scholarly journals Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex

1997 ◽  
Vol 321 (3) ◽  
pp. 729-735 ◽  
Author(s):  
Hiroshi DEGUCHI ◽  
Hiroyuki TAKEYA ◽  
Esteban C. GABAZZA ◽  
Junji NISHIOKA ◽  
Koji SUZUKI
Keyword(s):  
Dissociation Constants ◽  
Solid Phase ◽  
Factor Xa ◽  
Complex Activity ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Direct Binding ◽  
Kringle 2 ◽  
Prothrombin Activation

The kringle 2 domain of prothrombin has been shown to interact with factor Va during the activation of prothrombin by the prothrombinase complex composed of factor Xa, factor Va, negatively charged phospholipids and Ca2+ ions. However, contradictory results have been reported about the role of the kringle 1 domain of prothrombin during the assembly of the prothrombinase complex. In an attempt to clarify the role of the kringle 1 domain of prothrombin, its effect on the activation of prothrombin by the prothrombinase complex and its direct binding to human factor Va were assessed. Comparative evaluation with the effects caused by other prothrombin structural components [a fragment 1 (γ-carboxyglutamic acid and kringle 1 domains), a kringle 2 domain and a catalytic protease domain] was also performed. In the presence of factor Va, each kringle 1 and kringle 2 fragment significantly inhibited the factor Xa-catalysed prothrombin activation in the absence of phospholipids. However, in the absence of both factor Va and phospholipids, kringle 2 fragment, but not kringle 1 fragment, inhibited prothrombin activation. Evaluation of the molecular interaction of the kringle domains with factor Va in assays with solid-phase phospholipid vesicles showed that each kringle 1 and kringle 2 fragment inhibited the prothrombinase complex activity. Assessment of the direct binding of prothrombin and each kringle domain of prothrombin with factor Va by fluorescence polarization showed that prothrombin, kringle 1 and kringle 2 fragments bind directly to factor Va with dissociation constants of 1.9ŷ0.1, 2.3ŷ0.1 and 2.0ŷ0.4 ƁM (meansŷS.D.) respectively. These findings suggest that both kringle 1 and 2 domains of prothrombin interact with factor Va during the assembly of the prothrombinase complex.

Abstract 30: Tissue Factor Pathway Inhibitor α Inhibits Prothrombinase via a Three-Step Mechanism

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Alan E Mast
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Direct Binding ◽  
Affinity Interaction

TFPIα inhibits early forms of the prothrombinase complex (factor Xa (FXa), factor Va (FVa)), though the inhibitory mechanism is not entirely understood. One step of inhibition is a high affinity interaction between a TFPIα C-terminal basic region (BR) (252-LIKTKRKRKK-261) and an acidic region (AR) present in FXa-activated and platelet-released forms of FVa. We investigated two additional potential mechanistic steps: (1) binding of the second Kunitz-type inhibitory domain (K2) of TFPIα to the FXa active site; and (2) the function of uncharged residues L252, I253, and T255 within the BR, which are evolutionarily conserved, suggesting they have activity. Direct inhibition of FXa was investigated using TFPIα with an altered K2 (TFPI-R107A) incapable of binding FXa. TFPI-R107A inhibited purified prothrombinase 17-fold weaker than TFPIα (IC50 = 30.6nM vs. 1.8nM) and did not inhibit FXa-initiated thrombin generation in platelet-rich plasma (PRP). Therefore, direct binding of FXa and K2 is required for efficient inhibition of prothrombinase under physiological conditions. Similarly, the role of L252, I253, and T255 was investigated by substituting them with alanine (TFPI-AAKA). The IC50 for prothrombinase inhibition by TFPI-AAKA was 10.4nM, and it had reduced inhibitory activity in PRP, revealing that these residues are also required for efficient prothrombinase inhibition. The role of L252, I253, and T255 was further probed using the peptide LIKTKRKRKK, which inhibited purified prothrombinase (IC50 = 1.0μM) and thrombin generation in PRP at 1μM. AAKAKRKRKK had very little activity in either assay (~20% prothrombinase inhibition with 225μM peptide), but bound the FVa AR equivalently to LIKTKRKRKK (K d = 5.9nM and 6.0nM, respectively). Thus, the basic residues are responsible for AR binding, while a second step, mediated by L252-T255, is necessary for inhibitory activity. These residues may be necessary for displacement of FXa from FVa, as proposed by Bunce et al. We propose that prothrombinase inhibition by TFPIα involves three steps: (1) the TFPIα BR basic residues bind the FVa AR; (2) residues L252-T255 block prothrombinase assembly; and (3) K2 binds the FXa active site. All three steps are required for physiologic inhibition of prothrombinase by TFPIα.

