Qualitative description of factors involved in the retraction and lysis of dilute whole blood clots and in the aggregation and retraction of platelets.

Abstract

Dilute whole blood clots were prepared by addition of thrombin to blood diluted 1:10 in phosphate buffer. The pH of this buffer was 7.4 and the ionic strength was 0.084. Though the ionic strength was low, there was no hemolysis of red corpuscles due to the contribution to the osmotic gradient by plasma salts and proteins. In the standard assay the clot was formed by addition of thrombin at 4 degrees C then incubated at 37 degrees C. Retraction and lysis of these clots were inhibited by removal of platelets and by increasing concentrations of purified thrombin. Retraction and lysis were also inhibited by inactivation of any one of the following factors: gammaM globulin, complement components C4 and 3, and (in the case of lysis) plasminogen. Evidence that some of the above serum factors were adsorbed to the platelet membrane was obtained by aggregation of washed platelets by antisera to these factors (i.e. fibrinogen, gammaM, and C4 or C3). These platelets were not aggregated by antisera to other serum proteins (by albumin, transferrin, gammaG globulin). These and other studies suggested that platelets, thrombin, fibrinogen, gammaM globulin (cold agglutinin), complement components, and plasminogen influenced and facilitated retraction and lysis of clots. These studies also suggested that platelets and some of these factors were physically associated. Because of this physical association, and because of the fact that clot retraction is associated with aggregation and retraction of platelets, we extended the above observations to include a study of the effect of these same serum factors on serum-induced aggregation and retraction of washed platelets. (Other terms which have been in use in the past to describe serum-induced platelet aggregation and retraction have included those such as platelet "fusion" and "viscous metamorphosis," neither of which fully described the phenomena.)Platelet aggregation and retraction induced by serum was markedly accelerated by addition of increasing concentrations of thrombin and (or) cold agglutinin. Hirudin and antisera to gammaM globulin inhibited seruminduced aggregation and retraction of platelets. Reconstitution of inactivated serum with purified C4, 3, and 5 and thrombin restored its capacity to induce aggregation and retraction of platelets.Therefore, we postulated that platelet aggregation and retraction were necessary for clot retraction and that platelet aggregation and clot retraction facilitated clot lysis. More specifically we postulated that thrombin, in addition to catalyzing clot formation, also modified the platelet membrane such that gammaM globulin (cold agglutinin) and complement components can act on the platelet membrane leading to (a) aggregation and retraction of the platelets, (b) retraction of the clot, and (c) to the activation of plasminogen either on the surface of the platelet by C8i and (or) by release of platelet activators of plasminogen.