Abstract

A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases whose members include Rac, CDC42 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-nucleotide 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 spectrometry analysis to identify novel Rac binding GEFs from platelet lysates. Recombinant GST-Rac fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with human platelet lysates. Platelet lysate proteins associated with the different GST-Rac preparations (GTP-bound, GDP-bound or nucleotide-free) were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by mass spectrometry. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we identified three novel Rac-associated platelet GEFs including P-REX1, a G-βγ protein and phosphatidylinositol (3,4,5)-triphosphate regulated GEF previously found in neutrophils and neurons (

Welch, H.C.E et al.,
Cell
108
;
809
–822,
2002
). PREX-1 is a 196kDa peptide composed of 1659 amino that specifically activates Rac in neutrophils. In addition to the DH-PH exchange domain, PREX-1 also contains tandem PDZ and DEP domains as well as significant similarity over its C-terminal half to Inositol Polyphosphate 4-Phosphatase. Western analysis with anti-P-REX1 antibody (gift of H.C. Welch, Barbraham Institute, U.K.) showed that PREX-1 is present in purified whole platelet lysates. We have also shown that platelet PREX-1 specifically associates with GST-Rac by affinity pull-down experiments. Functional studies to characterize the exchange activity of platelet PREX-1 are ongoing. PREX-1 is the only known GEF that is directly regulated by G-βγ protein (
Hill, K. et al.,
J Biol Chem
280
(6):
4166
–4173,
2005
) and represents a novel pathway for platelet G protein coupled receptor signaling through Rac in human platelets.

Author notes

Disclosure: No relevant conflicts of interest to declare.