LAT is required for tyrosine phosphorylation of phospholipase C gamma 2 and platelet activation by the collagen receptor GPVI

In the present study we have addressed the role of the linker for activatio n of T cells (LAT) in the regulation of phospholipase C gamma 2 (PLC gamma 2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosin e phosphorylated in human platelets heavily in response to collagen, collag en-related peptide (CRP), and Fc gamma RIIA cross-linking but only weakly i n response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platele ts, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-l inked agammaglobulinemia (XLA) human platelets. Using mice engineered to la ck the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosph orylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphoryl ation of PLC gamma 2 was substantially reduced in LAT-deficient platelets b ut was not completely inhibited. The reduction in phosphorylation of PLC ga mma 2 was associated with marked inhibition of formation of phosphatidic ac id, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a su bstrate of protein kinase C, and expression of P-selectin in response to CR P, whereas these parameters were not altered in response to thrombin. Activ ation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhi bited. These results demonstrate that LAT tyrosine phosphorylation occurs d ownstream of Syk and is independent of the adapter SLP-76, and they establi sh a major role for LAT in the phosphorylation and activation of PLC gamma 2, leading to downstream responses such as alpha-granule secretion and acti vation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of L AT and that there is a minor, LAT-independent pathway of tyrosine phosphory lation of PLC gamma 2. We propose a model in which LAT and SLP-76 are requi red for PLC gamma 2 phosphorylation but are regulated through independent p athways downstream of Syk.