Modulation of Prothrombinse Assembly and Activity by Phosphotidylethanolamine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4207-4207
Author(s):  
Rinku Majumder ◽  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
Barry R. Lentz ◽  
William H. Kane
Keyword(s):  
Binding Sites ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Naturally Occurring ◽  
Early Phases

Abstract Abstract 4207 Constituents of naturally occurring phospholipid membranes regulate the activity of the prothrombinase complex. In the present study we demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PC:PE) bind factor Va with high affinity (Kd ∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a functional prothrombinase complex though thrombin generation at saturating factor Va concentrations is reduced approximately 60-70% compared to membranes containing 5% or more PS. The presence of PE markedly enhances the catalytic efficiency of the prothrombinase complex on membranes containing 1% PS with only modest effects on membranes containing 5% or more PS. The effect of PE on factor Va membrane binding appears to be due to direct interactions between PE and factor Va rather than to changes in membrane surface packing. Finally, we find that soluble C6PE is able to bind to factor Va (Kd ∼6.5 uM) and factor Xa (Kd ∼ 91 uM). We also show that soluble C6PE is able to stimulate formation of a partially active factor Va-factor Xa complex capable of catalyzing conversion of prothrombin to thrombin in the absence of a membrane surface. We further demonstrate that C6PE and C6PS binding sites in factor Xa are linked, as binding of one lipid enhances the binding and activity of the other. These findings provide important new insights into the role of PE in assembly of the prothrombinase complex that are relevant to understanding the activity of factor Xa on the surface of platelets particularly in the early phases of hemostasis when the concentration of PS may be limiting. Disclosures: No relevant conflicts of interest to declare.

Cholesterol and Phosphatidylethanolamine Enhance the Binding of Factor Va and Factor Xa to Phospholipid Vesicles and Promote Assembly of the Prothrombinase Complex on Membranes Containing Low Concentrations of Phosphatidylserine.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1712-1712
Author(s):  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
William H. Kane
Keyword(s):  
Phospholipid Composition ◽  
Factor Xa ◽  
Phospholipid Vesicles ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Kd Values ◽  
Direct Binding ◽  
Prothrombinase Activity

