Amino Acid Region 1000-1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

作者：Wiencek Joesph R; Na Mahesheema; Hirbawi Jamila; Kalafatis Michael^*

来源：Journal of Biological Chemistry, 2013, 288(52): 37026-37038.

DOI：10.1074/jbc.M113.462374

• Coagulation Factors
• Phospholipid Vesicle
• Protein Chemistry
• Prothrombin
• Thrombin
• Factor V
• Factor Va
• Factor Xa
• Prothrombinase
• Prothrombin

Single chain factor V (fV) circulates as an M-r 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(B9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(B9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, K-D(app) values for fV(B9) (0.9 nm), fVa(B9) (0.4 nm), and fV(B9/Q3) (0.7 nm) were similar to the affinity of fVa(WT) for fXa (0.3 nm). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(B9/Q3) had clotting activity comparable with fVa(WT). The k(cat) value of prothrombinase assembled with fV(B9/Q3) was minimally affected, whereas the K-m value of the reaction was increased 57-fold compared with the K-m value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

• 出版日期2013-12-27