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.

Evidence That the COOH-Terminal Region of Factor Va Heavy Chain Is Involved in the Regulation of Prothrombinase Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1025-1025
Author(s):  
Jamila Hirbawi ◽  
Melissa A. Blum ◽  
Michael A. Bukys ◽  
Tivadar Orban ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Thrombin Generation ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Sds Page ◽  
Carboxyl Terminal ◽  
Prothrombin Activation

Abstract 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. Incorporation of fVa into the prothrombinase complex results in 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. It has been demonstrated that elimination of the carboxyl terminal portion of the heavy chain of fVa by proteolytic enzymes results in a cofactor molecule with decreased clotting activity and slightly increased to normal chromogenic activity. In addition, we have previously shown that the carboxyl terminal portion of the heavy chain of fVa is involved in the interaction of the cofactor with prothrombin. To further ascertain the importance of this region of the molecule for cofactor activity we used PCR based methods to produce recombinant fVa molecules with several portions of the COOH-terminus deleted. Recombinant fV653 has amino acids 653–709 deleted, recombinant fV696 has amino acid residues 680–696 deleted, recombinant fV680 has amino acid residues 653–680 deleted, while recombinant fV709 has amino acid residues 680–709 missing. 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 Russell’s Viper Venom factor V activator (RVV-V activator). Thrombin generation was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. While fVa653 and fVa680 were devoid of clotting activity, fVa696 and fVa709 had reduced clotting activities compared to fVaWT and plasma-derived fVa. This level of clotting activity was similar to the clotting activity of a fV molecule that was treated with thrombin and human neutrophil elastase (HNE) resulting in fVaHNE. fVaHNE is cleaved at Ala677/Thr678 resulting in a cofactor with a shorter heavy chain. Further analyses revealed that all mutant recombinant molecules as well as fVaHNE have similar KD values for fXa when compared to plasma fVa and fVaWT. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with fVa696, fVa709, or fVaHNE was slower resulting in accumulation of meizothrombin. This data confirm our previous findings and suggest that this region on the heavy chain of fVa contribute to cofactor function. A logical explanation for these findings is that the COOH-terminus of the heavy chain of fVa participates in the regulation of the rates of appearance/disappearance of meizothrombin. Increased persistence of meizothrombin in the reaction mixture can explain the slower clotting times since it is well known that meizothrombin has poor clotting activity. Thus at a given time point there will be more meizothrombin present in a sample where prothrombinase was assembled with fVa709, or fVa696, or fVaHNE than in a sample where prothrombinase was formed with fVaWT. Overall the data suggests that the COOH-terminal portion of the factor Va heavy chain contributes to the appropriate orientation of prothrombin with respect to the catalytic site of fXa resulting in efficient cleavages at Arg320 /Arg271 and competent thrombin formation.

Amino Acids Asp695 and Tyr696 of Factor Va Are Essential for Optimal Rate of Prothrombin Activation by Prothrombinase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3163-3163
Author(s):  
Jamila Hirbawi ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Catalytic Efficiency ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Sds Page ◽  
Prothrombin Activation

Abstract Abstract 3163 Poster Board III-101 Blood coagulation is initiated after vascular injury, promoting formation of the 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 Ca2+. 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 intermediate, meizothrombin, that is enzymatically active with increased chromogenic substrate activity, but yields poor clotting activity. Factor Va is composed of heavy and light chains that play a crucial role during thrombin formation. Portions of the fVa heavy chain have been found to interact with proexosite 1 (pro1) of prothrombin and influence prothrombinase activity. It has been recently demonstrated that deletion of the COOH-terminal region of the factor Va heavy chain causes accumulation of meizothrombin due to delayed cleavage of prothrombin at Arg271. Site-directed mutagenesis was performed to generate recombinant mutant molecules in order to identify the specific amino acids of this COOH-terminal region that regulate cleavage. Mutants with the 695DYDY698→DFDY(fVaDFDY), KFDY(fVaKFDY),DEDE(fVaDEDE),DFDF(fVaDFDF) substitutions were constructed. These recombinant molecules along with wild type factor V (fVWT) were transiently expressed in COS7 cells purified to homogeneity and assessed for their capability to promote prothrombin activation. Prothrombin activation 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 fVaKFDY and fVaDFDY was delayed, while prothrombinase assembled with fVaDEDE and fVaDFDF had no significant effects when compared to fVaWT. Two- stage clotting assays (PT times) revealed that fVaKFDY and fVaDFDY both had reduced clotting activity when compared to fVaWT, while fVaDEDE and fVaDFDF gave similar clotting results as fVaWT. Determination of kcat values for prothrombinase assembled with the various recombinant molecules revealed that prothrombinase assembled fVaKFDY and fVaDFDY had a 20% increase catalytic efficiency as compared with prothrombinase assembled with fVaWT, while fVaDEDE and fVaDFDF gave values that were comparable to prothrombinase assembled with fVaWT. Comparison of the rate of cleavage of two recombinant prothrombin mutant molecules, rMZ that can't be cleaved at Arg271 and rPII that can't be cleaved at Arg320, by prothrombinase assembled with the mutant fVa molecules resulted in normal cleavage of rMZ by all the mutants. Cleavage of rPII, however, was impaired when fVaKFDY and fVaDFDY were incorporated into prothrombinase. The data presented suggests that the 695DY696 portion of the acidic cluster found in the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate interaction during thrombus formation. Disclosures No relevant conflicts of interest to declare.