Abstract Platelet membranes are composed of cholesterol (CH) and phospholipids including phosphatidylethanolamine (PE), sphingomeylin (SM), phosphatidylcholine (PC) and phosphatidylserine (PS). Phospholipid vesicles containing 20–25% PS have been used extensively to characterize assembly and regulation of the prothrombinase complex. However, the concentration of PS on the surface of activated platelets has been determined to be only 4–13%. Smeets et al (Thromb. Res. 81:419, 1996) demonstrated that CH and PE in the presence of 0–10% PS have stimulating effects on prothrombinase activity, while SM has an inhibitory one. To further investigate the roles of CH, PE and SM in the binding of factor Va and Xa to phospholipid vesicles and assembly of the prothrombinase complex, vesicles consisting of 1–25% DOPS plus 30% CH, 30% DOPE and/or 20% SM, were prepared for prothrombinase assays as a function of DOPS concentration. Compared to individual effect of DOPS alone or DOPS plus CH, PE or SM on prothrombinase activity, a synergistic effect was observed on vesicles containing CH, DOPE and SM, which was significant in the range of 1–15% DOPS. The PS concentration required for half-maximal rates of thrombin generation was 5.3% for vesicles containing CH, DOPE and SM, compared to 10.6, 11.2, 8.3 and 12.6% for vesicles containing DOPS alone, plus CH, plus DOPE or plus SM, respectively. This demonstrates that the requirement for PS is substantially decreased in the presence of CH, PE and SM. In order to further define the mechanisms for this effect, direct binding of factor Va to vesicles containing 30% CH, 30% PE and 20% SM was investigated using a fluorescence resonance energy transfer assay. These binding experiments demonstrated that factor Va bound to vesicles containing 1, 5, 10, and 20% DOPS with Kd values of 2.88±0.43 nM, 1.15±0.17 nM, 1.16±0.13 nM, and 1.18±0.19 nM, respectively. At low concentrations of PS (≤ 5%), CH, DOPE and SM increased the binding affinity of factor Va 2 to 6-fold compared to vesicles containing only DOPS. When prothrombinase activity was measured as a function of factor Va concentration on vesicles containing 5, 10, and 20% DOPS, K1/2Va values of 0.63±0.08nM, 0.38±0.05nM, and 0.37±0.07nM were observed which were comparable to the Kd’s determined in direct factor Va binding experiments. These results indicate that factor Va binds tightly to phospholipid vesicles mimicking the phospholipid composition of activated platelets even at very low concentrations of PS. When direct binding of factor Xa to vesicles containing 5, 10, and 20% DOPS plus CH, DOPE and SM was examined, the Kd values were 70.5±6.8 nM, 62.6±6.6 nM, and 49.6±4.0 nM, respectively. When the interaction of factor Xa with membranes was investigated using prothrombinase assays, K1/2Xa values of 0.12±0.03nM, 0.04±0.01nM, and 0.01±0.01nM were observed. In conclusion, the presence of CH and PE increases the affinity of factor Va and factor Xa binding to phospholipid vesicles containing physiologic concentrations of PS. The binding of factor Va to vesicles containing 1% PS is not sufficient for assembly and function of the prothrombinase complex, suggesting that higher concentrations of PS are needed to support the binding of factor Xa and prothrombin.

Mutation of the Hydrophobic Residues in Factor Va2 C1 Domain Affects the Phosphatidylserine Mediated Prothrombin Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1733-1733
Author(s):  
Rinku Majumder ◽  
Mary Ann Quinn-Allen ◽  
William H. Kane ◽  
Barry R. Lentz
Keyword(s):  
Factor Xa ◽  
Binding Pocket ◽  
Regulatory Site ◽  
Wild Type ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Essentially Normal ◽  
Tryptophan Residues ◽  
C1 Domain ◽  
Prothrombin Activation

Abstract Tightly associated factors Va and Xa serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. The binding of factor Va to PS membranes regulates assembly of the prothrombinase complex. The C-terminal domain (C2) of factor Va (residues 2037–2196 in human factor Va) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp2063/Trp2064 (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent results have shown that mutating these Trp residues abolishes FVa2-membrane binding, but does not affect the assembly and activity of the prothrombinase in the presence of 25% PS membranes or soluble C6PS. Our data also indicates that there is another site on factor Va2 that might be specific for PS or C6PS and might serve as a regulatory site for assembly or activity of the FVa2-FXa complex. A pair of solvent exposed amino acids, Tyr1956/Leu1957 in the C1 domain is located analogously to the critical Trp residues in C2. Recently, we showed that prothrombin activation in the presence of the factor Va mutant (Y1956, L1957) A was markedly impaired on phospholipid vesicles containing 10% or less PS but was essentially normal on vesicles containing 25% PS (Saleh, M., Peng, W., Quinn-Allen, MA., Macedo-Ribeiro, S., Fuentes-Prior, P., Bode,W. and Kane, WH. Thromb. Haemost. 2004,91:16–27). Our work aims to test the hypothesis that the PS regulatory site in Va2 is located analogously to the C6PS binding site in the C2 domain. We have used factor Va mutants with mutations in either the C1 domain, (Y1956, L1957) A or in both the C1 and C2 domains, (Y1956, L1957, W2063, W2064) A. We determine the rate of thrombin formation in the presence of 400 mM C6PS and wild type, C1 and C1C2 mutated factor Va2 to be 170, 12 and 11 nM/S−1/M−1, respectively. Mutations in the C1 and C1C2 domains of factor Va2 reduced the rate of activation of prothrombin to thrombin by 14–15 fold. We have also determined the effect of these mutations on the assembly of factors Xa–Va2 complex by monitoring the change in fluorescence of dansyl-glutamyl-glycyl-arginyl-chloromethylketone (DEGR-CK)-Xa with the addition of wild type, C1 and C1C2 mutated factor Va2 in the presence of 400 mM C6PS. Our data shows that the Kd’s of factor Xa with factor Va2 (wild type, C1 mutant and C1C2 mutant) are 3, 564, 624 nM respectively. Our results support the hypothesis that a PS regulatory site is located in the C1 domain of factor Va and includes residues Tyr1956 and Leu1957.

