Monoclonal antibody light chain with prothrombinase activity

Prothrombin is the precursor of thrombin, a central enzyme in coagulation. Autoantibodies to prothrombin are associated with thromboembolism, but the mechanisms by which the antibodies modulate the coagulation processes are not understood. We screened a panel of 34 monoclonal antibody light chains isolated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identified, and one of the light chains was characterized further. The prothrombinase activity eluted from a gel-filtration column run in denaturing solvent (6 M guanidine hydrochloride) at the characteristic positions of the light chain dimer and monomer. A constant level of catalytic activity was observed across the width of the light chain monomer peak, assessed as the cleavage of IEGR-methylcoumarinamide, a peptide substrate corresponding to residues 268-271 of prothrombin. Hydrolysis of this peptide by the light chain was saturable and consistent with Michaelis-Menten-Henri kinetics (K(m) 103 microM; k(cat) of 2.62 x 10(-)(2)/min). Four cleavage sites in prothrombin were identified by N-terminal sequencing of the fragments: Arg(155)-Ser(156), Arg(271)-Thr(272), Arg(284)-Thr(285), and Arg(393)-Ser(394). The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under conditions that readily permitted detectable prothrombin cleavage. Two prothrombin fragments (M(r) 55 000 and 38 000), were isolated by anion-exchange chromatography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroanilide. Conversion of fibrinogen to fibrin was accelerated by the prothrombin fragments generated by the light chain. These finding suggest a novel mechanism whereby antibodies can induce a procoagulant state, i.e., prothrombin activation via cleavage of the molecule.     

A control switch for prothrombinase: characterization of a hirudin-like pentapeptide from the COOH terminus of factor Va heavy chain that regulates the rate and pathway for prothrombin activation

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg(271) and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg(320) (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695-699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg(320). These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg(320) and Arg(271), respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg(271) of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.     

Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process

Prothrombinase cleaves prothrombin at Arg(271) and Arg(320) to produce thrombin. The kinetics of cleavage of five recombinant prothrombins were measured: wild-type prothrombin (WT-II), R155A/R284A/R271A prothrombin (rMZ-II), R155A/R284A/R320A prothrombin (rP2-II), S525C prothrombin labeled with fluorescein (WT-II-F*), and R155A/R284A/R271A/S525C prothrombin labeled with fluorescein (rMZ-II-F*). rMZ-II and rP2-II are cleaved only at Arg(320) and Arg(271), respectively, to yield the intermediates meizothrombin and prethrombin-2, respectively. WT-II-F* and rMZ-II-F* were labeled at Cys(525) with fluorescein; cleavage was monitored by enhanced fluorescence. Activation kinetics of WT-II, rMZ-II, and rP2-II indicated that the catalytic efficiency of cleavage at Arg(320) was increased by 30,000-fold by the cofactor factor Va, as was the conversion of prothrombin to thrombin. However, factor Va increased cleavage at Arg(271) only by 34-fold. Although WT-II competitively inhibited cleavage of WT-II-F*, rMZ-II or rP2-II did not inhibit completely even at saturating concentrations. However, rMZ-II and rP2-II together inhibited WT-II-F* cleavage competitively. Both WT-II and rMZ-II competitively inhibited rMZ-II-F* cleavage, whereas rP2-II did not. A model of prothrombin activation that includes two equilibrating forms of prothrombinase, each recognizing one of the cleavage sites, is quantitatively consistent with all of the experimental observations. Therefore, we conclude that the kinetics of prothrombin activation can be described by a "ping-pong"-like mechanism.     

Role of sequence and position of the cleavage sites in prothrombin activation

In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences ²⁶¹DEDSDRAIEGRTATSEYQT²⁷⁹ and ³¹⁰RELLESYIDGRIVEGSDAE³²⁸ were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation.     

MASP-1 Induced Clotting – The First Model of Prothrombin Activation by MASP-1

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.     

Fibrinogen-sepharose interaction with prothrombin, prethrombin 1, prethrombin 2 and thrombin

Binding of prothrombin, prethrombin 1, prethrombin 2 and thrombin to fibrinogen-Sepharose was studied. Thrombin and prethrombin 2 bound to fibrinogen-Sepharose, while prethrombin 1 and prothrombin did not. Bound thrombin and prethrombin 2 were recovered from the column by eluting with 0.1 M NaCl/0.05 M Tris-HCl buffer (pH 7.4). The affinity of thrombin and prethrombin 2 to fibrinogen-Sepharose depended on ionic strength and reached a maximum at 50 mm concentration. Prethrombin 2 interacts with fibrinogen as well as thrombin; and prothrombin fragment 1.2 is not important in the formation of this complex. Thus, prethrombin 2, which is a precursor of thrombin without measurable enzymatic activity and which lacks the single cleavage at Arg-322-Ile-323 present in thrombin, has the same or very similar structural conformation as thrombin and has the same macromolecular substrate recognition site. These results confirm the earlier results that active center is not necessary in fibrinogen-thrombin interaction.     