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.

A Peptide Region from Factor Va Increases Factor Xa Efficiency for Cleavage of Prothrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3977-3977
Author(s):  
Melissa A. Blum ◽  
Daniel O. Beck ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Membrane Surface ◽  
Factor Xa ◽  
Amino Acid Region ◽  
Factor Va ◽  
Prothrombinase Activity ◽  
Prothrombin Activation ◽  
Acid Region

Abstract The procoagulant enzymatic complex, prothrombinase, which is required for normal hemostasis, is composed of the enzyme, factor Xa, the protein cofactor, factor Va, associated on a cell surface in the presence of divalent metal ions. Incorporation of factor Va into prothrombinase and its interaction with factor Xa increases the catalytic efficiency of the enzyme by five orders of magnitude as compared to factor Xa alone. While the importance of the contribution of factor Va to the activity of factor Xa for rapid thrombin formation by prothrombinase at the place of vascular injury has been long established, the consequence of the interaction of the cofactor with the members of prothrombinase and the molecular mechanism by which factor Va accelerates prothrombin activation remains an enigma. Prothrombin is activated following two cleavages (Arg271/Arg320). Depending on the order of peptide bond cleavage different intermediates are formed. Factor Xa alone cleaves prothrombin sequentially, first at Arg271 to produce fragment 1•2 and prethrombin-2, followed by cleavage at Arg320 to produce fragment 1•2 and thrombin. The prothrombinase complex catalyzes the activation of prothrombin following the opposite pathway (Arg320 followed by Arg271), resulting in a formation of an active intermediate (meizothrombin) and a 300,000-fold increase in the rate of the overall reaction compared with the rate of prothrombin activation observed with factor Xa alone. We have shown that amino acid region 307–348 of factor Va heavy chain is critical for cofactor activity. A peptide containing this amino acid sequence (42 amino acids, N42R) was found to interact with fluorescently labeled factor Xa and to inhibit prothrombinase activity. Our present data show that N42R can be cross-linked to the heavy chain of membrane-bound factor Xa in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have also demonstrated that amino acid region 323–331 from N42R (AP4′) contains a binding site for factor Xa of factor Va heavy chain. Our present data show that a peptide containing amino acid residues 317–326 (AP3) inhibited both prothrombinase activity and the high affinity interaction of factor Va with factor Xa on the membrane surface. Moreover, we have found using site directed mutagenesis and recombinant factor Va that amino acids at the NH2-terminal end of AP4′ (i.e. residues 323–325, Glu-Tyr-Phe) are responsible for the inhibitory effect of AP3 and AP4′ and are crucial for the interaction of factor Va with factor Xa. A tripeptide with this sequence inhibited prothrombinase activity in an assay using a fluorescent thrombin inhibitor. To identify the effect of these peptides on factor Xa’s ability to cleave and activate prothrombin, we studied prothrombin activation by gel electrophoresis. The data demonstrated that several peptides that inhibited both the factor Va-factor Xa interaction on the membrane surface and prothrombinase activity, had the ability to accelerate cleavage of prothrombin by factor Xa alone, in the absence of factor Va. Specifically, N42R and AP3 were found to increase the rate of prothrombin consumption by factor Xa by approximately four-fold when compared to factor Xa acting alone. Both peptides induced acceleration in prethrombin-2 formation suggesting an increased in the rate of cleavage of prothrombin at Arg271. These data suggest that the binding of factor Va to factor Xa through amino acid region 323–331 alone produces an effect on factor Xa that increases its potency for cleavage at Arg271.