The Role of Phospholipids and Factor Va in the Mechanism of Prothrombin Activation

1979 ◽  
Author(s):  
J. Rosing ◽  
G. Tans ◽  
J.W.P. Govers-Riemslag ◽  
R.F.A. Zwaal ◽  
H.C. Hemker
Keyword(s):  
Kinetic Parameters ◽  
Factor Xa ◽  
Clotting Factor ◽  
Factor V ◽  
Factor X ◽  
Factor Va ◽  
Prothrombin Activation ◽  
Thrombin Formation

The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

scholarly journals The Factor V Activation Paradox

2004 ◽  
Vol 279 (19) ◽  
pp. 19580-19591 ◽  
Author(s):  
Thomas Orfeo ◽  
Nicole Brufatto ◽  
Michael E. Nesheim ◽  
Hung Xu ◽  
Saulius Butenas ◽  
...  
Keyword(s):  
Rate Constants ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Reaction Pathways ◽  
Factor V ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activation Products ◽  
Initial Activation ◽  
Prothrombin Activation

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

Studies On The Interaction Between Factor Va And Factor Xa

1981 ◽  
Author(s):  
M Lindhout ◽  
J Govers-Riemslag ◽  
J Rosing ◽  
H Hemker
Keyword(s):  
Factor Xa ◽  
Factor V ◽  
Molecular Weights ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
A Value ◽  
Wide Range ◽  
Carboxyglutamic Acid ◽  
Polypeptide Chains ◽  
Prothrombin Activation

Thrombin activated bovine factor V is composed of two polypeptide chains with molecular weights 94,000 and 80,000. The two polypeptide chains are complexed via Ca2+ions.Factor Va enhances the rate of thrombin formation by drastically increasing the Vmax of the prothrombin activation. We have undertaken a study of the interactions of factor Va with the different components of the prothrombinase complex (e.g. factor Xa and prothrombin), in order to get more insight in the mode of action of factor Va.Our kinetic experiments in solution show that the functional enzyme in the prothrombinase complex is a equimolar complex of factor Va and factor Xa. The dissociationconstant, as determined over a wide range of prothrombin concentrations, has a value of 3×10-9M.For the stimulating effect of factor Va on the prothrombin activation by factor Xa in solution, the presence of Ca2+ions is required. The dissociationconstant of the Va-Xa complex was found to be independent of the Ca2+ concentration. In order to reveal whether an interaction between Ca2+ and γ- carboxyglutamic acid residues is responsible for the observed Ca2+ requirement, identical experiments were carried out with decarboxyfactor Xa and decarboxyprothrombin. The isolated polypeptide chains of factor Va have, in the presence or absence of factor Va, no effect on the kinetic parameters of the prothrombin activation. This let us conclude that there is no interaction between factor Xa and the separate polypeptide chains of factor Va.The affinity of factor Xa for negatively charged phospholipid or stimulated bloodplatelets is greatly enhanced by the presence of factor Va. Our Kd value measured for the Xa-Va complex in combination with reported dissociationconstants of factor Xa-phospholipid and Factor Va-phospholipid complexes give a quantitative explanation for the above mentioned effect of factor Va on the binding of factor Xa to phospholipid membranes.

