Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y 12 receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor LY294002 and PI3K␤ inhibitor TGX-221 indicating an essential role of PI3K␤ in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca 2؉ mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3K␣ inhibitor PIK75, but were not affected by PI3K␥ inhibitor AS252424 and PI3K␦ inhibitor IC87114. Consistently, GPVI-induced integrin ␣ IIb ␤ 3 activation of PI3K␥ ؊/؊ and PI3K␦ ؊/؊ platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on G i stimulation through P2Y 12 receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3K␤ plays an essential role in GPVI-mediated platelet aggregation and Akt activation.Glycoprotein VI (GPVI) 2 is a platelet collagen receptor that is constitutively associated with Fc receptor-␥ chain (1-4). Fc receptor-␥ chain is phosphorylated by Src family kinase on tyrosine residue of its immunoreceptor tyrosine-based activation motif upon collagen ligation to GPVI and the tyrosine kinase Syk (spleen tyrosine kinase) binds to the immunoreceptor tyrosine-based activation motif and becomes autophosphorylated (5-10). Tyrosine phosphorylation of Syk leads to phosphorylation of several adaptor proteins such as linker for T-cell activation and Src homology 2-containing leukocyte protein 76, recruitment of Bruton tyrosine kinase, and activation of phosphoinositide 3-kinase (PI3K) (11)(12)(13)(14)(15). This phosphorylation process leads to tyrosine phosphorylation and activation of phospholipase C␥ 2 (16), which leads to intracellular calcium mobilization and protein kinase C (PKC) activation.Akt is a 57-kDa serine/threonine kinase that plays an important role in mediating the anti-apoptotic effect of many growth factors (17-19). Akt contains a pleckstrin homology domain adjacent to a centrally located catalytic domain that is connected to a short C-terminal tail (20). Both translocation of Akt to cell membranes and p...

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin αIIbβ3) is activated by agonist-mediated G q stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5h-dimethyl-bis-(o-aminophenoxy)ethane-N,N,Nh,Nh -tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADPand U46619-induced aggregation and anti-αIIbβ3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially

Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A 2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, ␣,␤-methylene adenosine 5-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADPmediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca 2؉ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. ␣,␤-Methylene adenosine 5-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.ADP was the first low molecular weight agent recognized to cause platelet aggregation (1, 2). It is stored in the dense granules of human platelets and is an important platelet agonist as evidenced by the fact that patients with defective ADP storage have bleeding tendencies (3, 4). Activation of platelets by ADP follows a defined sequence. The first event, shape change, occurs when discoid shaped resting cells are rapidly converted to spiculated spheres. Shape change is followed by platelet aggregation and granule secretion which releases more ADP as well as many other substances (5). Acting extracellularly, ADP causes a number of intracellular events including rapid calcium influx (6, 7), mobilization of intracellular calcium stores (8), and inhibition of adenylyl cyclase (9). An increase in intracellular Ca 2ϩ may be due to an increase in inositol trisphosphate (10 -13) but this finding remains controversial (14, 15). In addition, arachidonic acid, liberated from platelet membranes due to activation of phospholipase A 2 , is converted to thromboxane A 2 , itself a powerful platelet agonist. Despite this knowledge the exact identity of platelet ADP receptors responsible for functional responses of ADP are not fully defined and the mechanism by which aggregation occurs is still under investigation.The idea that ADP's effects on platelets are receptor-mediated was indicated by the finding that ATP shows true competitive inhibition of ADP-...

SummaryHuman platelets express two distinct G protein-coupled ADP receptors, one coupled to phospholipase C through Gq, P2Y1, and the other to inhibition of adenylyl cyclase through Gi, P2TAC. We have recently shown that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Previous studies have tested whether ADP causes a decrease in the basal cAMP level and this reduction promotes platelet aggregation, but did not study the effect of decreased cAMP levels when the Gq pathway is selectively activated. Since we are now aware that platelet aggregation requires activation of two receptors, we investigated whether the function of P2TAC receptor activation, leading to inhibition of platelet adenylyl cyclase, could be replaced by direct inhibition of adenylyl cyclase, when Gq pathway is also activated, a possibility that has not been addressed to date. In the present study, we supplemented the P2Y1 mediated Gq signaling pathway with inhibition of the platelet adenylyl cyclase by using SQ22536 or dideoxyadenosine, or by selective activation of the α2A adrenoceptors with epinephrine. Although SQ22536, dideoxyadenosine, and epinephrine reduced the cAMP levels, only epinephrine could mimic the P2TAC receptor mediated signaling events, suggesting that reduction in basal cAMP levels does not directly contribute to ADP-induced platelet activation. Adenosine-5’-phosphate-3’-phosphosulfate, a P2Y1 receptor antagonist, completely blocked ADP-induced inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation suggesting that P2TAC-mediated activation of Gi (or other G proteins) does not activate phospholipase C. These results suggest that a signaling event downstream from Gi, independent of the inhibition of platelet adenylyl cyclase, contributes to αIIbβ3 activation.

Tyrosine phosphorylation of phospholipase C␥2 (PLC␥2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of nonreceptor tyrosine kinases can phosphorylate PLC␥ in vitro, the specific kinase(s) controlling BCR-dependent PLC␥ activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLC␥2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLC␥2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLC␥2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btkdeficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr 753 and Tyr 759 . Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLC␥2 carboxyl-terminal sites, Tyr 1197 and Tyr 1217 , was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLC␥2 SH2-SH3 linker.Signals generated by the pre-B and mature B cell receptors are essential for B cell development, activation, and maintenance of mature B cell populations (1). Engagement of the BCR 1 initiates the formation of a lipid-raft associated signaling complex, or "signalosome," containing tyrosine and serine/threonine kinases, adapter molecules, and lipid hydrolases including phospholipase C␥ isoforms. Together, these events promote a series of downstream signals including a sustained increase in intracellular calcium concentrations. PLC␥ is essential for antigen receptor-mediated calcium mobilization (2-4). Activated PLC␥ hydrolyzes its substrate, phosphatidylinositol 4,5-bisphosphate, generating the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (5, 6). IP 3 acts to open intracellular calcium stores promoting an initial, transient rise in intracellular calcium. Depletion of intracellular calcium stores triggers the opening of plasma membrane store-operated calcium channels (7), resulting in an influx of extracellular calcium and a secondary, sustained calcium signal. The amplitude and duration of this sustained calcium signal is a key determinant of the specific transcription program initiated in response to antigen receptor engagement (8 -10).The most abundantly expressed PLC␥ isoform in B lineage cells is PLC␥2. Chicken B lymphoma cells lacking PLC␥2 are unable to generate IP 3 in response to BCR engagement, resulting in the abrogation of receptor-mediated calcium mobilization (11). Similarly, mice deficient in...