Effects of activation peptide bond cleavage and fragment 2 interactions on the pathway of exosite I expression during activation of human prethrombin 1 to thrombin

Activation of prothrombin (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg271-Thr272 and Arg320-Ile321, resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates fragment 1 and generates the zymogen analog, prethrombin 1 (Pre 1). The properties of exosite I on Pre 1 and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin(54-65) ([5F]Hir(54-65)-(SO3-)). Prethrombin 2 (Pre 2), formed by factor Xa cleavage of Pre 1 at Arg271-Thr272, had the same affinity for hirudin(54-65) peptides as Pre 1 in the absence or presence of near-saturating fragment 2 (F2). Pre 2 and thrombin also had indistinguishable affinities for F2. By contrast, cleavage of Pre 1 at Arg320-Ile321, to form active meizothrombin des-fragment 1 MzT(-F1), showed a 11- to 20-fold increase in affinity for hirudin(54-65), indistinguishable from the 13- to 20-fold increase seen for conversion of Pre 2 to thrombin. Thus, factor Xa cleavage of Pre 1 at Arg271-Thr272 does not effect exosite I expression, whereas cleavage at Arg320-Ile321 results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre 1 activation intermediates is not modulated by F2, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre 1 activation intermediates and the previously demonstrated role of (pro)exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre 1 activation pathway.     

Incorporation of factor Va into prothrombinase is required for coordinated cleavage of prothrombin by factor Xa

Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320. Factor Va, along with phospholipid and Ca2+, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg320. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor VaFF/MI). The present studies were undertaken to determine whether loss of activity can be attributed to selective loss of efficiency at one or both of the two prothrombin-activating cleavage sites. Kinetic constants for the overall activation of prothrombin by prothrombinase assembled with saturating concentrations of recombinant mutant factor Va were calculated, prothrombin activation was assessed by SDS-PAGE, and rate constants for both cleavages were analyzed from the time course of the concentration of meizothrombin. Prothrombinase assembled with factor VaFF/MI had decreased k(cat) for prothrombin activation with Km remaining unaffected. Prothrombinase assembled with saturating concentrations of factor VaFF/MI showed significantly lower rate for cleavage of plasma-derived prothrombin at Arg320 than prothrombinase assembled with saturating concentrations of wild type factor Va. These results were corroborated by analysis of cleavage of recombinant prothrombin mutants rMz-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A), which can be cleaved only at Arg320 or Arg271, respectively. Time courses of these mutants indicated that mutations in the factor Xa binding site of factor Va reduce rates for both bonds. These data indicate that the interaction of factor Xa with the heavy chain of factor Va strongly influences the catalytic activity of the enzyme resulting in increased rates for both prothrombin-activating cleavages.     

Temperature dependence of the thrombin-catalyzed proteolysis of prothrombin

Measurement of the temperature-dependence of thrombin-catalyzed cleavage of the Arg(155)-Ser(156) and Arg(284)-Thr(285) peptide bonds in prothrombin and prothrombin-derived substrates has yielded Arrhenius parameters that are far too large for classical mechanistic interpretation in terms of a simple hydrolytic reaction. Such a difference from the kinetic behavior exhibited in trypsin- and chymotrypsin-catalyzed proteolysis of peptide bonds is attributed to contributions by enzyme exosite interactions as well as enzyme conformational equilibria to the magnitudes of the experimentally determined Arrhenius parameters. Although the pre-exponential factor and the energy of activation deduced from the temperature-dependence of rate constants for proteolysis by thrombin cannot be accorded the usual mechanistic significance, their evaluation serves a valuable role by highlighting the existence of contributions other than those emanating from simple peptide hydrolysis to the kinetics of proteolysis by thrombin and presumably other enzymes of the blood coagulation system.     

Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site.

