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).