Platelet GPIb-IX-V-dependent signaling.

Although the signaling pathways related to GPIb-IX-V have not been fully elucidated, an accumulating body of evidence suggests that phospholipase C (PLC)gamma2 activation, subsequent Ca++ release and oscillations constitute an essential signal transduction pathway related to GPIb-IX-V. Src family kinases are required for PLCgamma2 activation, while FcR gamma-chain/Fc gammaRIIA may be dispensable for PLCgamma2 activation. Although PI-3K serves to potentiate various signaling events culminating in alpha(IIb)beta3 activation, PI-3K activity may be dispensable for Src-PLCgamma2 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 PLCgamma2 tyrosine phosphorylation occurs exclusively in GEMs.

Cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1) in platelets exposed to high shear stress.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130 kDa transmembrane glycoprotein that belongs to the immunoglobulin superfamily and is expressed on the surface of endothelial cells, platelets, and other blood cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its functional role in platelets remains to be elucidated. In this study, we examined whether PECAM-1 underwent changes in platelets exposed to high shear stress. Platelet PECAM-1 was cleaved under high shear stress and was released into the extracellular fluid as a fragment with an approximate molecular weight of 118 kDa. The cleavage was inhibited by an anti-VWF MoAb, but not by recombinant VWF A1 domains. These findings suggest that the GPIb-VWF interaction is involved in PECAM-1 cleavage under high shear stress, and that the cleavage is independent of GPIb clustering by VWF multimers. Furthermore, EGTA or calpeptin inhibited PECAM-1 cleavage. This finding provides evidence for the involvement of calpain in PECAM-1 cleavage. Flow-cytometric analysis revealed that PECAM-1 expression on the platelet surface was decreased under high shear stress. This reduction occurred exclusively in a specific population of platelets, which corresponded to platelet-derived microparticles (PMP). In conclusion, PECAM-1 cleavage under high shear stress is closely related to the activation of calpain and the process of PMP formation mediated by the GPIb-VWF interaction.

Rac, a small guanosine triphosphate-binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin alpha(2)beta(1).

In this study, the receptors and signals involved in collagen-induced platelet spreading were examined. It was found that platelet spreading on collagen (presenting a polygon shape with a number of filopodialike projections) was inhibited by the anti-integrin alpha(2) antibody, suggesting the involvement of integrin alpha(2)beta(1) in this process. Studies with a glutathione-S-transferase fusion protein that binds specifically to activated Rac and in vitro p21-activated kinase (PAK) kinase assays revealed that Rac and PAK were activated during this collagen-activated process. Platelet spreading on collagen-coated surfaces was inhibited strongly by PP1 (a Src family kinase inhibitor) or weakly by wortmannin (a phosphatidylinositol 3-kinase [PI3-kinase] inhibitor) but not at all by Y-27632 (a Rho kinase inhibitor). The surfaces coated with anti-integrin alpha(2)beta(1) antibodies also induced platelet spreading (presenting an almost complete round shape) and activation of Rac and PAK, although more slowly than collagen-coated surfaces. The antibody-induced responses were strongly inhibited by PP1 or wortmannin but not by Y-27632. The same concentration of Y-27632 inhibited collagen-induced shape change of platelets in suspension. These findings suggest that Rac and/or PAK activation, but not Rho, may play certain roles in platelet spreading via integrin alpha(2)beta(1) and that Src family kinases and PI3-kinase participate in these processes. Furthermore, the difference between spreading on collagen and the anti-integrin antibody suggests the involvement of other receptor(s) (in addition to the integrin alpha(2)beta(1)) for collagen-induced spreading, the most likely candidate being glycoprotein VI.

C-terminal peptide of thrombospondin-1 induces platelet aggregation through the Fc receptor gamma-chain-associated signaling pathway and by agglutination.