Controlling the Pathway for Prothrombin Activation by Prothrombinase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1695-1695
Author(s):  
Michael A. Bukys ◽  
Paul Y. Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amino Acid Region ◽  
Factor Va ◽  
Initial Cleavage ◽  
Membrane Bound ◽  
Prothrombin Activation ◽  
Thrombin Formation

Abstract 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.

The Specific Contribution of Amino Acids 334 and 335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1027-1027
Author(s):  
Melissa A. Blum ◽  
Tivadar Orban ◽  
Daniel O. Beck ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Factor Va ◽  
Cofactor Activity ◽  
Type Factor

Abstract The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.

Inhibition of Factor Xa in Prothrombinase Is Enhanced by Covalent Linkage of Antithrombin to Heparin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1613-1613
Author(s):  
Sanjay Patel ◽  
Leslie R. Berry ◽  
Lesley Smith ◽  
Anthony Chan
Keyword(s):  
Rate Constants ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Complex Mixtures ◽  
Free State ◽  
Phospholipid Vesicles ◽  
Covalent Linkage ◽  
Rate Difference ◽  
Prothrombinase Complex ◽  
Factor Va

Abstract The rate of prothrombin to thrombin conversion by factor Xa (Xa) is enhanced when Xa is incorporated into the surface-bound prothrombinase complex. However, in comparison to the free state, Xa within the prothrombinase complex is afforded protection from antithrombin + heparin (AT+H) inactivation. We have shown that, unlike AT+H, a covalent conjugate of AT and H (ATH) can neutralize fibrin-bound thrombin. In this study, AT+H and ATH were compared in their reaction with Xa +/− prothrombinase complex. Mixtures of CaCl2, phospholipid vesicles, factor Va (Va) and prothrombin in TSP buffer, were combined with Xa. Following addition of either AT+H or ATH, time samples were neutralized with Na2EDTA + polybrene + substrate (S-2222) and residual Xa activity measured. Second order rate constants (k2) were calculated from plots of activity versus time. Results were compared to corresponding experiments with Xa alone. AT+H inactivation of Xa in prothrombinase occurred at a k2 (x 107 M−1min−1) of 2.34 +/− 0.09. In contrast, neutralization of free Xa by AT+H was significantly faster (k2 = 8.34 +/− 0.18, p = 0.03). Reaction with ATH showed no significant rate difference for Xa inhibition in either the complexed or free states (18.5 +/− 3.3 and 16.3 +/− 3.7, respectively). Intriguingly, the rates achieved for ATH inhibition of complexed and free Xa were significantly greater than that for AT+H with free Xa (p=0.03 and p=0.02, respectively). We conclude that covalent complexes of AT and H do not encounter resistance in the inhibition of Xa in prothrombinase, as seen for non-covalent AT+H mixtures. Thus, it is possible for ATH to effectively inhibit the propagation phase of thrombin generation and thus dampen thrombin production via neutralization of Xa in prothrombinase.

scholarly journals The Action of a Synthetic Pentasaccharide on Thrombin Generation in Whole Plasma

1989 ◽  
Vol 61 (03) ◽  
pp. 397-401 ◽  
Author(s):  
S Béguin ◽  
J Choay ◽  
H C Hemker
Keyword(s):  
Binding Site ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Prothrombinase Activity

SummaryWe investigated the effect on thrombin generation in plasma of the pentasaccharide that represent the AT II/binding site in heparin. This compound has no effect on the breakdown of thrombin in plasma. It dose-dependently inhibits the formation of thrombin in both the intrinsic and the extrinsic pathway. If coagulation is triggered by the complete prothrombinase complex (phospholipid – factor Va – factor Xa) under conditions in which the large majority of factor Xa is bound to the complex, the inhibition of prothrombinase activity is only minor. If no factor Va is present or if the prothrombinase activity is triggered by adding complete tenase (PL-FVIIIa-FIXa) or incomplete tenase (PLFIXa) to the plasma the inhibition by pentasaccharide is of the same magnitude as that in the intrinsic or extrinsic system.We conclude that the pentasaccharide inhibits blood coagulation by katalysing the inactivation of free factor Xa. In contrast to classical heparin it does inhibit the peak of thrombin formation in platelet rich plasma, probably because it is less subject to inactivation by heparin binding proteins from platelets than classical heparin is.

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.

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.

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