Frederick A. Dombrose

Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3.

The relationship between the appearance of membrane-associated factor V-like activity (platelet factor 1, PF1) and phospholipid-like catalytic activity (platelet factor 3, PF3) has been examined, in vitro, in collagen-stimulated, human platelets. Both activities increased 7 fold upon collagen treatment relative to stirred controls. After sedimentation of stimulated platelets, 31% of total PF1 and 41% of PF3 remained in the supernatant fraction. PF1 eluted from a Sepharose CL-4B column in the same void volume fractions as PF3, phospholipid, and vesicular particles. These fractions had roughly 100 fold (lipid basis) or 1000 fold (protein basis) enhanced specific activity when compared to the stimulated platelet suspension. Freeze-fracture electron microscopy demonstrated that these void volume fractions contain two populations of membranous vesicles (80-200 nm and 400-600 nm in diameter). Upon centrifugation of the void volume fractions, PF1 and PF3 activities, phosphate-containing material, and ultraviolet-absorbing material all sedimented at the same rate, indicating that PF1 and PF3 are activities associated with one or both of the platelet-derived vesicle populations. Finally, we examined the effects of inhibitors on the appearance of PF1, PF3, platelet factor 4, total intrinsic factor V activity, and serotonin as well as on platelet aggregation. These studies suggest that the collagen-stimulated release of PF1 and PF3 is not coupled to either platelet aggregation or PF4 release but is probably a separate phase of the release reaction.

Association of factor V activity with membranous vesicles released from human platelets: Requirement for platelet stimulation

The membrane-associated factor V-like activity (platelet factor 1, PF1) and the phospholipid-like catalytic surface activity (platelet factor 3, PF3) were studied in human platelets from normal and two factor V-deficient donors. Collagen stimulation or mechanical disruption of gel-filtered platelets was necessary for the expression of significant amounts of PF1 and PF3. Stimulation was also necessary for the uptake of factor V or Va by PF1-deficient platelets from the factor V-deficient donors. The activity of PF1 was also generated by association of factor V or Va with membrane-rich fractions obtained by gel filtration of the supernatant from collagen-stimulated or frozen-thawed PF1-deficient platelets. The amount of PF1 obtained by such all-or-none binding experiments was directly proportional to the amount of PF3 already expressed in the platelet preparation. These data have been summarized in terms of a hypothesis which views PF1 and PF3 to be activities associated with membranous vesicles released from platelets only after stimulation.

Differentiation of factor V-like coagulant activity from catalytic phospholipid-like surface activity in membrane fractions derived from human platelets

Abstract To investigate the role of the platelet membrane as a catalytic surface in thrombin generation, a relatively physiologic clotting assay was devised that was based on the kaolin-activated partial thromboplastin time. Frozen-and-thawed lysed platelets from normal and Factor V-deficient donors were used as a model for maximally activated normal or V-deficient platelets. An aqueous dispersion of brain phospholipids was employed as an analogue of the lipid portion of a platelet membrane. This test system made it possible to distinguish quantitatively between the contributions of an intrinsic platelet-associated Factor V-like activity (platelet factor 1 or PF1) and phospholipid-like catalytic surface activity (platelet factor 3 or PF3). The degree to which a particular membrane preparation shortened the clotting time was related to its overall catalytic surface activity which was the sum of the contributions of PF1 and PF3. In platelet preparations, both PF1 and PF3 were associated with a membrane-rich fraction that was not easily sedimentable at 12,000g-min. The log-log slope obtained for dilutions of a membrane preparation was taken as catalytic surface effectiveness and was dependent upon the parametric level of Factor V in the test system. The observed dependence was different for each membrane preparation. Effectiveness appeared to be a combined function of both the availability and occupancy of Factor V binding sites as well as the specific affinity of such sites for Factor V(Va).