PLATELET-DERIVED MICROVESICLES AND ACTIVATED PLATELETS EXPRESS FACTOR XA ACTIVITY

Activated platelets and platelet-derived microvesicles demonstrate procoagulant properties. It is known that following stimulation, negatively charged phospholipids and factor Va become located on their surfaces. The aim of this study was to see whether activated platelets and platelet-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 in 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 thrombin receptor agonist peptide SFLLRN. Microvesicles and activated platelets were separated by centrifugation. Flow cytometry using a monoclonal antibody against factor Xa demonstrated the presence of factor Xa on the surface of the activated platelets. In addition, platelet-derived microvesicles and activated platelets demonstrated factor Xa activity on their surfaces detected directly by splitting of the chromogenic 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 inhibiting at the factor Xa level, as well as by leupeptin which also inhibited the thrombin-chromogenic substrate interaction as such. The microvesicles and the activated remnant platelets also gave a significantly shortened clotting time in a factor X deficient plasma without any other initiators. These data demonstrate that stimulated platelets and platelet-derived microvesicles express some membrane bound factor Xa activity.