Identification of the epitope(s) of the inhibitor.

Purified human factor V (600 nM) was incubated with 15 nM thrombin for 10 minutes at 37°C in TBS, 5 mM CaCl₂, followed by the addition of 30 nM hirudin and treated with APC and PCPS. At the end of the incubation the samples were mixed with the total immunoglobulin fraction isolated from patient plasma. Immunoreactive fragments were precipitated with a solution containing protein G–Sepharose. Elution of fragments from the protein G–Sepharose beads was accomplished with a solution of 10% SDS and analysis of the fragments was performed on a 5% to 15% SDS-PAGE. Following transfer to nitrocellulose immunoreactive fragments were revealed with anti-factor V antibodies. (A) Staining with αHFVa_HC#17, which recognizes an epitope on the heavy chain of factor Va between amino acid residues 307-506. (B) Staining with αHFVa_LC#9, which recognizes an epitope on the light chain of the cofactor. (C) Staining with a polyclonal antihuman factor V; the antibody is directed against the whole factor V molecule and has epitopes on both chains of the cofactor as well as on the B region of the molecule. Lane 1, factor Va; lane 2, factor Va in the presence of EDTA prior to immunoprecipitation; lane 3, factor Va with APC; lane 4, factor Va with APC in the presence of EDTA prior to immunoprecipitation; lane 5, factor Va in the presence of APC and PCPS; lane 6, factor Va with APC and PCPS in the presence of EDTA prior to immunoprecipitation.