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Sriram Krishnaswamy - One of the best experts on this subject based on the ideXlab platform.
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fate of membrane bound reactants and products during the activation of human Prothrombin by Prothrombinase
Journal of Biological Chemistry, 2008Co-Authors: Parvathi Kamath, Sriram KrishnaswamyAbstract:Abstract 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 Arg320 yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg271, 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 Arg271. 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 Arg271, accumulates on the surface because of a ∼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.
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human platelets and endothelial cells differentially regulate the pathway for Prothrombin cleavage by Prothrombinase
Blood, 2007Co-Authors: Harlan N Bradford, Sriram Krishnaswamy, Dezhi Shang, Long X ZhengAbstract:Human Prothrombinase assembled on synthetic membranes composed of phosphatidylcholine and phosphatidylserine (PCPS) catalyzes thrombin formation almost exclusively by sequential cleavage of Prothrombin at Arg 320 to yield the protease meizothrombin (mIIa) as an intermediate which is then further cleaved at Arg 271 . This cleavage pathway arises because Arg 320 in intact Prothrombin is cleaved ∼30-fold faster than Arg 271 . When Prothrombin lacks γ-carboxyglutamic acid modifications (desGlaII), product formation is modestly decreased but bond cleavage largely occurs in the opposite order, yielding the zymogen, prethrombin 2 (P2), as an intermediate. This results from a reduction in the rate of cleavage at Arg 320 in intact Prothrombin and a partly compensating gain of function in cleavage at Arg 271 . Thus, membrane binding and/or other interactions mediated by γ-carboxyglutamic acids in the substrate play a major role in modulating the pathway for cleavage and whether the intermediate is a zymogen or protease. We now extend these approaches to evaluate Prothrombinase function on activated platelets and human umbilical vein endothelial cells (HUVECs) at the physiologic concentration of Prothrombin. Prothrombin activation was detected by measuring the appearance of product(s) with proteolytic activity and by analysis of the cleavage process by SDS-PAGE and western blot analysis quantitatively imaged by infrared fluorescence. This approach was validated by documenting equivalence in the progress curves obtained by western blot imaging or following staining of total protein in a purified system with PCPS membranes. Prothrombin cleavage was assessed using human platelets (10 8 /ml), activated with thrombin and studied with saturating concentrations of Va and a limiting concentration of Xa. The pattern of Prothrombin cleavage, intermediate accumulation and product formation was clearly distinct from that observed with PCPS membranes, indicating substantial flux towards thrombin formation via initial cleavage at Arg 271 followed by cleavage at Arg 320 producing the zymogen P2 as an intermediate. Transient formation of mIIa from initial cleavage at Arg 320 was undetectable. Thus, Prothrombinase assembled on thrombin activated platelets cleaves Prothrombin in a way that is reminiscent of the cleavage of desGlaII rather than fully carboxylated Prothrombin seen with PCPS membranes. In contrast, equivalent studies with Prothrombinase assembled on thrombin activated HUVECs produced cleavage patterns consistent with significant flux towards thrombin formation via initial cleavage of Prothrombin at Arg 320 yielding mIIa as an intermediate. The use of desGlaII with HUVECs yielded lower rates and cleavage patterns similar to those obtained with fully carboxylated Prothrombin in the platelet reactions. Our results document that the cleavage pathway for thrombin formation is dependent on cell type. Because mIIa is a protease with a different spectrum of activities from thrombin, its formation restricted to the vessel wall suggests an important regulatory role for the modulation of the pathway of Prothrombin cleavage. Such bimodal regulation of the pathway for Prothrombin cleavage on HUVECs and platelets suggests differential roles of Prothrombinase assembled on platelets versus endothelium in regulating the hemostatic response to vascular injury.
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active site independent recognition of substrates and product by bovine Prothrombinase a fluorescence resonance energy transfer study
Journal of Biological Chemistry, 2004Co-Authors: Danilo S Boskovic, Thomas Troxler, Sriram KrishnaswamyAbstract:Abstract The conversion of Prothrombin to thrombin is catalyzed by Prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of Prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with Prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green488 at the active site of Prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to Prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of Prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to Prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with Prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of Prothrombin activation and product release.
