Abstract 25: Platelet Activation by GPVI, GPIb-V-IX and aIIbß3 Requires PLC?2 but Not PLC?1

Abstract

Receptor-mediated cellular activation requires phospholipase C (PLC) activity to generate second messengers that elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are grouped into structurally distinct β, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2), which are critical for cellular activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2; however, their relative roles in platelet activation are poorly understood. Receptors that use PLCγ activity to activate platelets include the glycoprotein (GP) VI collagen receptor, GPIb-V-IX receptor for von Willebrand Factor (VWF), and integrin αIIbβ3 receptor for fibrinogen (Fg). While PLCγ2 is critical for platelet responses to GPVI-specific stimuli and in VWF-dependent thrombus formation, its role in αIIbβ3-dependent responses is controversial, suggesting that PLCγ1 may compensate for PLCγ2, when absent, in αIIbβ3-mediated outside-in signaling. Assessment of the role of PLCγ1 in platelet activation is hindered by the early embryonic lethality of PLCγ1-deficient (PLCγ1-/-) mice. We obtained platelets from newly-generated conditionally PLCγ1-/- mice and compared their responses to αIIbβ3-, GPVI-, and GPIb-V-IX-specific stimuli with those of platelets from wild-type (WT), PLCγ2-/-, and PLCγ1-/-/PLCγ2-/- mice. We found that platelet spreading on immobilized Fg, collagen-induced aggregation, and VWF-dependent thrombus formation are severely impaired in PLCγ2-/- or PLCγ1-/-/PLCγ2-/- mice. Defective spreading on immobilized Fg was overcome by addition of exogenous ADP, suggesting that PLCγ2 is required for granule secretion in response to αIIbβ3 ligation. Consistent with this hypothesis, αIIbβ3-mediated granule release was impaired in the absence of PLCγ2. In contrast, platelets from PLCγ1-/- mice spread and released granule contents normally on Fg, exhibited near normal levels of GPVI-dependent platelet aggregation, and formed near normal thrombi over immobilized collagen/VWF. These results establish that PLCγ2 is indispensable for platelet secretion downstream of adhesive interactions mediated by GPVI, GPIb-V-IX, and αIIbβ3, and demonstrate that PLCγ1 plays little role in these processes.