Hirugen, a synthetic dodecapeptide corresponding to the carboxyl-terminal amino acids 53-64 of hirudin, binds within a deep groove in thrombin that contains a cationic region referred to as the anion-binding exosite. This region is important in many of the binary interactions of thrombin with macromolecular substrates and cofactors. Fluorescein-labeled hirugen was used to probe which steps in the prothrombin activation process generate this anion-binding exosite. Two activation cleavage sites exist in bovine prothrombin. Cleavage at Arg274-Thr275 releases the activation fragments to generate the thrombin precursor, prethrombin 2. Cleavage of prothrombin within a disulfide loop at Arg323-Ile324 leads to formation of meizothrombin with no loss of peptide material but with formation of amidolytic activity. Cleavage of the same bond in prethrombin 2 generates thrombin. Hirugen, labeled at the amino terminus with fluorescein isothiocyanate, does not bind to prothrombin but does bind to thrombin (Kd = 9.6 +/- 1.2 x 10(-8) M), prethrombin 2 (Kd = 1.3 +/- 0.1 x 10(-7) M), thrombin-fragment-2 complex (Kd = 1.1 +/- 0.2 x 10(-6) M), and meizothrombin (Kd = 1.6 +/- 0.5 x 10(-8) M). Prothrombin fragment-2 and hirugen both bind independently to thrombin. A ternary complex can form with hirugen and fragment-2 and either thrombin or prethrombin 2, suggesting that fragment-2 and hirugen bind to discrete sites. Hirugen also alters the active site conformation of thrombin as detected by modulation of synthetic substrate hydrolytic activity. These studies suggest that conformational changes, rather than alleviating steric hindrance, are responsible for the formation of the hirugen-binding site during prothrombin activation. Furthermore, this conformational change can be effected by the cleavage of either of the two bonds required for activation of prothrombin.     

Interaction of α-thrombin and prethrombin 2, with phosphatidylserine-containing monolayers

Prothrombin activation complex is located at a phospholipid surface on activated platelets. To see whether the thrombin domain of the molecule plays a role in the interaction with lipids, we investigated the direct interaction of human α-thrombin and its precursor prethrombin 2 with phospholipid monolayers of varous compositions (PS/PC). Adsorption of the labeled proteins was determined by surface radioactive measurements. Penetrations of the proteins in the lipid layer was inferred from capacitance variation of the monolayer measured by a palarography. Disulfide bridges reduced at the electrode were determined by cycle voltametry.In all the cases studied, although in different manners thrombin was found both to adsorb and penetrate the lipid layer, whereas prethrombin 2 did not penetrate pure phosphatidylcholine (PC). In the case of thrombin but not of prethrombin 2, penetration is accompanied by S-S reduction which is maximum at 10 per cent of phosphatidylserine (PS). This indicate a different orientation for prethrombin 2 and thrombin in the lipid layer. This observation might be of importance for the comprehension of the architecture of the prothrombin might be of for the regulation of thrombin formation within the complex.     

Regulated Cleavage of Prothrombin by Prothrombinase: REPOSITIONING A CLEAVAGE SITE REVEALS THE UNIQUE KINETIC BEHAVIOR OF THE ACTION OF PROTHROMBINASE ON ITS COMPOUND SUBSTRATE*?

Prothrombinase converts prothrombin to thrombin via cleavage at Arg³²⁰ followed by cleavage at Arg²⁷¹. Exosite-dependent binding of prothrombin to prothrombinase facilitates active site docking by Arg³²⁰ and initial cleavage at this site. Precise positioning of the Arg³²⁰ site for cleavage is implied by essentially normal cleavage at Arg³²⁰ in recombinant prothrombin variants bearing additional Arg side chains either one or two residues away. However, mutation of Arg³²⁰ to Gln reveals that prothrombinase can cleave prothrombin following Arg side chains shifted by as many as two residues N-terminal to the 320 position at near normal rates. Further repositioning leads to a loss in cleavage at this region with an abrupt shift toward slow cleavage at Arg²⁷¹. In contrast, the binding constant for the active site docking step is strongly dependent on the sequence preceding the scissile bond as well as position. Large effects on binding only yield minor changes in rate until the binding constant passes a threshold value. This behavior is expected for a substrate that can engage the enzyme through mutually exclusive active site docking reactions followed by cleavage to yield different products. Cleavage site specificity as well as the ordered action of prothrombinase on its compound substrate is regulated by the thermodynamics of active site engagement of the individual sites as well as competition between alternate cleavage sites for active site docking.     

Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase

Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg(320) yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg(271), yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg(271). We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg(271), accumulates on the surface because of a approximately 20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.     

Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.     

