Prothrombin Derivatives Research Articles

An assay to measure levels of factor Xa inhibitors in blood and plasma

Unlabelled Box BackgroundRivaroxaban and apixaban are the most commonly used anti‐factor (F) Xa direct oral anticoagulants (DOAC), with indications for prevention of stroke in nonvalvular atrial fibrillation as well as treatment and prevention of venous thromboembolism. However, lacking is accessibility to a detection method that is able to quantify low levels of anti‐FXa DOACs. ObjectiveWe report a new assay that measures anti‐FXa DOAC levels in plasma and whole blood. MethodsThis is achieved by the use of a prothrombin derivative that is labeled with a fluorescent probe (Flu‐II), which then acts as the macromolecular substrate to measure residual FXa activity. The Flu‐II cleavage is then initiated by the addition of a solution containing FXa, FVa, and phospholipid vesicles composed of 75% PC and 25% PS (PCPS) vesicles with calcium, in the presence of hirudin to prevent feedback activity by the native thrombin generated. The Flu‐II cleavage is monitored by fluorescence in real time where the initial rate of fluorescence change is inversely proportional to DOAC levels. ResultsIn plasma systems, the assay demonstrates dose‐response between 0 and 5 nmol/L rivaroxaban and between 0 and 10 nmol/L apixaban. Corn trypsin inhibitor did not affect this assay. With individual plasma samples, the assay showed excellent consistency and reproducibility. From 2 μL of whole blood, the assay showed dose‐response between 0 and 2 nmol/L of DOACs in the final mixture of 100 μL, thus representing up to 100 nmol/L in circulating blood. ConclusionThe assay is ideal for rapidly and accurately measuring DOAC levels in plasma and blood, demonstrating its potential for point‐of‐care applications.

Identification and characterization of a factor Va-binding site on human prothrombin fragment 2

The fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of prothrombin. The extent to which the F2-FVa interaction affects the overall thrombin generation, however, is uncertain. To study this interaction, nuclear magnetic resonance spectroscopy of recombinant F2 was used to identify seven residues within F2 that are significantly responsive to FVa binding. The functional role of this region in interacting with FVa during prothrombin activation was verified by the FVa-dependent inhibition of thrombin generation using peptides that mimic the same region of F2. Because six of the seven residues were within a 9-residue span, these were mutated to generate a prothrombin derivative (PT6). These mutations led to a decreased affinity for FVa as determined by surface plasmon resonance. When thrombin generation by an array of FXa containing prothrombinase components was monitored, a 54% decrease in thrombin generation was observed with PT6 compared with the wild-type, only when FVa was present. The functional significance of the specific low-affinity binding between F2 and FVa is discussed within the context of a dynamic model of molecular interactions between prothrombin and FVa engaging multiple contact sites.

Detection of Prethrombin 1 in Human Blood Plasma

Background: Prethrombin 1 is one of prothrombin derivatives which appears in the presence of thrombin in vitro. However, existence of prethrombin 1 in vivo remained questionable. The aim of present work was to detect of prethrombin 1 in vivo at abdominal aortic aneurysm, hip replacement after fracture and stroke. Methodology: Blood plasma samples of patients with abdominal aortic aneurysm, hip replacement after fracture, stroke, and patients which were treated with warfarine were collected. Detection of prethrombin 1 was completed using three independent approaches: combination of prothrombin index and ecamulin (ecarin) index; the APTT-test modified by addition of exogenous prothrombin; Western-blotting of blood plasma using polyclonal antibody to prothrombin. Results: Prethrombin 1 presence in patients’ blood plasma at studied pathologies was proven. Inactive prothrombin (descarboxy-prothrombin), but not prethrombin 1 was found in blood plasma of patients which were treated with warfarine. Developed approach can be used for testing the prethrombin 1 in clinical diagnostics. Conclusion: Prethrombin 1 at different pathologies, accompanied by coagulation disorders was detected in patients’ blood plasma directly.

