PLATELET-DERIVED MICROVESICLES AND ACTIVATED PLATELETS EXPRESS FACTORXA ACTIVITY

Activated platelets and platelet-derived microvesicles demonstrate pro coagulant properties. It is known that following stimulation, negative ly charged phospholipids and factor Va become located on their surface s. The aim of this study was to see whether activated platelets and pl atelet-derived microvesicles also expressed some factor Xa activity on their surfaces in a system where factor Xa did not come from external sources. In order to study this question, flow cytometry, as well as the use of a chromogenic substrate to factor Xa and a clotting assay i n a factor X depleted plasma, were applied. A prothrombinase assay was also applied using prothrombin, CaCl2 and a chromogenic substrate to thrombin. The platelets were gel-filtered or washed, suspended in Tris -buffered saline, and activated by calcium ionophore A23187 or the thr ombin receptor agonist peptide SFLLRN. Microvesicles and activated pla telets were separated by centrifugation. Flow cytometry using a monocl onal antibody against factor Xa demonstrated the presence of factor Xa on the surface of the activated platelets. In addition, platelet-deri ved microvesicles and activated platelets demonstrated factor Xa activ ity on their surfaces detected directly by splitting of the chromogeni c substrate to factor Xa, or by the prothrombinase assay. The thrombin generation in the last assay could be inhibited by a selective factor Xa inhibitor (recombinant tick anticoagulant peptide (rTAP)), soybean trypsin inhibitor, and antithrombin III plus LMW-heparin, all inhibit ing at the factor Xa level, as well as by leupeptin which also inhibit ed the thrombin-chromogenic substrate interaction as such. The microve sicles and the activated remnant platelets also gave a significantly s hortened clotting time in a factor X deficient plasma without any othe r initiators. These data demonstrate that stimulated platelets and pla telet-derived microvesicles express some membrane bound factor Xa acti vity.