Abstract
Tightly associated factors Va and Xa serve as the essential prothrombin-activating complex whose assembly is triggered by occupancy of phosphatidylserine (PS) regulatory sites on both proteins. Factor Va C2 domain contains a binding site for soluble, short chain PS (C6PS) that includes the indole moieties of Trp2063/Trp2064 at the apex of a loop (“spike-1”) (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent data show that there is a C6PS site in the factor Va2 C1 domain that serves as a regulatory site for assembly and/or activity of the FVa2-FXa complex (Majumder R, Quinn-Allen MA, Kane WH & Lentz BR. Manuscript in Preparation). This C6PS-binding site also involves aromatic and hydrophobic residues (Tyr1956/Tyr1957) located in a homologous loop whose apex is termed “spike 3”. In order to identify the amino acid residues in the C1 domain that contribute to the PS-mediated cofactor activity of factor Va2, charged and hydrophobic residues predicted to be exposed in FVa2-C1 domain were mutated to alanine in clusters of 1–3 mutations per construct. The resultant 20 mutants (R1880A, D1892A, (K1896,E1899)A, (F1900,L1901,Y1903)A, (E1905,R1907)A, Y1917A, (E1923,K1924)A, (K1941,E1942)A, (K1954,H1955)A, (Y1956,L1957)A, Y1956A, L1957A, K1958A, E1964A, K1980A, D1995A, R2019A, (R2023,R2027)A, R2023A, R2027A,) and factor V wild type were expressed in Cos-7 cells followed by activation with thrombin, partial purification and concentration using HiTrap SP HP columns. The specific activities of all factor Va mutants were greater than 70% of wild type, with concentrations in the 1.5-7μM range. Recently it has been shown that two mutants (Y1956, L1957)A and (R2023,R2027)A showed decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS (Saleh M, Peng W, Quinn-Allen MA, Macedo-Ribeiro S, Fuentes-Prior P, Bode W & Kane WH. Thromb. Haemost.2004, 91:16–27). Here we report the rate of prothrombin activation in the presence of 1 nM factor Xa, 5 nM factorVa2 (mutants and wild type) and 400 mM C6PS. Enhancement of cofactor activity (E) of factor Va-C1 wild type and mutants by C6PS was measured using the following equation ( Zhai X, Srivastava A, Drummond DC, Daleke D and Lentz BR. Biochemistry. 2002, 41: 5675–84):
\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[E=\ (r_{Xa.Va.PL}/r_{Xa.PL})/(r_{Xa.Va}/r_{Xa})\] \end{document}
Here, rXa·Va·PL is the rate of prothrombin activation measured as the initial slope of the rate of change of normalized DAPA fluorescence with time by enzyme in the presence of factor Va and lipid, and other terms are defined analogously. The cofactor activities of (Y1956, L1957) A, Y1956A and L1957A were drastically reduced (values are 1.1, 4.2 and 5.1 respectively) relative to the cofactor activity of the wild type factor Va2 (15). The cofactor activities of (R2023, R2027) A, E1964A and (K1954, H1955) A were also reduced but to a lesser extent (values are 8, 10.6 and 12 respectively). We plan to monitor the binding of these mutants to C6PS and to factor Xa in the presence of C6PS in order to determine the role of these mutations on the assembly and activity of prothrombinase. Supported by grants from the NHLBI (HL43106 to W. Kane and HL 072827 to BRL).
The Primary Structure of Antithrombin-III