Long-Range Allosteric Linkage Between Exosites Reciprocally Regulates the Zymogenicity of Prothrombin Derivatives

The binding of ligands to anion binding exosite I (ABEI) and exosite II (ABEII) on prothrombin (II) derivatives plays an integral role in regulating their function. These exosites are located on opposite faces of the proteinase domain and exhibit unique binding specificities. Fragment 1.2 (F12) binds to ABEII and acts as a zymogen-promoting allosteric ligand. Conversely, Na+ and active site ligands stabilize the proteinase state. Here, we investigated the allosteric linkage between ABEI and the Na+ binding site, active site or ABEII using multiple ABEI ligands and prothrombin derivatives differentially poised along the zymogen to proteinase continuum. Prethrombin 2 (P2) represents the most zymogen-like state that differs from thrombin (IIa) because it is not cleaved at the R320 site. To mimic the zymogen-like character of P2 in a cleaved IIa molecule, residues responsible for N-terminal insertion and proteinase formation (IVE) were swapped with TAT to produce IIaTAT, a mutant with vastly diminished proteolytic activity. Alanine was substituted with the catalytic serine residue in IIa (IIaS195A) to represent the proteinase without a ligand at its active site. The thermodynamics of interactions between the thrombin inhibitor and ABEI ligand hirugen (Hir) and the various reference states was assessed using isothermal titration calorimetry (ITC). Titration of Hir into P2, IIaTAT or IIaS195A revealed thermodynamically more favorable binding to proteinase-like IIaS195A in comparison to zymogen-like P2 or IIaTAT. Binding of Hir to IIaS195A was affected by the concentration of Na+ at constant ionic strength. Global analysis done in the presence of increasing concentrations of Na+ revealed a 5-fold increase in Hir binding affinity when IIaS195A is ligated with Na+; demonstrating positive allosteric linkage between ABEI and Na+ binding. Using a truncated Staphylocoagulase variant that binds ABEI without N-terminus insertion (SC13-325), we found that SC13-325 binding alone promoted active site opening and fluorescent inhibitor incorporation in zymogen-like P2. These data reinforce the observation that ABEI ligands promote a proteinase-like state in prothrombin derivatives through positive allosteric linkage with the Na+ binding and active sites. Interestingly, inhibitor incorporation and ITC studies both showed that SC13-325 interacts poorly with II, but strongly with P2 despite both species being zymogens. These findings imply that the ABEII ligand F12, which is produced upon cleavage of II at R271 to form P2, may display negative allosteric linkage with ABEI. Titration of SC13-325 into pre-formed complexes of P2/F12 revealed a drastic reduction in affinity of SC13-325 for P2 when F12 is bound to ABEII. Thus, F12 binding at ABEII negatively affects ABEI binding. Further studies used soluble thrombomodulin (sTM) as the most physiologically pertinent ligand for ABEI. ITC and global analysis of the binding of F12 to P2 in the presence of different concentrations of sTM revealed a ~1200-fold decrease in binding affinity and enthalpy for either the binding of F12 to P2 bound to sTM or the binding of sTM to P2 bound to F12. These data illustrate the strong negative linkage associated with the binding of protein ligands to ABEI and ABEII which yields the appearance of competitive and mutually exclusive binding at the two sites despite the fact that they are on opposite sides of the proteinase domain. Allosteric linkage between ligand binding at the two exosites is centered on the ability of F12 binding to ABEII and favor zymogen-like forms and ligand binding to ABEI to favor proteinase-like forms. Thus, allosteric linkage among exosites is vital to the interconversion of prothrombin species along the zymogen to proteinase spectrum. These ligand-dependent conformational shifts and associated changes in function are likely to greatly contribute to the dynamic roles that IIa plays during coagulation. DisclosuresNo relevant conflicts of interest to declare.

