Although sphingosine 1-phosphate (Sph-1-P) is reportedly involved in diverse cellular processes and the physiological roles of this bioactive sphingolipid have been strongly suggested, few studies have revealed the presence of Sph-1-P in human samples, including body fluids and cells, under physiological conditions. In this study, we identified Sph-1-P as a normal constituent of human plasma and serum. The Sph-1-P levels in plasma and serum were 191+/-79 and 484+/-82 pmol/ml (mean+/-SD, n=8), respectively. Furthermore, when Sph-1-P was measured in paired plasma and serum samples obtained from 6 healthy adults, the serum Sph-1-P/plasma Sph-1-P ratio was found to be 2.65+/-1.26 (mean+/-SD). It is most likely that the source of discharged Sph-1-P during blood clotting is platelets, because platelets abundantly store Sph-1-P compared with other blood cells, and release part of their stored Sph-1-P extracellularly upon stimulation. We also studied Sph-1-P-related metabolism in plasma. [3H]Sph was stable and not metabolized at all in plasma, but was rapidly incorporated into platelets and metabolized mainly to Sph-1-P in platelet-rich plasma. [3H]Sph-1-P was found to be unchanged in plasma, revealing that plasma does not contain the enzymes needed for Sph-1-P degradation. In summary, platelets can convert Sph into Sph-1-P, and are storage sites for the latter in the blood. In view of the diverse biological effects of Sph-1-P, the release of Sph-1-P from activated platelets may be involved in a variety of physiological and pathophysiological processes, including thrombosis, hemostasis, atherosclerosis and wound healing.

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.

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.

Introductionvon Willebrand factor (vWF) is a multimeric protein that mediates platelet adhesion to exposed subendothelium at sites of vascular injury. The adhesive property of vWF is tightly regulated so that plasma vWF does not normally interact with circulating platelets. However, after vWF is activated by binding to damaged vessel walls, it serves to bridge the constituents of subendothelium to glycoprotein Ib (GPIb) on the membrane of circulating platelets. 1,2 Although much is known about the molecular basis of the interaction between vWF and GPIb, little has been clarified about the intracellular signal transduction pathway in GPIb-mediated platelet activation.Events related to protein-tyrosine phosphorylation have emerged as important signals mediated by GPIb. [3][4][5][6][7] GPIb-mediated platelet activation in response to vWF plus botrocetin, shear stress, or vWF from patients with von Willebrand disease type IIb induces tyrosine phosphorylation of multiple proteins, [3][4][5] suggesting that the binding of vWF to GPIb causes the activation of tyrosine kinases. Studies have shown that the activation of Syk and Src and their association can be induced by GPIb stimulation. [6][7][8][9][10] To date, there is no report showing that tyrosine residues within the GPIb molecule can be phosphorylated, nor has GPIb itself intrinsic kinase activity. Thus, how clustering of GPIb induced by vWF mediates the activation of tyrosine kinases such as Src and Syk has become an important issue. Because Syk is activated by engagement of its tandem SH2 domains with phosphorylated tyrosine residues in proteins containing the immunoreceptor tyrosine-based activation motif (ITAM), 11 it is of interest whether ITAM-containing transmembrane molecules are also involved in GPIb signaling. Two ITAM-containing proteins have been identified in platelets, the low-affinity receptor for immunoglobulin (Ig) G, Fc␥RIIA, and the Fc receptor ␥-chain (FcR ␥-chain). Of particular note is the finding that Fc␥RIIA is physically associated with GPIb and that it mediates some signaling events. 12,13 However, the level of Fc␥RIIA tyrosine phosphorylation induced by GPIb clustering is much lower than that inducible by Fc␥RIIA clustering. Further, murine platelets lacking Fc␥RIIA on their membrane appear to have normal responses to GPIbrelated signals. Thus, to what extent Fc␥RIIA contributes to the GPIb signaling pathway remains to be determined.The other ITAM-containing protein, FcR ␥-chain, has been recognized for its role in signaling related to the high-affinity receptors for IgE (Fc⑀RI) 14 and IgG (Fc␥RI), 15 and the low-affinity IgG receptor (Fc␥RIII). 16 In platelets, FcR ␥-chain is colocalized with GPVI and is critical for GPVI-mediated platelet activation. 17,18 Clustering and activation of GPVI induces tyrosine phosphorylation of FcR ␥-chain ITAM, 19 which then facilitates the recruitment of Syk, resulting in its activation. 20 Moreover, FcR ␥-chain is required for tyrosine phosphorylation of phospholipase C ␥2 (PLC␥2). 20 Recently, Falati...

Summary. Although the signaling pathways related to GPIb-IX-V have not been fully elucidated, an accumulating body of evidence suggests that phospholipase C (PLC)c2 activation, subsequent Ca ++ release and oscillations constitute an essential signal transduction pathway related to GPIb-IX-V. Src family kinases are required for PLCc2 activation, while FcRc-chain/ FccRIIA may be dispensable for PLCc2 activation. Although PI-3K serves to potentiate various signaling events culminating in a IIb b 3 activation, PI-3K activity may be dispensable for SrcPLCc2 activation in GPIb-IX-V-mediated signaling. Glycosphingolipid-enriched microdomains (GEMs) appear to provide platforms for the signal transduction pathway related to GIb-IX-V, as the interaction between GPIb-IX-V and Src or PLCc2 tyrosine phosphorylation occurs exclusively in GEMs.