DNA replication in a polymerase I deficient mutant and the identification of DNA polymerases II and 3 in Bacillus subtilis

The partial amino acid sequences at the amino terminal of prothrombin and the intermediates of activation have been determined. These data indicate that the products of the first step of activation, whether derived from the action of factor Xa or thrombin, are identical. The data also show that the activation of prothrombin proceeds by the sequential cleavage of the amino terminal region of prothrombin and the intermediates, and confirm the mechanism of prothrombin activation as: NH₂-Prothrombin-COOH $\text{Xa or thrombin}$ NH₂-Intermediate 3 + Intermediate 1-COOH; NH₂-Intermediate 1-COOH $\text{Xa}$ NH₂-Intermediate 4 + Intermediate 2-COOH; NH₂-Intermediate 2-COOH $\text{Xa}$ NH₂-A chain α-thrombin -S-S-B chain α-thrombin-COOH.Previous reports from this laboratory have demonstrated that the activation of prothrombin proceeds through several single-chain intermediates prior to the appearance of thrombin activity. (1) Subsequent studies have sequence of the prothrombin molecule can be deduced from the sequences of its activation intermediates and we are continuing our studies toward this goal.     

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.     

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 NH₂-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.     

Activation of prothrombin Barcelona evidence for active high molecular weight intermediates

Prothrombin Barcelona is a varient of human prothrombin. Its activation by factor Xa is characterized by two abnormal features. The first one is the absence of one of the two Xa-catalyzed cleavages, and the second one the generation of a thrombin-like activity responsible for the thrombin-catalyzed cleavages on the prothrombin molecule and for the activity toward small substrates but without clotting activity.The molecular species exhibiting the thrombin-like activity have been characterized Diisopropyl phosphofluoridate incorporation experiments show that upon activation, two high molecular weight intermediates bearing an active site are formed: one has the molecular weight of prothrombin, the other of prethrombin 1. In the presence of hirudin, only the first intermediate is formed due to a single cleavage by factor Xa which is therefore responsible for the unmasking of the active site.     

Thrombin receptor activating peptides induce Ca2+ mobilization,barrier dysfunction,prostaglandin synthesis,and platelet-derived growth factor mRNA expression in cultured endothelium

Endothelial cell activation by thrombin is a key event in wound healing, Inflammation, and hemostasis. To better define thrombin-endothelial cell interactions we synthesized several peptides of varying length corresponding to the initial 14 amino acid sequence of the cloned human platelet thrombin receptor after cleavage at an arginine⁴¹ site (R/SFLLRNPNDKYEPF). Thrombin receptor activating peptides (TRAPs) as short as 5 amino acids induced significant levels of PGl₂ synthesis and expression of PDGF mRNA in human endothelium and produced dose-dependent cellular contraction and permeability of confluent human umbilical vein and bovine pulmonary artery endothelial monolayers. To explore whether TRAPs utilized similar signal transducing pathways as α-thrombin to accomplish endothelial cell activation, phospholipase C production of the Ca²⁺ secretagogue IP₃ was measured and detected 10 seconds after either TRAP 7 or α-thrombin. Furthermore, TRAPs ranging from 5-14 residues induced significant dose-dependent incsreases in Fura-2 fluorescence indicative of Ca²⁺ ₁ mobilization. These results indicate that thrombin-mediated proteolytic cleavage of the human and bovine thrombin receptor initiates stimulus/coupling respones such phospholipase C activation, Ca²⁺ mobilization, and protein kinase C activation. The functional consequence of this cellular activation via the cleaved receptor is enhanced cellular contraction, barrier dysfunction, PGI₂ synthesis, and expression of PDGF mRNA. © 1993 Wiley-Liss, Inc.     

Isolation and characterization of the vitamin K dependent domain of human prothrombin

Chymotryptic cleavage of human prothrombin produces two fragments, prothrombin (des 1–44) previously characterized and peptide 1–41. This peptide has been purified by barium citrate adsorption and Sephadex G100 chromatography. It contains the 10 γ-carboxyglutamic residues of prothrombin. Added to a prothrombin activation mixture containing FXa, phospholipid and Ca⁺⁺, peptide 1–41 inhibits thrombin generation with the same potency as prothrombin fragment 1. Ca⁺⁺ produces a 20 % quenching of the intrinsic fluorescence of the peptide. So do Mn⁺⁺ and Mg⁺⁺ although to a lesser extent. Phospholipid enhances the Ca⁺⁺ induced quenching by a factor of 1.7 and shifts the midpoint of the transition from 0.34 to 0.46 mM Ca⁺⁺. The major difference with titration curves obtained with prothrombin F1 is the absence of cooperativity. Hence peptide 1–44 has retained some of the prothrombin properties to interact with Ca⁺⁺ and phospholipid and represents the vitamin K dependent domain of the molecule. The presence of the remaining part of F1 (residues 44–155) however is necessary for the expression of cooperativity.