A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42 (fillapodia), Rac1 (lamellapodia) and RhoA (focal adhesions). 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 used an affinity binding technique followed by mass spectroscopy to identify novel Rho family binding GEFs in platelet lysates. Recombinant GST-Rho fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with clarified human platelet lysates. Platelet lysate proteins associated with the different GST-Rho preparations were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by Mass Spectroscopy. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we found Cool-1 and Cool-2, two closely related GEFs that regulate Rac1 and CDC42 activation in nucleated cells. Cool-1 has not been previously described in platelets. Homodimeric Cool-1 specifically activates CDC42, whereas Cool-2 is capable of activating Rac1 as a homodimer or CDC42 as a monomer. The substrate specificity of Cool-2 is regulated by complex interactions with p21-activated kinases (PAK) and Gb/g proteins. In nucleated cells, Cool-1 and Cool-2 are localized to focal adhesions by the GPCR-kinase-interacting proteins 1 and 2 (GIT1 and GIT2). GIT binding to the Cool proteins has been shown to inhibit their GEF activity. Here we show that Cool-1 is present only in the membrane fraction of resting platelet lysates while Cool-2 is present in both the membrane and cytoplasmic fractions. Upon activation with thrombin, about half the membrane bound Cool-1 and all the membrane bound Cool- 2 migrate to the cytoplasm. Precipitation experiments in thrombin activated platelets with anti-GIT1 and anti-GIT2 antibodies show GIT-1 avidly binds Cool-2 whereas GIT2 binds a small amount of Cool-1. These data suggest a complex mechanism of CDC42 and Rac1 activation in platelets by the Cool proteins. Based on our findings and the current understanding of the regulation of Cool proteins in nucleated cells, we hypothesize that early after thrombin activation, the CDC42 and Rac1 GEF activities of Cool-1 and Cool-2 result in fillapodia (CDC42) and lamellapodia (Rac1) formation. As integrant mediated focal adhesions are formed, the GEF activities of Cool-1 and Cool-2 are inhibited by the scaffolding proteins GIT2 and GIT1, respectively. In this manner, Rac1 and CDC42 mediated fillapodia and lamellapodia formation may be regulated by the extent of integrin engagement and platelet spreading.

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