rate of inactivation of factor Xa and prothrombin fragment 2 had no effect at all. From this we conclude that, in addition to the thrombin part of the prothrombin molecule, the fragment 1 region also exhibits a rather high affinity for heparin.

Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism.

The kinetics of the activation of human prothrombin catalyzed by human prothrombinase was studied using the fluorescent alpha-thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Prothrombinase proteolytically activates prothrombin to alpha-thrombin by cleavages at Arg273-Thr274 (bond A) and Arg322-Ile323 (bond B). The differential fluorescence properties of DAPA complexed with the intermediates and products of human prothrombin activation were exploited to study the kinetics of the individual bond cleavages in the zymogen. When the catalyst was composed of prothrombinase (human factor Xa, human factor Va, synthetic phospholipid vesicles, and calcium ion), initial velocity studies of alpha-thrombin formation indicated that the kinetic constants for the cleavage of bonds A or B were similar to the constants that were obtained for the overall reaction (bonds A + B). The progress of the reaction was also monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results indicated that the activation of human prothrombin catalyzed by prothrombinase proceeded exclusively via the formation of meizothrombin (bond B-cleaved) as an intermediate. Kinetic studies of the cofactor dependence of the rates of cleavage of the individual bonds indicated that, in the absence of the cofactor, cleavage at bond B would constitute the rate-limiting step in prothrombin activation. Progress curves for prothrombin activation catalyzed by prothrombinase and monitored using the fluorophore DAPA were typified by the appearance of a transient maximum, indicating the formation of meizothrombin as an intermediate. When factor Xa alone was the catalyst, progress curves were characterized by an initial burst phase, suggesting the rapid production of prethrombin 2 (bond A-cleaved) followed by its slow conversion to alpha-thrombin. Gel electrophoresis followed by autoradiography was used to confirm these results. Collectively, the results indicate that the activation of human prothrombin via the formation of meizothrombin as an intermediate is a consequence of the association of the cofactor, human factor Va, with the enzyme, human factor Xa, on the phospholipid surface.

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.

Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin.

Staphylocoagulase with a molecular weight of 64,000 and subspecies ranging in molecular weight from 36,000 to 64,000 were purified by affinity column chromatography on bovine prothrombin-Sepharose 4B from the culture filtrates of the Staphylococcus aureus strains, st-213 and 104. The samples containing all molecular species from both strains had the same NH2-terminal sequence, Ile-Val-Thr-Lys-Asp-Tyr-Ser-Lys-Glu-, implying that the molecular heterogeneity was due to proteolytic degradation to some extent of the COOH-terminal portion during cultivation or purification. Staphylocoagulase (Mr = 64,000) from strain st-213 formed an active complex, "staphylothrombin," with human prothrombin in a molar ratio of 1 to 1.1. Staphylothrombin was unstable at 37 degrees C and some portions of staphylocoagulase in the complex were rapidly degraded into small fragments, together with the fragmentation of prothrombin into prethrombin 1 and prothrombin fragment 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent fluorography for the products of prothrombin activation by staphylocoagulase in the presence of [3H]diisopropylphosphofluoridate (DFP) demonstrated the formation of a DFP-sensitive active site in the prothrombin molecule, and no cleavage of the Arg-Ile bond linking the A and B chains of alpha-thrombin was found. The enzymatic properties including the pH-dependency of the activity, substrate specificity and behavior towards thrombin inhibitors of staphylothrombin differed from those of alpha-thrombin, although the active site titration of staphylothrombin with p-nitrophenyl-p'-guanidinobenzoate showed 0.95 +/- 0.2 mol of active site/mol of enzyme.

Quantitation of prothrombin activation products in human urine.

Urine samples obtained from 37 normal individuals have been screened for the presence of prothrombin activation products using radioimmunoassays developed for fragment 1, fragment 2 and prothrombin derivatives bearing the thrombin region. The cross-reacting materials detected in urine were isolated by affinity chromatography on insolubilized antibodies, and analysed by SDS polyacrylamide gel electrophoresis. The only prothrombin derivatives detected were fragment 1 (37/37) and fragment 2 (22/37). The mean values of daily urinary fragment 1 and fragment 2 excretion were respectively 13.4 nM and 1.5 nM. The excretion of prothrombin derivatives has been quantitated in 14 normal pregnant women, during the third trimester of gestation. The mean values of urinary excretion were 47.2 nM per day for fragment 1 (P less than 0.005) and 6.4 nM per day for fragment 2 (P less than 0.05). The significant increase in fragment 1 and fragment 2 excretion observed in a condition known to be associated with the so-called hypercoagulable state suggest that the measurement of prothrombin derivatives in urine could be a useful tool for the non-invasive detection of thromboembolic diseases or prethrombotic states.

Binding of the products of prothrombin activation to human platelets.

Binding of prothrombin and activation intermediates 1 and 2 to human platelets was tested with 125-I-labeled protein preparations. None of these precursors of thrombin bound to platelets under conditions in which high affinity binding of thrombin was observed,nor did they cause platelet aggregation or serotonin release. The molecular conformation required for binding to platelets as well as for induction of platelet aggregation and release is present, therefore, only after the final step in prothrombin activation.

Activation of prothrombin with (taipan snake) venom

Complete activation of prothrombin with taipan snake () venom yields thrombin and two fragments, fragment-1 and fragment-2, which by the criteria of ion-exchange chromatography and SDS gel electrophoresis are indistinguishable from the products of Xa-catalyzed prothrombin activation. Activation in the presence of diisopropylfluorophosphate with either taipan venom or Xa results in the formation of thrombin-sensitive fragment 1·2, a single polypeptide composed of fragments-1 and 2. Whereas the rate of venom-catalyzed activation is enhanced 20–30 times by phospholipid vesicles, Factor V is without effect in either the presence or absence of phospholipid. The venom activity is not inhibited by diisopropylfluorophosphate, phenylmethylsulfonylfluoride, soybean trypsin inhibitor, p-hydroxymercuribenzoate or iodoacetate. The venom does not hydrolyze N2-toluene-p-sulfonyl-L-arginine methyl ester, N2-benzoyl-L-arginine ethyl ester or N-acetyl-L-tyrosine ethyl ester. The rate of venom-catalyzed prothrombin activation is not affected by plasma factor Xa inhibitor plus heparin.