The contribution of amino acid region Asp695-Tyr698 of factor V to procofactor activation and factor Va function

被引:41
作者
Beck, DO
Bukys, MA
Singh, LS
Szabo, KA
Kalafatis, M
机构
[1] Cleveland State Univ, Dept Chem, Cleveland, OH 44115 USA
[2] Cleveland Clin Fdn, Lerner Res Inst, Dept Mol Cardiol, Cleveland, OH 44195 USA
关键词
D O I
10.1074/jbc.M306850200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690 699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC50 values of similar to12 and similar to10 muM, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K-i values of similar to6.3 and similar to5.3 muM, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp- Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC50 of 1.6 muM (with a K-D for prothrombin of 850 nM), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --> Lys, Tyr(696) --> Phe, Asp(697) --> Lys, and Tyr(698) --> Phe (factor V-2K2F) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa ( factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695 698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.
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页码:3084 / 3095
页数:12
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