Efficient Thrombin Generation Requires Molecular Phosphatidylserine, Not a Membrane Surface.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1022-1022
Author(s):  
Rinku Majumder ◽  
Gabriel E. Weinreb ◽  
Barry R. Lentz
Keyword(s):  
Reaction Pathway ◽  
Membrane Surface ◽  
Factor Xa ◽  
Elastic Light Scattering ◽  
Complex Assembly ◽  
Peptide Bonds ◽  
Prothrombinase Complex ◽  
Pyrene Fluorescence ◽  
Factor Va

Abstract Activation of prothrombin to thrombin is catalyzed by “prothrombinase” complex, traditionally viewed as factor Xa (FXa) in complex with factor Va (FVa) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through “channeling” (activation without release of an intermediate). We ask here whether the PS molecule itself, not the membrane surface, is sufficient to produce fully active human “prothrombinase” complex in solution. Both FXa and FVa bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FXa and FVa2 (light isoform of FVa), these proteins form a tight (Kd = 0.6 ±0.09 nM at 37°C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels show that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FXa and one FVa2, further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2x108 M− 1sec− 1) and via the same reaction pathway (50–60% channeling, rest via meizothrombin intermediate) as activation catalyzed by a complex assembled on PS-containing membranes (4.4x108 M− 1sec− 1). These results question the accepted role of PS-membranes as providing “dimensionality-reduction” and favor a regulatory role for platelet-membrane-exposed PS.

The Regulatory Function of Amino Acid Region 659–663 of Factor Va on Prothrombinase during Thrombin Formation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2012-2012
Author(s):  
Jamila Hirbawi ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Binding Sites ◽  
Factor Xa ◽  
Prothrombinase Complex ◽  
Amino Acid Region ◽  
Factor Va ◽  
Acidic Amino Acids ◽  
Prothrombin Activation ◽  
Acid Region

Abstract Following vascular injury, the process of hemostasis facilitates the generation of thrombin, which in turn allows the formation of a fibrin clot. Without the proper regulation of this process, serious life threatening conditions, such as DVT (deep vein thrombosis), can occur. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. The incorporation of factor Va (fVa) into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of factor Xa (fXa) for thrombin generation. Factor Va is composed of heavy and light chains. The light chain of the cofactor contains the binding sites of the cofactor to the membrane surface while the heavy chain contains binding sites for the other components of prothrombinase. Portions of the fVa heavy chain have been found to act as fXa binding sites. It has been demonstrated that the COOH-terminal region of factor Va contains cluster of acidic amino acids that are crucial for its cofactor activity. More specifically, amino acid region 695–698 from fVa heavy chain regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. The COOH-terminal portion of the heavy chain also contains another cluster of acidic amino acids (encompassing residues 659–663). Site-directed mutagenesis was performed to generate a factor V (fV) molecule with region Asp659-Asp663 (fV663) deleted. We have also constructed mutant molecules with regions Lys680-Arg709 and Asp659-Asp663 (fV663+709)) deleted from the COOH-terminal region of the heavy chain. Finally, a mutant molecule containing point mutations in region Asp659-Asp663 where the five amino acids in this sequence are mutated to all lysines (fV5K), was also constructed. These recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells and assessed for their capability to promote prothrombin activation following activation by thrombin. Prothrombin activation by prothrombinase assembled with the mutant molecules was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with the recombinant mutant molecules was slower. Two-stage clotting assays revealed that FV663+709, fVa5K, and fVa663 all had reduced clotting activities compared to fVaWT and plasma-derived fVa. Kinetic analyses demonstrated that Kd values for fXa of all the mutants were similar to fVaWT. However, kcat values for the various molecules varied. The kcat values for prothrombinase assembled with fVa5K, and fVa663 were 10-fold reduced when compared to the values obtained with prothrombinase assembled with fVaWT, while prothrombinase assembled with fVa663+709 had a kcat value that was sligtly lower than that of fVaWT. Our data suggest that amino acid region 659–663 from fV plays a crucial role for fVa cofactor acivity and overall the data demonstrate that acidic amino acids from the COOH-terminus of the factor Va heavy chain play a preeminent role in proper prothrombinase complex assembly and function, resulting in competent thrombin formation. These data assign an important regulatory role of the acidic COOH-terminal region of fVa to the activity of factor Xa within prothrombinase. Finally, our data aid in further studies that may lead to the development of small synthetic molecules that could be used as anticoagulants in individuals with thrombotic tendencies.

