Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cγ2 regulation in platelets

Abstract

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…GPVI recognises collagen as a dimer, and a proportion of GPVI exists as a dimer on resting platelets, with dimer levels increasing upon platelet activation [10, 11]. GPVI has been reported to be lipid raft-associated [12–14] and more recently to be a component of a distinct type of microdomain formed by tetraspanin transmembrane proteins [15]. However, it is currently unknown how GPVI lateral mobility in the plasma membrane and compartmentalisation into membrane microdomains regulates its activation.…”

Rho and Rho-kinase Mediate Thrombin-induced Phosphatidylinositol 4-Phosphate 5-Kinase Trafficking in Platelets

“…Lipid rafts are found in platelets (36,37), although their functional role is just beginning to be elucidated (18,38). Rozelle et al (18) have shown that lipid rafts in Ref52 cells are the preferred platforms for membrane-linked actin polymerization mediated by in situ PIP 2 synthesis.…”

“…3A) (36, 39). The soluble fractions containing nonraft-associated membrane proteins and cytosolic proteins were identified by blotting for cytosolic p42/44 ERK (extracellular signal-regulated kinase) (not shown) (38). In the cell fractions derived from either resting or TRAP-stimulated platelets, we tested for PIP5K.…”

“…Briefly, PIP5K activity was assayed in 50-l reactions containing 50 mM Tris, pH 7.5, 0.1 mM NaCl, 10 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 142 g/ml lipid substrate, 4.2 mM ATP, and [␥- Lipid Raft Preparation-Lipid rafts were isolated by density through a step gradient of 0 -40% (w/v) sucrose as described previously (37)(38)(39). Briefly, resting or stimulated platelets (2 ϫ 10 8 , in 100 l of HEPESTyrode's buffer) were lysed in 200 l of ice-cold buffer (20 mM Tris, 150 mM NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, 1 g/ml pepstatin A, pH 8.3) containing 1% Brij 58.…”

The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLCγ2 signalling cascade

“…In addition, and with respect to the marked phosphorylation of LAT determined in Pam3CSK4-treated platelets, it has been shown that LAT is not involved in  IIa  3 outside-in signalling mediated PLC2 activation and Ca 2+ -mobilization [42].…”