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Platelet factor XIII contributes to the hemostatic process : factor XIII interactions at the platelet surface and with the internal platelet skeleton Serrano, Katherine

Abstract

During blood clot formation, the transglutaminase, FXIII, functions to chemically crosslink fibrin and α[sub 2]-antiplasmin to fibrin polymers. This strengthens and stabilizes the clot. Activated platelets accelerate these crosslinking reactions via the presentation of surfacebound active FXIII. The platelet cytoplasm contains approximately half of the FXIII content circulating in blood, yet the function of cytoplasmic FXIII is poorly understood at present. This thesis investigated the function of platelet FXIII in processes both outside and inside the platelet by studying the interactions of FXIII with fibrin(ogen) and with platelet cytoskeletal proteins. Surface associations determined using specific antibodies and flow cytometry indicate that FXIIIa association with platelets, occurred at early stages of activation, was partially reversible, correlated significantly with fibrinogen expression, and was enhanced after alkylation of the active-site cysteine. Immunoblotting experiments indicate that surface-associated fibrin(ogen) was crosslinked but not directly to the platelet surface. In aggregometry experiments, FXIIIa slightly enhanced the rate although not the final amount of aggregation. FXIII was present in the cytoskeletal fractions of the platelet lysate separated with centrifugation. When these fractions were immobilized on nitrocellulose membranes, activated but not resting FXIII bound to cytoskeletal proteins. With fluorescence microscopy, an actin polymerization-dependent, transient translocation of FXIII from a diffuse homogeneous distribution throughout the cytoplasm to the platelet periphery was observed upon platelet activation suggesting an association with cytoskeletal proteins. There was also a measurable increase in the transglutaminase activity o f the cytoskeletal fractions of activated platelets. Immunoblotting analysis of such cytoskeletal fractions identified that the focal adhesion proteins, filamin and vinculin were crosslinked upon platelet activation. In summary, this study has demonstrated several new factors that affect the interactions of FXIII on the platelet surface, which could affect the promotion of fibrin polymer crosslinking by platelets. In addition to its role on the outside of platelets in stabilizing the fibrin clot, a role for FXIII on the inside of platelets in stabilizing focal adhesions can now be proposed. It appears that FXIII may enable clot retraction by strengthening structural elements required for such a process on both sides of the platelet plasma membrane.

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