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CONTRIBUTION OF THE Prothrombin FRAGMENT 2 DOMAIN TO THE FUNCTION OF FACTOR VA IN THE ProthrombinASE COMPLEX
Biochemistry, 1997Co-Authors: Sriram Krishnaswamy, Randall K. WalkerAbstract:: The Prothrombinase complex assembles through reversible interactions between factor Xa, factor Va and acidic phospholipid-containing membranes in the presence of calcium ions. This complex catalyses the conversion of Prothrombin to thrombin through two proteolytic steps. We have used prethrombin 2 as a substrate analog for the first cleavage reaction of Prothrombin activation (cleavage at Arg323-Ile324) catalyzed by the Prothrombinase complex and have also relied on the known ability of prethrombin 2 to interact tightly but reversibly with fragment 2 or fragment 1.2. The kinetics of cleavage at Arg323-Ile324 have been assessed with these substrate analogs to investigate the contribution of cofactor-substrate interactions mediated by the fragment 2 domain to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the Prothrombinase complex. Initial velocity measurements indicated that the rate of prethrombin 2 cleavage by the factor Xa-PCPS binary complex was increased by a factor of approximately 1300 upon the addition of saturating concentrations of factor Va to assemble Prothrombinase. Although the measured initial velocity was higher when either fragment 2 or fragment 1.2 was present, the factor Va-dependent enhancement in initial rate (2600- and 1500-fold) was comparable in each case. Steady state kinetic constants were obtained using prethrombin 2, prethrombin 2 plus fragment 2, and prethrombin 2 plus fragment 1.2 as substrates. For each substrate, the addition of saturating concentrations of factor Va to the Xa-PCPS binary complex led to increases in catalytic efficiency of between 1000 and 9000-fold. The kcat/Km for prethrombin 2 cleavage by Prothrombinase was essentially identical to that obtained for prethrombin 2 saturated with fragment 2. Thus, comparable accelerating effects of factor Va are observed independent of the presence of the fragment 2 domain in the substrate. The results indicate that interactions between factor Va and the substrate mediated by the fragment 2 domain do not contribute in a dominant way to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the Prothrombinase complex.
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the activation of Prothrombin by the Prothrombinase complex the contribution of the substrate membrane interaction to catalysis
Journal of Biological Chemistry, 1994Co-Authors: R K Walker, Sriram KrishnaswamyAbstract:Abstract The conversion of Prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the Prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled Prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using 125I-Prothrombin revealed that the activation reaction proceeded through the ordered cleavage at Arg323-Ile324 followed by cleavage at Art274-Thr275 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg323-Ile324 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg274-Thr275, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin 2 plus fragment 1.2 or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg323-Ile324 by Prothrombinase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg274-Thr275 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during Prothrombin activation. Proteolytic removal of the membrane binding fragment 1 domain of the substrates, meizothrombin and prethrombin 2-fragment 1.2, had no effect on the cleavage at Arg274-Thr275 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg323-Ile324. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg323-Ile324, which leads to the conversion of Prothrombin to meizothrombin. In contrast, the substrate-membrane interaction mediated by the fragment 1 domain has no detectable effect on the second cleavage reaction at Arg274-Thr275 which is required for the conversion of meizothrombin to thrombin.
Alireza R Rezaie - One of the best experts on this subject based on the ideXlab platform.
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proexosite 1 dependent recognition and activation of Prothrombin by taipan venom
Journal of Biological Chemistry, 2004Co-Authors: Lin Chen, Alireza R RezaieAbstract:Abstract An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate Prothrombin to thrombin. To determine whether, similar to Prothrombinase, taipan venom utilizes proexosite-1 on Prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the Km and kcat values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr63-sulfated hirudin54-65 peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a KD of ∼2 μm, it is ineffective in inhibiting the activation of mutant zymogens (KD > 4-30 μm). Interestingly, an ∼50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of Prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to Prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.
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proexosite 1 on Prothrombin is a factor va dependent recognition site for the Prothrombinase complex
Journal of Biological Chemistry, 2003Co-Authors: Lin Chen, Likui Yang, Alireza R RezaieAbstract:Abstract Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of Prothrombin recognition by factor Xa in the Prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (Prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the Prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the Prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54–65) peptide suggested that although the peptide inhibits the Prothrombinase activation of the wild type zymogen with a KD of 0.5–0.7 μm, it is ineffective in inhibiting the activation of mutant zymogens (KD = 2–30 μm). These results suggest that basic residues of proexosite-1 on Prothrombin are factor Va-dependent recognition sites for factor Xa in the Prothrombinase complex.
Michael Kalafatis - One of the best experts on this subject based on the ideXlab platform.
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Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function.
ACS omega, 2017Co-Authors: Jamila Hirbawi, Michael KalafatisAbstract:Human factor Va (hfVa) is the important regulatory subunit of Prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg701 of hfVa for human Prothrombin (hPro) activation by Prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by Prothrombinase. The difference between the two cofactor molecules was also found within the Asn700–Arg701 dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp–Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution 700NR701→DE. rhfVNR→DE together with the wild-type molecule (rhfVWT) were expressed in COS7 cells, purified, and tested for their capability to function within Prothrombinase. Kinetic studies showed that the Kd of rhfVaNR→DE for human fXa as well as the kcat and Km of Prothrombinase made with rhfVaNR→DE for hPro activation were similar to the values obtained following hPro acti...