Meizothrombin Preparation and Its Role in Fibrin Formation and Platelet Aggregation

Meizothrombin (MT) is one of prothrombin derivatives which appears in haemostasis activation area. However, its role in haemostasis regulation isn’t clear. We studied the role of MT in fibrin formation, platelet activation and aggregation. A new effective method of obtaining MT from native human prothrombin was developed using immobilised prothrombin activator from Echis multisquamatis venom. The protein was stable and electrophoretically pure. Platelet-rich plasma for aggregation study and gel-sieved platelets for flow-cytometry were separated from blood of healthy donors. It was shown that MT transformed fibrinogen to fibrin and activated clotting factor XIII. MT didn’t activate gel-sieved intact platelets, but in platelet-rich plasma, increased platelet aggregation induced by ADP, collagen and adrenalin.

Effect of Zymogen Domains and Active Site Occupation on Activation of Prothrombin by von Willebrand Factor-binding Protein

Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(2)-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1-263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1-474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an ∼100-fold enhancement in affinity. The affinities of vWbp(1-263) or vWbp(1-474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1-263) and vWbp(1-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.

In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

Membrane-Dependent Interaction of Factor Xa and Prothrombin with Factor Va in the Prothrombinase Complex

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.

Fate of Membrane-Bound Reactants and Products during Prothrombin Activation by Prothrombinase.

The activation of prothrombin by the prothrombinase complex yields the protease thrombin (IIa) and the activation peptide fragment 1.2 (F1.2). IIa and F1.2 are established to interact noncovalently through the fragment 2 (F2) domain. Since membrane binding is mediated by the fragment 1 domain, the interaction between IIa and F1.2 could allow the newly produced protease to remain bound to the membrane surface and modulate its biological functions. In this case, the feedback action of thrombin to cleave the Arg155-Ser156 site between the F1 and F2 domains would be expected to play a key regulatory role in releasing newly formed thrombin into solution. We have investigated the ability of F1.2 to localize nascent IIa to the membrane surface and the role of IIa-mediated feedback cleavages in regulating this phenomenon. We used a recombinant prothrombin mutant in which the two thrombin cleavage sites were rendered uncleavable by mutation to Gln (IImut), a prothrombin derivative (IIS195A) that yields catalytically inactive product, and a prothrombin derivative IImut/Q271, which yields stable meizothrombin. The binding of IIa and prethrombin 2 (P2) produced from IIS195A to F1.2, examined by isothermal titration calorimetry, yielded: F1.2 plus IIaS195A (Kd 4.5±0.3 μM, n 0.93±0.02, ΔH −13.6±0.4 kcal.mol-1), and F1.2 plus P2S195A (Kd 156±11 nM, n 1.17±0.01, ΔH −28.6±0.2 kcal.mol-1). The relatively weak interaction between IIaS195A and F1.2 indicates that only a modest fraction of IIa will be bound to F1.2 even at the highest possible concentrations achievable in vivo. In contrast, binding of F1.2 to the zymogen P2 is of considerably higher affinity, implying that conversion of zymogen to protease results in a ∼25-fold change in affinity for F1.2 binding. Membrane retention of products during prothrombin activation catalyzed by prothrombinase was studied at the physiologic prothrombin concentration by assessing the fate of membrane-bound species using light scattering correlated with progress curves for cleavage established by SDS-PAGE and quantitative densitometry. In the absence of steps taken to inhibit thrombin produced in situ, prothrombin cleavage by prothrombinase and the associated cleavage of substrate and products by thrombin was accompanied by a rapid decrease in light scattering to a level expected for membrane-bound F1. Surprisingly, similar light scattering profiles were observed in the activation of IImut and IIS195A even in the absence of feedback proteolysis of F1.2. In these cases, the light scattering signal decreased to a level expected for F1.2 bound to membranes and correlated with the appearance of F1.2. In contrast, activation studies with IImut/Q271 revealed that formation of stable meizothrombin as a limiting product produced essentially no decrease in light scattering indicating that this species remains membrane-bound. Thus, covalent linkage with the F1 domain allows meizothrombin to remain membrane bound with potentially modulated function. In addition, the higher affinity interaction between P2 and F1.2 likely plays a very significant role in influencing prothrombinase function under conditions when P2 is produced as an intermediate. In contrast, as a result of the much weaker interaction between IIa and F1.2, membrane binding mediated by F1.2 likely plays a minor role in localizing IIa to the membrane surface during activation of physiological concentrations of prothrombin irrespective of feedback reactions catalyzed by newly formed thrombin.

