Cooperative Regulation of the Activity of Factor Xa within Prothrombinase by Discrete Amino Acid Regions from Factor Va Heavy Chain

Abstract

The prothrombinase complex catalyzes the activation of prothrombin to α-thrombin. We have repetitively shown that amino acid region 695DYDY698 from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. We have also recently demonstrated that amino acid region 334DY335 is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334–335 and 695−698 as follows: factor V3K (334DY335 → KF and 695DYDY698 → KFKF), factor VKF/4A (334DY335 → KF and 695DYDY698 → AAAA), and factor V6A (334DY335 → AA and 695DYDY698 → AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va3K, factor VaK4/4A, and factor Va6A had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor VaWt) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va3K had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor VaWt, while prothrombinase assembled with saturating concentrations of factor VaKF/4A and factor Va6A had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334–335 together with amino acid region 695−698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa.

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

“…The data presented herein demonstrate for the first time that the basic amino acid region composed of residues 1000–1008 and located in the middle portion of the B domain of fV is a dynamic regulator for the binding of the active cofactor to fXa within prothrombinase. We have shown that three out of the six amino acid residues from the heavy chain of fVa that are involved in fXa binding are acidic in nature ( 17 , 18 , 22 , 34 , 51 ). Our previous work with recombinant fVa molecules containing specific point mutations of these amino acids demonstrates a significant decrease in affinity for fXa when the residues were mutated two by two and a dramatic decrease in affinity (50-fold) when four amino acids were mutated simultaneously ( 17 , 18 ).…”

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

“…To quantify the interaction between the two sets of double mutations (S478A/L480A and S478A/Q481A) and to confirm their apparent synergistic detrimental effect on prothrombinase function for activation of rFII SLQ3AAA , we have further calculated the difference in free energy of the transition state analog (⌬⌬G int ) for the triple mutant as described previously by our laboratory (75,76). The large positive value of ⌬⌬G int (ϩ2.4 kcal/mol) for the combination of the mutations at Leu 480 and Gln 481 together with the sizable 55-fold decrease in the secondorder rate constant of prothrombinase for rFII SLQ3AAA activation signify that there is a deficiency in recognition between prothrombinase and rFII SLQ3AAA .…”