Association of coagulation factor V with the platelet cytoskeleton.

Abstract

Triton-insoluble cytoskeletons from thrombin-activated platelets have been shown to contain Factor Va.This conclusion is based on the following evidence (a) a monoclonal antibody to Factor V inhibits the cytoskeleton's ability to potentiate the Factor Xa-catalyzed activation of prothrombin and the ability of cytoskeletons to correct the clotting defect of Factor V-deficient plasma; (b) the properties of cytoskeletal-associated Factor Va and purified Factor Va are similar, for example, both factors are inhibited by EDTA and heat but not by proteolytic enzyme inhibitors such as DFP; (c) cytoskeletal Factor V is most likely in the activated form, Factor Va, since further treatment of the washed cytoskeletons with thrombin does not increase its Factor V coagulant activity.Furthermore, cytoskeletons bind Factor Xa with a dissociation constant of 10.4 X 10"' M, a value more consistent for platelet Factor Va than for Factor V. Finally, it is postulated that Factor V arises on the platelet cytoskeleton from a site on the surface of the platelet because: (a) the time course for cytoskeleton formation is identical with the time course for appearance of Factor Va coagulant activity in activated platelet suspensions; (6) cytoskeletons prepared from platelets inhibited from secreting Factor V and ['4C]serotonin with metabolic inhibitors (antimycin A (9 m), 2-deoxyglucose (30 m ~) , and gluconolactone (40 m ~) )contained no Factor V activity; (c) whole thrombin-activated platelets and their corresponding cytoskeletons bind Factor Xa to the same extent (equal number of binding sites and same dissociation constant); and (d) Factor V-deficient platelets regained Factor Va on their cytoskeletons only when Factor V was added to the whole platelet suspension prior to thrombin activation.Platelets, like most eukaryotic cells, contain a complex network of internal fibrils upon which rests the plasma membrane (1).This internal network in various cells differs and is composed of distinct classes of fibrils which include thin, intermediate, and thick filaments (2).The term "skeleton" was applied to this internal fibrillar network by Yu et al.(3) in their study of red cells and later the term "cytoskeleton" was adopted by Brown et al. (4) to describe a similar structure in cultured embryo fibroblasts.The total cytoskeleton of most