Further Evidence for Two Functional Forms of Prothrombinase Each Specific for Either of the Two Prothrombin Activation Cleavages

Previous work showed that prothrombin derivatives cleavable only at Arg-320 (rMZ) or Arg-271 (rP2) are partial, rather than competitive, inhibitors of prothrombin activation by prothrombinase. A "ping-pong"-like model, which posits two equilibrating forms of prothrombinase, explained the inhibition pattern. The present studies were undertaken to further investigate this putative mechanism. Two models were developed, one allowing for one form of the enzyme and the other allowing for two forms. Both models also allowed channeling and ratcheting. The models were fit to full time courses of prothrombin, meizothrombin, prethrombin-2, and the B-chain. In the absence of ratcheting and channeling, neither model fits the data. In their presence, however, both models fit very well, and thus they could not be distinguished. Therefore, inhibition of rMZ activation by rP2 was studied. Inhibition was partial and the two-form model fit the data with randomly distributed residuals, whereas the one-form model did not. Initial rates of fluorescein-labeled prothrombin cleavage in the presence of various prothrombin derivatives reported by Brufatto and Nesheim (Brufatto, N., and Nesheim, M. E. (2003) J. Biol. Chem. 278, 6755-6764) were also analyzed using the two models. The two-form model fit the partial inhibition data well, whereas the one-form model did not. In addition, prothrombin at varying concentrations was activated, and subsequently, the initial rates were plotted with respect to the initial prothrombin concentration. When compared with the expected initial rates as determined by the simulation of the models, the two-form model fit the observed rates better than the one-form model. The results obtained here further support the existence of two functional forms of prothrombinase.

Restricted Active Site Docking by Enzyme-bound Substrate Enforces the Ordered Cleavage of Prothrombin by Prothrombinase

The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.

Activated protein C.

Protein C is a vitamin K-dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to down-regulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor-1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APC's direct effects on various cell types, it is clear that APC's molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells.

Ordered Cleavage of Prothrombin by Prothrombinase Results from Constrained Binding and Preferential Active Site Engagement by One of the Two Cleavage Sites in Prothrombin.

