CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin αIIbβ3 in platelets

Abstract

Second messenger-mediated inside-out activation of integrin ␣IIb␤3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent ␣IIb␤3-mediated aggregation of CalDAG-GEFI-deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for ␣IIb␤3 activation in platelets activated with a PAR4 receptorspecific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI-deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI-deficient platelets, however, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for ␣IIb␤3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the G␣i-coupled receptor for ADP, P2Y12. Independent roles for CalDAG-GEFI and PKC/G␣i signaling were also observed for PAR4p-induced activation of the small GTPase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and ␣IIb␤3 activation in mouse platelets activated through the PAR4 receptor. (Blood. 2008;112:1696-1703)

Integrins in immunity

“…In platelets, rapid chemokine induction of Rap1 occurs via CalDAG-GEF1, whereas more sustained activation is via PKC (Cifuni et al, 2008). There also seems to be fast versus slow routes to Rap1 activation in lymphocytes, as chemokines activate Rap1 within seconds, which lasts a few minutes, whereas a more sustained level of Rap1 activation occurs through the TCR (Katagiri et al, 2002;Shimonaka et al, 2003).…”

Protein Kinase C-θ Is Required for Murine Neutrophil Recruitment and Adhesion Strengthening under Flow

“…In other cell types, PKC isoforms have been shown to be involved in the activation of different integrins (16)(17)(18)(19). PKCs have been grouped into the conventional, novel, and atypical isoforms.…”

Ras guanyl nucleotide releasing protein 2 affects cell viability and cell–matrix adhesion in ECV304 endothelial cells

“…RasGRP2 has potential calcium-binding EF-hands that may be involved in Rap1 activation. 4,5 The increase in Rap1 activation in ECV304rasgrp2 cells was suppressed by BAPTA-AM pre-treatment. This suppression, albeit to a lesser extent, was also observed in ECVmock cells, which have low levels of endogenous RasGRP2 (Fig.…”

“…1,2 Because RasGRP2 cannot bind DAG, it fails to translocate to membranes in vivo in response to DAG analogs. 3 RasGRP2, also called CalDAG-GEFI, plays a role in controlling T-cell and platelet adhesion through Rap1 activation, 4,5 and is involved in integrin-independent neutrophil chemotaxis. 6 RasGRP2 has been reported to be associated with Huntington disease and immune-mediated thrombocytopenia and thrombosis.…”