Identification of the factor V molecule present in plasma.

Citrated plasma (100 μL) from a pool of normal plasma (A) and from patient I3 (Castoldi et al5, B) was diluted 10-fold in a buffer containing 5 mM CaCl₂ and treated with phospholipid vesicles and APC (5 nM) as described.2,3 Immunoreactive fragments were detected using the monoclonal antibody αHFVa_HC17 under nonreducing conditions.2 (A) Normal pool plasma; (B) plasma from patient I3 (Catoldi et al5). Lane 1, plasma immediately following clot formation and the addition of APC (5 nM, ∼20 sec); lanes 3-5 plasma at 5, 10, 20, and 30 min following the addition of APC. Roman numerals on the right indicate the following: (i) the heavy chain of factor Va following clot formation; (ii) the M_r 75 000 fragment deriving from cleavage of the heavy chain of factor Va at Arg506 (amino acid residues 1-506); (iii) the M_r 60 000 fragment that derives from factor Va following cleavage at Arg306 (amino acid residues 307-709); (iv) the M_r 30 000 fragment derived from normal factor Va following cleavage at Arg506 and Arg306 (amino acid residues 307-506). The position of the immunoglobulin G (IgG) molecules (nonreduced) is also shown.