Reconstitution of membrane-associated Factor V-like activity (PF1) in human platelets isolated from severe F.V-deficient donors was assessed following incubations in citrated normal platelet free plasma. Coagulant activities were measured using: a one-stage prothrombin time, the Stypven time and a modified KAPTT in which the kaolin particles were removed from the plasma prior to recalcification. The supernatant from 3x frozen-and-thawed lysed normal platelets was used as a standard for 100% PF1 and 100% PF3. Normal platelets gel filtered or centrifuge-washed in the presence of adenosine, theophylline and PGEj had <1% available PFI and PF3, whereas platelets from severe F.V-deficient donors isolated by the same procedure had no PF1 and the same amount of PF3. The supernatant from 3x frozen-and-thawed lysed F.V-deficient platelets also had no PF1 but had 100% total PF3. When gel filtered, PFl-deficient platelets were incubated in normal plasma, they acquired about 1% PF1 which did not change following freezing-and-thawing. By contrast, PFI-deficent platelets washed in the absence of these inhibitors had 15-20 times more available PF3 and acquired about 15-20 times more total PFI after incubation in normal plasma. When either the crude frozen-and-thawed lysed membrane supernatant from these PFl-deficient platelets (100% available PF3) or a partially purified membrane-rich fraction from this supernatant was incubated with normal plasma, then 100% reconstitution of PFI was achieved. PF1 was also reconstituted in a purified system using all-or-none binding when the PF3-containing lipid-protein particles secreted by platelets, upon collagen induced aggregation, were incubated with purified human F.Va. Following incubation, these particles had the same amount of PFI as the PF3-containing particles from normal platelets. It is apparent that in human platelets a correlation exists between the amount of available PF3 and the capacity to reconstitute PFI.
