Factor XIII Associates with the Guanine Exchange Factors Cool-1 and Cool-2 in Human Platelets.

Abstract 4015

Poster Board III-951

A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42, Rac1 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We previously identified the presence of two homologous GEFs, Cool-1 and Cool-2, in human platelets. In nucleated cells, the activities and substrate specificities of the Cool GEFs are regulated by complex interactions with p21-activated kinases (PAK), Gb/g heterodimers, focal adhesion kinase, and the scaffolding proteins GIT-1 and GIT-2. The Cool GEFs are found at the sites of focal adhesions during spreading and migration in nucleated cells, however, little is known about the regulation or activity of Cool-1 or Cool-2 in platelets.

Co-immunoprecipitation experiments were performed with Cool-1 and Cool-2 antibodies using lysates from resting and thrombin stimulated human platelets. We analyzed the precipitated proteins by Mass Spectroscopy and found a number of structural and scaffold proteins bound to the Cool GEFs in the thrombin activated lysates. These cytoskeletal proteins include talin, multimerin, tubulin, filamin A, actin and fibrinogen. Interestingly, a large number of Factor XIII also co-immunoprecipitated with Cool-1 and Cool-2 in the thrombin activated platelet lysates. Western analysis of the co-immunoprecipated platelet proteins from thrombin, TRAP1 and TRAP4 demonstrated that the association of factor XIII with the Cool GEFs is calcium dependent. Inhibition of transglutaminase activity and presence of RGD peptide did not affect the association of Factor XIII with Cool-1 or Cool-2.

Factor XIII deficiency is commonly thought to result in bleeding due to the impaired crosslinking of fibrin. However, platelet factor XIII may also play an important role in cross linking platelet cytoskeleletal proteins such as actin and myosin. Consistent with that hypothesis is a recent report showing that platelet factor XIII deficiency results in decreased lamellapodia formation under static adhesive conditions. The association of Factor XIII with the Cool GEFs further supports investigating the potential for the transglutaminase to crosslink platelet cytoskeletal proteins.