The conversion of prothrombin to thrombin is a paradigm for proteolytic activation reactions wherein product is produced following cleavage at more than one site in the substrate. Human prothrombinase catalyzes thrombin formation by sequential cleavage of prothrombin at Arg320 followed by cleavage at Arg271. The largely ordered activation arises because Arg320 in intact prothrombin is hydrolyzed by prothrombinase with a Vmax that is ~30-fold greater than that for cleavage at Arg271 while substrate affinity is unchanged. Paradoxically, this phenomenon has been proposed to arise from the constrained binding of prothrombin to prothrombinase. The alternate proposal is that two interconverting forms of the enzyme differentially recognize and cleave the two sites in prothrombin. We have investigated substrate binding using prothrombinase assembled with a catalytically inactive recombinant factor Xa (XaS195A) in which Ser195 was replaced with Ala and a series of recombinant variants of prothrombin. The prothrombin variants included wild type prothrombin (IIWT) containing both cleavage sites, IIQ271 with Arg271 replaced with Gln and a single cleavable site at Arg320, IIQ320 with Arg320 replaced with Gln and a single cleavable site at Arg271 and the uncleavable IIQQ containing both Gln substitutions. Titration of XaS195A assembled into prothrombinase with increasing concentrations of the fluorescent active site probe p-aminobenzamidine (PAB) produced a saturable increase in fluorescence confirming the expected ability of XaS195A to bind ligands, such as PAB, at the active site despite its lack of catalytic activity. The addition of a saturating concentration of IIWT to reaction mixtures containing PAB and prothrombinase assembled with XaS195A resulted in a decrease in PAB fluorescence arising from the engagement of the active site by the substrate and the associated displacement of PAB. Only a minor change in PAB fluorescence was observed with IIQQ even though this uncleavable derivative binds to prothrombinase with the same affinity as IIWT. Thus, features of the substrate-enzyme interaction that determine affinity are independent of active site engagement by the substrate. Saturating concentrations of IIQ271 displaced PAB from the active site of XaS195A within prothrombinase to the same extent observed with IIWT while essentially no fluorescence decrease was observed with IIQ320. It follows that although all prothrombin derivatives bind with similar affinity to prothrombinase, elements flanking Arg320 can readily engage the active site of XaS195A within the enzyme complex while those flanking the Arg271 site cannot. Our equilibrium binding measurements establish a primary role for active site binding in determining the perceived rate constant for catalysis without influencing substrate affinity. The differential action of prothrombinase on the two cleavage sites in prothrombin likely arises from constraints imposed by active site-independent interactions that drive substrate affinity and permit active site docking by the Arg320 site in the substrate but not the Arg271 site. These constraints in active site engagement explain asymmetry in the recognition of the two bonds in intact prothrombin and its ordered cleavage by prothrombinase.

Binding of Substrate in Two Conformations to Human Prothrombinase Drives Consecutive Cleavage at Two Sites in Prothrombin

Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (II(WT)) contained both cleavage sites; II(Q271) contained a single cleavable site at Arg(320); II(Q320) and II(A320) contained a single cleavable site at Arg(271); and II(QQ) was resistant to cleavage. Cleavage at Arg(320) in II(Q271) could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or II(WT), whereas cleavage at Arg(271) in either II(Q320) or II(A320) was found to be approximately 30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an approximately 30-fold reduction in V(max). Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. II(Q320) was a competitive inhibitor of II(Q271) cleavage, and II(QQ) was a competitive inhibitor for each reaction with K(i) approximately K(m). The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.

The Substrate -Membrane Interaction Constrains Bond Presentation and Cleavage in the Action of Prothrombinase on Prothrombin.

Thrombin formation is catalyzed by human prothrombinase almost exclusively through sequential cleavage at Arg320 followed by cleavage at Arg271 in prothrombin. Activation site mutants in which the individual sites have been rendered uncleavable indicate that Arg271 in intact prothrombin is hydrolyzed ~26-fold more slowly than Arg320. This finding provides an adequate kinetic explanation for the observed pathway of prothrombin activation by prothrombinase via the formation of meizothrombin. However, deviations from this behavior are implied by findings in the absence of membranes or with prothrombin species that are defective in membrane binding. We have investigated the contribution of the substrate-membrane interaction to the individual cleavage reactions by comparing the action of prothrombinase on a series of recombinant prothrombin derivatives expressed in the presence or absence of reduced vitamin K to yield either fully-carboxylated (Gla) or un-carboxylated (desGla) protein. The variants included wild type prothrombin (IIWT) containing both cleavage sites, IIQ271 with a single cleavable site at Arg320 and IIQ320 with a single cleavable site at Arg271. Quantitative analysis of 4-carboxyglutamic acid content established the intended extent of carboxylation for all Gla and desGla isoforms. Analysis of prothrombin activation by prothrombinase indicated a ~10 fold reduction in the rate of active product formation with desGla-IIWT compared to Gla-IIWT. Surprisingly, analysis by SDS-PAGE and quantitative densitometry revealed only a modest decrease (~ 2.5-fold) in the rate of consumption of desGla-IIWT. However, marked differences were evident in the intermediates produced. Bond cleavage of desGla-IIWT proceeded with a major contribution from cleavage at Arg271 followed by Arg320, in an order opposite to that observed with Gla-IIWT. Thus, the reduced rate of active product formation with desGla-IIWT reflects, in large part, the formation of a zymogen intermediate as a result of an altered order in which bonds are cleaved by prothrombinase. Comparison of the rates of cleavage of individual bonds in Gla and desGla versions of IIQ271 and IIQ320 revealed the kinetic basis for this change. Elimination of membrane binding led to a ~5 fold reduction in the rate of cleavage at Arg320 but surprisingly yielded a ~10-fold enhancement in cleavage at Arg271. Lack of Gla is not uniformly deleterious to all reactions of prothrombin activation. Compensating rate effects on the individual reactions imply a change in the restricted presentation of cleavage sites to the active site of Xa within prothrombinase upon the loss of the substrate-membrane interaction. Thus, substrate-membrane interactions appear to work in concert with exosite binding to modulate bond cleavage order by maximizing accessibility of the Arg320 bond to the active site while restricting that of Arg271. Loss of the membrane anchoring component leads to increased accessibility of the Arg271 site and a concomitant decrease in the rate of cleavage at Arg320. These observations have major implications for prothrombinase function on cell surfaces with limited binding sites for prothrombin, as well as the impact of warfarin therapy on prothrombinase function in vivo.

