Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay

Abstract

Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC-resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC-resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC.

An Antifibrinolytic Mechanism Describing the Prothrombotic Effect Associated with Factor VLeiden

“…The samples were then diluted in sample preparation buffer (1% sodium dodecyl sulfate (SDS), 1% 2-mercaptoethanol, and 10% glycerol with bromphenol blue in 0.2 M Tris, pH 8.0) and subjected to SDS-PAGE using a 5-15% gradient gel (12). Following transfer to nitrocellulose (13), cleavage products of factor V were detected with MoAb FVa HC #6 (6,14), which recognizes an epitope on factor V between amino acid residues 307 and 506, and visualized using chemiluminescence (Chemiluminescence Detection Kit; DuPont NEN).…”

Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT 20210G/A) affecting the prothrombinase complex in a thrombophilic family

“…This doubly heterozygous condition offers the opportunity to evaluate the protein expression of the FV Y1702C allele, because plasmatic FV bearing the R506Q substitution is clearly recognizable on an immunoblot by its characteristic APC-mediated inactivation pattern. 40 The time course of APCmediated FVa inactivation in vitro showed a pattern indistinguishable from that of a FV R506Q homozygote ( Figure 4A), providing direct evidence for the impaired secretion of FV bearing the Y1702C substitution. However, to further investigate the possibility that FV molecules encoded by the FV Y1702C allele contribute to the abnormal FVa inactivation pattern observed in the propositus, family members who carry the FV Y1702C mutation as a single defect (II2, II5, and III1) were also studied as controls ( Figure 4B).…”

The contribution of amino acid residues 1508–1515 of factor V to light chain generation

“…The exact concentration of all mutants was determined by absorption at 280 n m and by ELISA as described [19]. The integrity of the molecules was verified by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) followed by immunoblotting with specific monoclonal antibodies against the heavy and light chains of FV (monoclonal antibodies αHFVa HC #17, which recognize an epitope on the heavy chain of the cofactor between amino acid residues 307 and 506, and αHFVa HC #9, which recognizes the light chain of FVa, provided by Dr Kenneth Mann, Department of Biochemistry, University of Vermont, Burlington, VT, USA) as described [5,20].…”