Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization

Abstract

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca 2؉ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP 3 ) and that treating platelets with the IP 3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca 2؉ flux and platelet shape change. Normal shape change was observed in G␣ q ؊/؊ mouse platelets, excluding a role for PLC␤ isoforms in this process. However, decreased shape change and Ca 2؉ mobilization were observed in mice lacking PLC␥2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca 2؉ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca 2؉ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-Fc␥RIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcR␥ chain. Taken together, these studies define an important role for PLC␥2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.

PLCγ1 is essential for early events in integrin signalling required for cell motility

“…4) was also suggested by other studies using an inhibitor of PI-PLC activity (U73122) or platelets deficient in the PLCγ2 isoform (Inoue et al, 2003;Mangin et al, 2003;Nakamura et al, 2001;Wonerow et al, 2003). The resulting cell phenotype was similar to that observed for deficiency in Src kinases in both cases, although it was more pronounced with U73122 than with disruption of PLCγ2 (Mangin et al, 2003;Wonerow et al, 2003).…”

“…The inhibition of Src kinase activity in platelets also resulted in reduced cell spreading and loss of PLCγ2 phosphorylation following the engagement of von Willebrand factor or α2β1 and αIIbβ3 integrins (Inoue et al, 2003;Mangin et al, 2003;Wonerow et al, 2003).…”

Phospholipase Cγ2 contributes to stable thrombus formation on VWF

“…PLCc2-deficient murine platelets adhere to, but spread poorly on, immobilized VWF Adhesive interactions between human GPIb (huGPIb) and huVWF can be examined under static conditions by ''activating'' VWF immobilized on glass or plastic surfaces with conformational modifiers such as ristocetin or botrocetin. Previous studies [16,21] have shown that botrocetin is also able to support the binding of huVWF to murine platelets, resulting in shape change and aggregation. As shown in Fig.…”

“…Consistent with this notion, we found that pharmacological inhibition of total PLC activity by U73122, a PLC antagonist [24] while not affecting stable adhesion, completely abolished thrombus formation by human platelets on VWF (data not shown). Since PLCb isoforms do not appear to be involved in GPIb-mediated platelet activation [16], it is likely that PLCc1, though expressed at much lower levels than is PLCc2 [12], may compensate for the absence of PLCc2 in supporting thrombus formation on VWF. In fact, PLCc1 has recently been shown to function downstream of GPVI following platelet exposure to collagen [12].…”

Bruton tyrosine kinase is essential for botrocetin/VWF-induced signaling and GPIb-dependent thrombus formation in vivo

“…2,8 Similarly, shear stress-independent, adhesiondependent GPIb-induced activation of ␣IIb␤3 apparently requires mobilization of internal stores but not Ca 2ϩ influx, ADP, or TxA2. 8,9 Despite these differences, GPIb-mediated signaling has been found to be PI3K 8,[10][11][12][13][14] and PLC␥2 dependent [14][15][16] in every system tested for these requirements. Because of the functional relationships between PI3K and Bruton tyrosine kinase (Btk), [17][18][19] Btk and PLC␥2 [20][21][22][23] activation, and the documented role of Btk in collagen-induced signaling in platelets, 19,24 we investigated the role(s) of Btk in TxA2 production induced by signaling elicited by bt/VWF/GPIb-mediated agglutination.…”