Activation of human platelets considerably enhanced their ability to accelerate factor Va inactivation by activated protein C (APC). The anticoagulant activity of platelet suspensions was markedly dependent on the kind of agonist used to activate platelets. APC-catalyzed factor Va inactivation in free solution was characterized by an apparent second-order rate constant of 2 x 10(5) (mol/L)-1 (seconds)-1. Nonstimulated platelets (2.4 x 10(8)/mL) and platelets stimulated with adenosine diphosphate or adrenalin accelerated factor Va inactivation fourfold. Rates of factor Va inactivation were increased 11-fold by thrombin-stimulated platelets, 29-fold after platelet stimulation with the Ca(2+)-ionophore A23187. At low platelet concentrations (3 x 10(7)/mL) only background levels of anticoagulant activity were observed in platelet suspensions that were nonstimulated or stimulated with thrombin or collagen. However, when such reaction mixtures were stirred during the activation procedure, platelet anticoagulant activity was increased more than 10-fold. Independent of platelet stimulation and stirring conditions, exogenously added purified plasma protein S increased platelet-dependent factor Va inactivation approximately twofold. Addition of a neutralizing antiprotein S antibody had little effect on the anticoagulant activity of platelets. This indicates that, under the reaction conditions tested, platelet- released protein S did not contribute to factor Va inactivation. Approximately 25% of the anticoagulant activity of stimulated platelet suspensions appeared to be associated with microparticles that were released on platelet activation. Such microparticles may provide an important source of anticoagulant activity. A similar distribution of procoagulant, ie, prothrombinase, activity between platelets and microparticles was observed for the same platelet suspensions. Because platelet stimulation and stirring also had the same overall effects on the ability of platelets and platelet microparticles to promote prothrombin activation and factor Va inactivation, it appears likely that the generation of potential platelet anticoagulant and procoagulant activities is coupled to the same platelet stimulation reactions.

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The mechanism by which activated protein C stimulates fibrinolysis was studied in a simple radiolabeled clot lysis assay system containing purified tissue-type plasminogen activator, bovine endothelial plasminogen activator inhibitor (PAI), plasminogen, 125I-fibrinogen and thrombin. Fibrinolysis was greatly enhanced by the addition of purified bovine activated protein C; however, in the absence of PAI, activated protein C did not stimulate clot lysis, thus implicating this inhibitor in the mechanism. In clot lysis assay systems containing washed human platelets as a source of PAI, bovine-activated protein C-dependent fibrinolysis was associated with a marked decrease in PAI activity as detected using reverse fibrin autography. Bovine-activated protein C also decreased PAI activity of whole blood and of serum. In contrast to the bovine molecule, human-activated protein C was much less profibrinolytic in these clot lysis assay systems and much less potent in causing the neutralization of PAI. This species specificity of activated protein C in clot lysis assays reflect the known in vivo profibrinolytic species specificity. When purified bovine-activated protein C was mixed with purified PAI, complex formation was demonstrated using immunoblotting techniques after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These observations suggest that a major mechanism for bovine protein C- dependent fibrinolysis in in vitro clot lysis assays involves a direct neutralization of PAI by activated protein C.

Gel filtered human platelets contaminated with less than 0.02% of plasma protein S contained 490 ng of protein S antigen per 3 X 10(8) platelets, equivalent to 2.5% of protein S in whole blood. Three patients with heterozygous plasma protein S deficiency, a congenital disorder associated with venous thrombotic disease, had platelet protein S antigen levels that were 40% of the mean platelet level in ten normal volunteers. In immunoblotting analysis, platelet protein S was indistinguishable from plasma protein S. Thrombin stimulation of platelets caused release of 63% of total protein S antigen and this release was abolished when platelets were preincubated with metabolic inhibitors. Thrombin effected limited proteolysis of platelet protein S and this reaction was inhibited by calcium ions. Immunofluorescent staining of platelets using protein S antibodies demonstrated that protein S colocalized with fibrinogen, an established alpha-granule protein. Thus, human platelets contain protein S in alpha granules that can be released by thrombin stimulation. The released protein S may bind to stimulated platelets and thereby promote and localize the anticoagulant activity of activated protein C on the platelet surface.

A family with a history of severe recurrent venous thromboembolic disease was studied to determine if a plasma protein deficiency could account for observed disease. Protein S levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his mother, his aunt, and his cousin who were clinically affected had 17% to 65% of the control levels of protein S antigen (normal range, 71% to 147%). Since three of these patients were receiving oral anticoagulant therapy, the ratios of protein S to prothrombin, factor X, and protein C in these patients were compared with values for a group of orally anticoagulated controls. These results suggested that protein S is half-normal in all family members with thrombotic disease. Other proteins known to be associated with familial thrombotic disease, including antithrombin III, plasminogen, fibrinogen, and protein C, were normal. Because plasma protein S serves as a cofactor for the anticoagulant activity of activated protein C and because protein C deficiency is associated with recurrent thrombotic disease, it is suggested that recurrent thrombotic disease in this family is the result of an inherited deficiency of protein S.