Contribution of Amino Acid Region 334−335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase†

Melissa A. Barhoover; Tivadar Orban; Daniel O. Beck; Michael A. Bukys; Michael Kalafatis

doi:10.1021/bi800057r

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

Blood ◽

10.1182/blood.v104.11.3977.3977 ◽

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.

Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase†‡

Biochemistry ◽

10.1021/bi800593k ◽

2008 ◽

Vol 47 (30) ◽

pp. 7963-7974 ◽

Cited By ~ 6

Author(s):

Jamila Hirbawi ◽

Michael A. Bukys ◽

Melissa A. Barhoover ◽

Evrim Erdogan ◽

Michael Kalafatis

Keyword(s):

Amino Acid ◽

Heavy Chain ◽

Terminal Amino Acid ◽

Amino Acid Region ◽

Factor Va ◽

Terminal Amino ◽

Acid Region

The Role of Amino Acids 659-663 of Factor Va Heavy Chain During Prothrombin Activation by Prothrombinase.

Blood ◽

10.1182/blood.v114.22.2131.2131 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2131-2131

Author(s):

Jamila Hirbawi ◽

Michael Kalafatis

Keyword(s):

Amino Acid ◽

Heavy Chain ◽

Anion Binding ◽

Prothrombinase Complex ◽

Amino Acid Region ◽

Factor Va ◽

Cofactor Activity ◽

Prothrombin Activation ◽

Acid Region

Abstract 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.

Contribution of Amino Acid Region 659−663 of Factor Va Heavy Chain to the Activity of Factor Xa within Prothrombinase,

Biochemistry ◽

10.1021/bi101097t ◽

2010 ◽

Vol 49 (39) ◽

pp. 8520-8534 ◽

Cited By ~ 6

Author(s):

Jamila Hirbawi ◽

John L. Vaughn ◽

Michael A. Bukys ◽

Hans L. Vos ◽

Michael Kalafatis

Keyword(s):

Amino Acid ◽

Heavy Chain ◽

Factor Xa ◽

Amino Acid Region ◽

Factor Va ◽

Acid Region

Controlling the Pathway for Prothrombin Activation by Prothrombinase.

Blood ◽

10.1182/blood.v108.11.1695.1695 ◽

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 ◽

10.1182/blood.v106.11.1027.1027 ◽

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.

The Contribution of Amino Acid Region Asp695-Tyr698of Factor V to Procofactor Activation and Factor Va Function

Journal of Biological Chemistry ◽

10.1074/jbc.m306850200 ◽

2003 ◽

Vol 279 (4) ◽

pp. 3084-3095 ◽

Cited By ~ 27

Author(s):

Daniel O. Beck ◽

Michael A. Bukys ◽

Lisam S. Singh ◽

Katalin A. Szabo ◽

Michael Kalafatis

Keyword(s):

Amino Acid ◽

Factor V ◽

Amino Acid Region ◽

Factor Va ◽

Acid Region

Identification of Sulfo-Tyrosines of Factor V.

Blood ◽

10.1182/blood.v104.11.1731.1731 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1731-1731

Author(s):

Evrim Erdogan ◽

Michael Kalafatis

Keyword(s):

Amino Acid ◽

Factor Xa ◽

Factor V ◽

Wild Type ◽

Single Chain ◽

Amino Acid Region ◽

Thrombin Cleavage ◽

Factor Va ◽

Cofactor Activity ◽

Acid Region

Abstract The factor Va molecule is the essential cofactor of the prothrombinase complex. This complex composed of factor Xa and factor Va assembled on a platelet membrane-surface in the presence of Ca2+ ions converts membrane-bound prothrombin to thrombin. Single chain factor V does not bind factor Xa. Single-chain factor V is cleaved by thrombin first at Arg709 followed by cleavages at Arg1018 and Arg1545 to produce the heavy and light chains of the active cofactor (factor Va) and two activation peptides. Efficient thrombin cleavage and activation of factor V is essential for cofactor function and requires tyrosine sulfation. Tyrosine sulfation of factor V also appears to regulate its activity. Seven tyrosine residues in factor V, Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, and Tyr1565 have been identified as potential sites of sulfation. However, which residues are sulfated and their contribution to procofactor activation and cofactor function still remain to be elucidated. Two of the sulfation sites Tyr696 and Tyr698 are located in the acidic amino acid region near to the first required thrombin cleavage site at Arg709. Recent data demonstrated that these residues are essential for factor V activation and cofactor activity. Another acidic amino acid region, 1490–1520 is adjacent to the thrombin cleavage site at Arg1545 required for light chain formation. This region also contains three potential sulfation sites at residues 1494, 1510, and 1515 and was shown to be required for optimum procofactor activation. To ascertain which of these three residues is important for procofactor activation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations 1493DY1494→AF, 1508DDY1510→AAF and 1514DY1515→AF. The clotting and cofactor activity of the 1493DY1494→AF and 1514DY1515→AF mutants was similar to the clotting activity observed with the wild type recombinant factor Va molecule following activation by thrombin or RVV-V activator. In contrast, under similar experimental conditions recombinant factor V with the substitution 1508DDY1510→AAF was deficient in its clotting activity and had impaired cofactor activity. Moreover, following prolonged incubation with thrombin, no light chain formation was observed in the factor V molecule bearing the 1508DDY1510→AAF mutation. Thus, amino acid residues 1508–1510 of factor V are required for thrombin interaction with the procofactor which in turn appears necessary for cleavage at Arg1545. Studies of sulfated proteins have shown that the effect of sulfo-tyrosines on protein structure/function can be preserved by replacing them with glutamic acid. To explicitly identify the sulfated tyrosines on the factor V molecule, we mutated Tyr696, Tyr698 and Tyr1510 to glutamic acid and transfected them into COS-7L cells. Expression was performed in the presence of media containing or devoid of sulfate. In the presence of sulfate, the cofactor and clotting activities of the DY696DY698→DEDE and DDY1510→DDE mutants, separately were similar to the wild type recombinant factor Va molecule. However, in the absence of sulfate, the wild type and the mutant recombinant factor V molecules had both impaired cofactor activity and clotting activity following their activation with thrombin. However, their respective activity was higher than the activity of the factor V molecule bearing the 1508DDY1510→AAF mutation. Our data suggest that residues 696, 698, and 1510 of factor V appear to be sulfated and might be important for procofactor activation and cofactor function.

