Inactivation of membrane-bound human factor Va by APC.

(A) Factor Va was made by incubating purified normal plasma factor V (50 nM) with α-thrombin (1 nM). The reaction was stopped with hirudin (2 nM). Following addition of phospholipid vesicles (50 μM) and normal APC (2.5 nM) the activity of factor Va was assessed in a clotting assay using factor V–deficient plasma (▪) at 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 30 minutes following the addition of APC; (●) factor Va was incubated with the purified immunoglobulin inhibitor (64 nM) and the inactivation by APC was performed under similar experimental conditions as described for normal factor Va. (B) Some of the samples assayed for cofactor activity in panel A were also analyzed on a 5% to 15% linear gradient SDS-PAGE as described in “Patient, materials, and methods.” Lane 1, factor Va control, no APC; lanes 2 through 5, factor Va incubated with APC at 5, 10, 20, and 30 minutes, respectively. (C) Prior to the addition of APC and PCPS vesicles, factor Va was incubated with the purified immunoglobulin solution from the patient's plasma. The arrows indicate the heavy chain of factor Va (a), the M_r 75 000 intermediate (b), that results from cleavage at Arg506 of the heavy chain, and the M_r 30 000 fragment (c) that derives from cleavage of the M_r 75 000 intermediate at Arg306. The open arrowhead indicates cleavage at Arg306 first and generation of a fragment containing the region 307-6791709 of the heavy chain of the cofactor.