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The Role of Amino Acids 659-663 of Factor Va Heavy Chain During Prothrombin Activation by Prothrombinase.
Blood, 2009Co-Authors: Jamila Hirbawi, Michael KalafatisAbstract:Abstract 2131 Poster Board II-108 The proteolytic conversion of Prothrombin to thrombin is catalyzed by the Prothrombinase complex composed of the enzyme, factor Xa (fXa), and the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. FXa alone can activate Prothrombin following sequential cleavages at Arg271 and Arg320 yielding the transient inactive intermediate prethrombin 2. However, the interaction of fVa with fXa on a membrane/cell surface in the presence of divalent metal ions and formation of the Prothrombinase complex results in the reversal of the order of cleavages and a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. A first cleavage of Prothrombin by Prothrombinase at Arg320 produces the active intermediate meizothrombin, while the second cleavage at Arg271 produces thrombin. Thrombin and Prothrombin contain two positively charged binding regions (anion binding exosite I, ABE-I and anion binding exosite II, ABE II), that are crucial for protein function. Initial cleavage of Prothrombin at Arg320 by Prothrombinase which is absolutely factor Va dependent, entirely exposes (pro)exosite I. FVa is required for the specific recognition of Prothrombinase by (pro)exosite I of Prothrombin. The COOH-terminal region of the heavy chain of fVa contains acidic amino acid clusters that are important for cofactor activity. We have investigated the role of amino acid region 659-663 that contains five consecutive acidic amino acid residues. To ascertain the function of this region, site-directed mutagenesis was performed. We have constructed a mutant molecule with this region deleted (fVD659-663) and a mutant molecule in which all five residues were mutated to lysine (fV5K, charge reversal). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to fVaWT. Kinetic analyses studying Prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all mutant molecules for factor plasma-derived fXa was similar to fVaWT. Gel electrophoresis analyzing plasma-derived and recombinant mutant Prothrombin activation demonstrated delayed cleavage of Prothrombin at both Arg320 and Arg271 by Prothrombinase assembled with the mutant molecules. Using recombinant Prothrombin molecules we determined that cleavage at Arg271 by Prothrombinase assembled with either fVaD659-663 or fVa5K was severely impaired compared to cleavage at Arg320 by Prothrombinase assembled with the same recombinant cofactor molecules, resulting in lingering of meizothrombin throughout the activation process. To ascertain the effect of the mutations of the fVa heavy chain on the cleavage at Arg271 alone following the transition that occurs after cleavage at Arg320, we compared the rate of cleavage of active-site blocked meizothrombin (FPR-meizothrombin) by Prothrombinase assembled with either fVaWT or fVaD659-663. The data demonstrate a delay for cleavage of FPR-meizothrombin at Arg271 by Prothrombinase assembled with fVaD659-663 as compared to the same reaction catalyzed by Prothrombinase assembled with fVaWT. Quantitative scanning densitometry of fragment 1•2-A demonstrated a ∼4-fold delay in cleavage of FPR-meizothrombin at Arg271 by Prothrombinase assembled with fVaD659-663, compared to cleavage at Arg271 by Prothrombinase assembled with fVaWT. Direct comparison between the rates of cleavage of FPR-meizothrombin by membrane-bound fXa alone or by Prothrombinase assembled with fVaRVVD659-663 do not show any significant differences. Thus, deletion of amino acid region 659-663 virtually eliminates the acceleration in the rate of cleavage at Arg271 of meizothrombin attributed to the interaction of fVa with fXa. These data demonstrate that amino acid sequence 659DDDED663 from the factor Va heavy chain, regulates meizothrombin concentration during activation of Prothrombin by Prothrombinase. Disclosures: No relevant conflicts of interest to declare.
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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
Journal of Biological Chemistry, 2006Co-Authors: Michael A. Bukys, Michael E. Nesheim, Michael KalafatisAbstract:Abstract Membrane-bound factor Xa alone catalyzes Prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs Prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (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 Arg320. 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 Arg320 and Arg271, 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 Arg271 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.
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Controlling the Pathway for Prothrombin Activation by Prothrombinase.