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

Blood ◽

10.1182/blood.v112.11.2012.2012 ◽

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.

Role of the N-Terminal Amino Acid Region of Factor Va Light Chain In Prothrombinase Assembly and Function.

Blood ◽

10.1182/blood.v116.21.1127.1127 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1127-1127

Author(s):

John L. Vaughn ◽

Jamila Hirbawi ◽

Michael A. Bukys ◽

Michael Kalafatis

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Light Chain ◽

Gel Electrophoresis ◽

Fold Increase ◽

Divalent Ions ◽

Amino Acid Region ◽

Factor Va ◽

And Function ◽

Acid Region

Abstract Abstract 1127 The timely activation of prothrombin (II) to thrombin (IIa) by the prothrombinase (IIase) complex is required for the maintenance of hemostasis. The IIase complex is composed of the enzyme factor Xa (fXa) and the cofactor factor Va (fVa) assembled on a membrane surface in the presence of divalent ions. While fXa alone is capable of activating II, the incorporation of fVa into the IIase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for IIa formation. FVa is generated following limited proteolysis of factor V (fV) by IIa at Arg709, Arg1018, and Arg1545, releasing a heavy chain (residues 1–709) and light chain (residues 1546–2196) that associate noncovalently through divalent ions. The A3 domain of the light chain of fVa has been implicated in the interaction of the cofactor with fXa. To determine the contribution of the A3 domain of fVa to IIase assembly and function, synthetic peptides representing residues 1546–1612 were screened for their ability to inhibit IIase. The peptide representing residues 1546–1558 strongly inhibited IIase with an IC50 of 50 μM. Additional fluorometric studies with overlapping pentapeptides from that region revealed that amino acids 1553–1558 were responsible for the observed inhibition. To verify the data from the overlapping pentapeptide studies, site-directed mutagenesis was used to generate a fVa mutant molecule with amino acids 1549–1558 deleted (fVaLCD1). FVaLCD1 demonstrated greatly reduced clotting activity in two-stage clotting assays. Gel electrophoresis of the IIase catalyzed activation of II using fVaLCD1 revealed a 50% reduction in the rate of prothrombin consumption. IIase assembled with fVaLCD1 exhibited a 5-fold increase in the apparent disassociation constant (Kdapp) for the enzyme-cofactor interaction and a 50% reduction in the turnover number (kcat) for the enzyme. To investigate the possibility that amino acids 1549–1558 are important for procofactor activation, the fVaLCD1 mutant was incubated with IIa and visualized with gel electrophoresis. The fVaLCD1 mutant exhibited a greatly reduced rate of IIa catalyzed activation. Additional recombinant fVa molecules with overlapping alanine mutations within the amino acid region 1548–1559 revealed that amino acids 1554–1555 were responsible for the observed effects in fVaLCD1. The recombinant fVa molecule with amino acids 1554–1555 mutated to alanines (fVaAA) demonstrated a greatly reduced clotting activity, an 8-fold increase in the Kdapp for the enzyme-cofactor interaction, and a 50% reduction in the kcat for the enzyme. To further investigate the mutational robustness of fVa against mutations in amino acids 1554–1555, a recombinant fVa molecule with both tyrosine residues in that region mutated conservatively to phenylalanines (fVaFF) was created. The more conservative fVaFF mutant, which differed from fVaWT only in the absence of side-chain hydroxyl moieties, exhibited normal clotting activity, kinetics, and procofactor activation. Altogether, the data demonstrate that amino acids 1554–1555 from the N-terminal region of the fVa light chain are important for IIase assembly and function because they constitute an interactive site for IIa and fXa. Disclosures: No relevant conflicts of interest to declare.