Ratcheting between Two Distinct Conformations of Substrate Drives the Sequential Cleavage of Prothrombin by Prothrombinase.

The conversion of human prothrombin to thrombin requires the cleavage of two peptide bonds. When catalyzed by prothrombinase, the reaction proceeds almost exclusively via initial cleavage at R320 followed by cleavage at R271 yielding meizothrombin (mIIa) as an intermediate. The scissile bonds are expected to be ~36 A apart in the zymogen. Yet, remarkably, evidence indicates that cleavage at these two sites is accomplished by a single type of exosite interaction that tethers the substrate to prothrombinase. The ability of prothrombinase to act on these spatially distinct sites, with such constraints, can be explained by a conformational change in the substrate following initial cleavage at R320. Cleavage at this site leads to internal salt bridge formation and a conformational transition to the proteinase. The role of the zymogen to proteinase transition in substrate cleavage was investigated by the use of a fully-carboxylated recombinant prothrombin derivative (IITAT) with the I-V-E sequence following R320 replaced with T-A-T. Thrombin produced by cleavage of IITAT exhibited ~0.2% of the catalytic activity observed with thrombin produced from wild type recombinant prothrombin (IIWT). IITAT can be cleaved at the R320 site but fails to undergo all subsequent conformational changes required for proteinase formation. SDS-PAGE and quantitative densitometry revealed that the action of prothrombinase on either IIWT or IITAT was consistent with ordered cleavage at R320 followed by cleavage at R271. The rates of consumption of IIWT and IITAT resulting from cleavage at R320 were equivalent. Cleavage at R320 in IITAT by prothrombinase is not detectably affected by the substituted P1′-P3′ sequence. The disappearance of IIWT was accompanied by a transient accumulation in mIIa that decreased to zero within 4 minutes while thrombin accumulated following a short delay. With IITAT, mIIa accumulated to higher levels and persisted for 45 minutes. Thrombin was produced with a lower rate and a longer delay phase. The findings imply a substantial decrease in the rate of the second cleavage reaction at R271 resulting from distant effects of the new P1′-P3′ residues following R320. The thrombin inhibitor, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA) had no effect on the cleavage reactions in IIWT. However, increasing concentrations of DAPA as high as 400 μM were found to systematically enhance the rate of thrombin formation from IITAT by correcting defective cleavage at R271. The data are consistent with the interpretation that IITAT can be cleaved normally at R320 but subsequent accessibility and cleavage at R271 is reduced because of a defect in internal salt bridge formation and the conformational change associated with proteinase formation. Rescue of this defect by high concentrations of DAPA likely relates to the stabilization of a proteinase-like conformation in the intermediate produced by cleavage of IITAT at R320. Our findings suggest an important role for the zymogen to proteinase transition in determining the sequential action of prothrombinase on the two sites in prothrombin. We propose that exosite-dependent tethering of two distinct conformations of the substrate to prothrombinase drives the presentation of distantly positioned cleavage sites to the active site of the enzyme and accounts for the sequential cleavage of prothrombin.

Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg(323)-Ile(324), followed by cleavage at Arg(274)-Thr(275). We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaDeltaF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaDeltaF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K(m). This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaDeltaF1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIaDeltaF1 to the enzyme. Specific recognition of mIIaDeltaF1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.

Prothrombin and Its Derivatives Stimulate Motility of Melanoma Cells

Several studies indicated that activation of the clotting system may promote the growth and the invasive behavior of tumor cells. In the present study, we evaluated the migratory response of various melanoma cell lines to several clotting factors and prothrombin derivatives (thrombin, fragment 1, fragment 2 and kringle 1 fragment). Prothrombin, thrombin and fragment 1 stimulated chemotaxis of the murine (K-1735 M2, X21) and human A375 (SM) melanoma cell lines. Prothrombin and prothrombin fragment 1 showed their maximal chemotactic activity at 0.5 approximately 1 microM. Chemotaxis induced by thrombin was inhibited by hirudin, but not that induced by prothrombin or fragment 1. Other clotting proteins and the fragment 2 and kringle 1 fragment of prothrombin did not elicit chemotactic activity. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. The K-1735 M2 cells also migrated in a concentration-dependent manner to substratum-bound insoluble prothrombin, thrombin or fragment 1. Ligand binding assays showed that both prothrombin and fragment 1 bound to K-1735 M2 cells with apparent Kds of 0.5 microM. This binding was inhibited by an excess concentration of unlabeled prothrombin and fragment 1 but not by similar concentrations of other prothrombin fragments. These findings suggest that prothrombin and its fragment 1 exert chemotactic activity on melanoma cells by different mechanisms and different binding sites from that induced by thrombin.

Rational Design, Recombinant Preparation, and in Vitro and in Vivo Characterization of Human Prothrombin-derived Hirudin Antagonists

A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.

Isolation and Characterization of Carinactivase, a Novel Prothrombin Activator in Echis carinatus Venom with a Unique Catalytic Mechanism

The venom of the viper Echis carinatus contains a metalloprotease, ecarin, that is a potent prothrombin activator. We here show that the venom is also rich in another prothrombin activator, which does not belong to any known category of prothrombin activators. The novel enzyme, designated carinactivase-1 (CA-1), consists of two subunits held together non-covalently but very tightly. One subunit is a 62-kDa polypeptide that has metalloprotease activity and is homologous to the single-chain enzyme ecarin; the other subunit of 25 kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and this subunit resembles the anticoagulant in the habu snake venom, IX/X-bp, that specifically binds the Gla domains of coagulation factors IX and X in a Ca2+-dependent fashion. The activation of prothrombin by CA-1 requires Ca2+ ions at millimolar concentrations and in the absence of Ca2+ ions this enzyme is virtually inactive. By contrast, activation by ecarin is completely independent of Ca2+ ions. CA-1, unlike ecarin, does not activate prothrombin derivatives, in which binding of Ca2+ ions has been perturbed, namely prethrombin-1 and acarboxyprothrombin. Furthermore, the isolated catalytic subunit, although its activity is greatly reduced as compared to that of the holoenzyme, no longer requires Ca2+ ions for the activation of prothrombin. Reconstitution with the non-catalytic 25-kDa subunit restores high level activity and the dependence on Ca2+ ions. Finally, prothrombin activation by CA-1 is inhibited by prothrombin fragment 1, and the isolated non-catalytic subunit is capable of binding fragment 1 in the presence of Ca2+ ions. From these observations, we postulate the following unique mechanism for the activation of prothrombin by CA-1. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the 25-kDa regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62-kDa catalytic subunit.