The Role of Amino Acids 700–701 of the Factor Va Heavy Chain During Prothrombin Activation by Factor Xa

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2203-2203
Author(s):  
Jamila Hirbawi ◽  
Paul Y Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Human Origin ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Sds Page ◽  
Carboxyl Terminal ◽  
Prothrombin Activation

Abstract Abstract 2203 Blood coagulation is initiated after vascular injury, promoting formation of the fibrin plug. The prothrombinase complex plays a crucial role during activation of prothrombin (Pro) to thrombin. The complex is composed of the enzyme, factor Xa (fXa), along with its non-enzymatic cofactor, factor Va (fVa), in the presence of calcium on a phospholipid surface. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Prothrombinase activates prothrombin through initial cleavage at Arg320 followed by cleavage at Arg271 to yield human alpha-thrombin. This pathway is responsible for the generation of a transient catalytically active intermediate, meizothrombin. Recent data has suggested a differential effect of bovine and human factor Va on prothrombin-1 (Pre-1) activation by prothrombinase. This difference was localized within the last ten amino acids from the carboxyl-terminal region of fVa heavy chain. The only amino acid difference between the two cofactor molecules is localized at position 700–701 where the Asn-Arg dipeptide in the fVa of human origin is replaced by the Asp-Glu sequence in the carboxyl-terminal region of the cofactor of bovine origin. We have therefore constructed a recombinant human mutant fVa molecule with these amino acids mutated to their bovine counterpart. We have created a recombinant fVa molecule with the mutation700NR701 →DE. This recombinant cofactor molecule (fVDE) along with wild type factor V (fVWT) were transiently expressed in COS7 cells, purified to homogeneity, and assessed for their capability to by assembled in prothrombinase and promote Pro activation. Thrombin generation was evaluated by SDS-PAGE in a system using all proteins of human origin and the kinetic parameters of the reactions were determined using a chromogenic substrate to assess for thrombin activity. Kinetic analyses revealed that the Kd of fVaDE for human fXa, as well as the kcat and Km values of prothrombinase assembled with fVaDE for human Pro activation were similar to the values obtained following Pro activation by prothrombinase assembled with fVaWT. Surprisingly, SDS-PAGE analyses of prothrombin activation time courses revealed that the overall rate of cleavage of Pro by prothrombinase assembled with fVaDE was significantly delayed with significant accumulation of the intermediate meizothrombin, and delayed thrombin generation when compared to the rate of activation of Pro by prothrombinase assembled with fVaWT. Two-stage clotting assays (PT times) also revealed that fVaDE had reduced clotting activity when compared to fVaWT. Comparison of the rate of cleavage of two recombinant Pro mutant molecules, rMZ-II a recombinant Pro molecule that cannot be cleaved at Arg271 and rP2-II a recombinant Pro molecule that cannot be cleaved at Arg320, by prothrombinase assembled with fVaDE demonstrated impaired rate of cleavage of both substrates when compared to the rate of cleavage of the mutant recombinant Pro molecules by prothrombinase assembled with fVaWT. These findings were verified by experiments using active-site blocked purified human meizothrombin (FPR-meizo). Prothrombinase assembled with fVaDE was considerably impaired in its ability to cleave FPR-meizo at Arg271 as compared to the ability of prothrombinase assembled with fVaWT for the same cleavage. In fact, gel electrophoresis analyses demonstrated that prothrombinase assembled with fVaDE cleaves FPR-meizo with a rate similar to the cleavage of FPR-meizo by fXa alone. All these data together strongly suggest that the 700NR701 portion of the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate recognition/interaction during Pro activation by prothrombinase and thus regulates the rates of thrombin formation locally at the place of vascular injury. Disclosures: No relevant conflicts of interest to declare.

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