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. Disclosures: No relevant conflicts of interest to declare.

Complex and Novel Associations of the Guanine Exchange Factors Cool- 1 and Cool-2 with the Scaffolding Proteins GIT1 and GIT2 in Human Platelets

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.

The ArfGAP Glo3 Is Required for the Generation of COPI Vesicles

The small GTPase Arf and coatomer (COPI) are required for the generation of retrograde transport vesicles. Arf activity is regulated by guanine exchange factors (ArfGEF) and GTPase-activating proteins (ArfGAPs). The ArfGAPs Gcs1 and Glo3 provide essential overlapping function for retrograde vesicular transport from the Golgi to the endoplasmic reticulum. We have identified Glo3 as a component of COPI vesicles. Furthermore, we find that a mutant version of the Glo3 protein exerts a negative effect on retrograde transport, even in the presence of the ArfGAP Gcs1. Finally, we present evidence supporting a role for ArfGAP protein in the generation of COPI retrograde transport vesicles.