Blood, 2006Co-Authors: Michael A. Bukys, Michael E. Nesheim, Michael KalafatisAbstract:Prothrombinase is the enzymatic complex responsible for timely thrombin formation. Activation of human Prothrombin is the consequence of two cleavages at Arg271 and Arg320 in Prothrombin by factor Xa. Membrane-bound factor Xa alone catalyzes Prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs Prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have previously shown 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) interacts with anion binding exosite I (ABE-I) of thrombin and inhibits Prothrombin activation by Prothrombinase. The peptide was found to be a competitive inhibitor of Prothrombinase with respect to substrate. According to the mode of inhibition, we postulated that the peptide binds Prothrombin in competition with the binding of the substrate to the enzyme, and inhibits Prothrombinase activity by substrate depletion. This mode of DYDYQ inhibition of Prothrombin activation by the factor Va-factor Xa complex is similar to that previously demonstrated for sulfated hirugen. 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 fully assembled Prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. Higher peptide concentrations were required to impair thrombin formation through the latter pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of Prothrombin by Prothrombinase at Arg320. These findings were corroborated by studying the kinetics of activation of recombinant mutant Prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271 respectively. Cleavage of rMZ-II by Prothrombinase was completely inhibited by low concentrations of DYDYQ while high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with thrombin formation by membrane-bound factor Xa alone in the absence of factor Va. Nonetheless, while the rate for cleavage at Arg271 of plasma-derived Prothrombin or rP2-II by membrane-bound factor Xa alone was significantly accelerated in the presence of DYDYQ, resulting in accumulation of prethrombin 2, the rate for cleavage at Arg320 of plasma-derived Prothrombin or rMZ-II by membrane-bound factor Xa alone was only moderately affected by the pentapeptide. Our data demonstrate that a pentapeptide mimicking amino acids 695–699 of the heavy chain of factor Va has opposing effects on membrane-bound factor Xa for Prothrombin activation, depending on the incorporation of factor Va in Prothrombinase. In the presence of the cofactor the peptide inhibits the rate of thrombin generation by specifically interfering with initial cleavage of Prothrombin at Arg320, while in the absence of factor Va the pentapeptide accelerates cleavage of Prothrombin by factor Xa at Arg271. Thus, the amino acid region spatially surrounding proexosite I in Prothrombin most likely has two interactive sites for the components of Prothrombinase, a factor Va interactive site and a factor Xa binding site.
Michio Sakai - One of the best experts on this subject based on the ideXlab platform.
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thrombosis from a Prothrombin mutation conveying antithrombin resistance
The New England Journal of Medicine, 2012Co-Authors: Yuhri Miyawaki, Atsuo Suzuki, Junko Fujita, Asuka Maki, Eriko Okuyama, Moe Murata, Akira Takagi, Takashi Murate, Shinji Kunishima, Michio SakaiAbstract:We identified a novel mechanism of hereditary thrombosis associated with antithrombin resistance, with a substitution of arginine for leucine at position 596 (p.Arg596Leu) in the gene encoding Prothrombin (called Prothrombin Yukuhashi). The mutant Prothrombin had moderately lower activity than wild-type Prothrombin in clotting assays, but the formation of thrombin-antithrombin complex was substantially impaired. A thrombin-generation assay revealed that the peak activity of the mutant Prothrombin was fairly low, but its inactivation was extremely slow in reconstituted plasma. The Leu596 substitution caused a gain-of-function mutation in the Prothrombin gene, resulting in resistance to antithrombin and susceptibility to thrombosis.
Lin Chen - One of the best experts on this subject based on the ideXlab platform.
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proexosite 1 dependent recognition and activation of Prothrombin by taipan venom
Journal of Biological Chemistry, 2004Co-Authors: Lin Chen, Alireza R RezaieAbstract:Abstract An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate Prothrombin to thrombin. To determine whether, similar to Prothrombinase, taipan venom utilizes proexosite-1 on Prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the Km and kcat values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr63-sulfated hirudin54-65 peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a KD of ∼2 μm, it is ineffective in inhibiting the activation of mutant zymogens (KD > 4-30 μm). Interestingly, an ∼50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of Prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to Prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.
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proexosite 1 on Prothrombin is a factor va dependent recognition site for the Prothrombinase complex
Journal of Biological Chemistry, 2003Co-Authors: Lin Chen, Likui Yang, Alireza R RezaieAbstract:Abstract Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of Prothrombin recognition by factor Xa in the Prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (Prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the Prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the Prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54–65) peptide suggested that although the peptide inhibits the Prothrombinase activation of the wild type zymogen with a KD of 0.5–0.7 μm, it is ineffective in inhibiting the activation of mutant zymogens (KD = 2–30 μm). These results suggest that basic residues of proexosite-1 on Prothrombin are factor Va-dependent recognition sites for factor Xa in the Prothrombinase complex.