A peptide from the C-terminal domain of thrombospondin-1 (Arg-Phe-Tyr-Val-Val-Met-Trp-Lys; known as 4N1-1) has been reported to induce platelet aggregation and to bind to the integrin-associated protein (IAP), which is also known as CD47. In this study, it was discovered that 4N1-1 or its derivative peptide, 4N1K, induces rapid phosphorylation of the Fc receptor (FcR) gamma chain, Syk, SLP-76, and phospholipase C gamma2 in human platelets. A specific inhibitor of Src family kinases, 4-amino-4-(4-methylphenyl)-7-(t-butyl) pyrazola[3,4-d]pyrimidine, prevented phosphorylation of these proteins, abolished platelet secretion, and reduced aggregation by approximately 50%. A similar inhibition of aggregation to 4N1-1 was obtained in the presence of Arg-Gly-Asp-Ser in mouse platelets deficient in FcR gamma chain or SLP-76 and in patients with type I Glanzmann thrombasthenia. These results show that 4N1-1 signals through a pathway similar to that used by the collagen receptor glycoprotein (GP) VI. The alphaIIbbeta3-independent aggregation induced by 4N1-1 was also observed in fixed platelets and platelets from patients with Bernard-Soulier syndrome, which are deficient in GPIbalpha. Surprisingly, the ability of 4N1-1 to stimulate aggregation and tyrosine phosphorylation was not altered in platelets pretreated with anti-IAP antibodies and in IAP-deficient mice. These results show that the C-terminal peptide of thrombospondin induces platelet aggregation through the FcR gamma-chain signaling pathway and through agglutination. The latter pathway is independent of signaling events and does not use GPIbalpha or alphaIIbbeta3. Neither of these pathways is mediated by IAP.

The role of ITAM- and ITIM-coupled receptors in platelet activation by collagen.

The major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor gamma-chain (FcR gamma-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.

Role of Fc receptor gamma-chain in platelet glycoprotein Ib-mediated signaling.

Interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in human platelets. This study found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fc receptor gamma-chain (FcR gamma-chain), Syk, linker for activation of T cells (LAT), and phospholipase C gamma2 (PLCgamma2). Pretreatment of platelets with 10 microM PP1 completely inhibited these tyrosine phosphorylation events. On GPIb stimulation, Src and Lyn formed a complex with FcR gamma-chain and Syk, suggesting that Src and Lyn are involved in FcR gamma-chain tyrosine phosphorylation and downstream signals. In spite of the PLCgamma2 tyrosine phosphorylation, however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production. In Brij 35 lysates, FcR gamma-chain was found to constitutively associate with GPIb. The number of GPIb expressed on FcR gamma-chain-deficient platelets was comparable to that of the wild-type, as assessed by flow cytometry. However, tyrosine phosphorylation of Syk, LAT, and PLCgamma2 in response to vWF plus botrocetin was significantly suppressed, suggesting that FcR gamma-chain mediates activation signals related to GPIb. Compared with the aggregation response of wild-type platelets, that of FcR gamma-chain-deficient platelets in response to vWF plus botrocetin was impaired, implying that FcR gamma-chain is required for the full activation of platelets mediated by GPIb. (Blood. 2001;97:3836-3845)

The snake venom toxin alboaggregin-A activates glycoprotein VI.

The glycoprotein (GP)-Ib-IX-V receptor complex has recently been reported to signal through a pathway similar to that used by the collagen receptor GPVI, with a critical role described for the Fc receptor gamma-chain. The evidence for this was based in part on studies with the GPIbalpha-selective snake venom toxin, alboaggregin-A. In the present study, it is reported that alboaggregin-A has activity at the collagen receptor GPVI in addition to GPIbalpha, and evidence is provided that this contributes to protein tyrosine phosphorylation, shape change, and GPIIb-IIIa-dependent aggregation. This may explain why responses to alboaggregin-A are distinct from those to von Willebrand factor-ristocetin. (Blood. 2001;97:3989-3991)

A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI.

The interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin and alboaggregin-A activate platelets by interacting with GPVI. In this study, we isolated from white-lipped tree viper (Trimeresurus albolabris) venom, alborhagin, which is functionally related to convulxin because it activates platelets but is structurally different and related to venom metalloproteinases. Alborhagin-induced platelet aggregation (EC50, <7.5 microg/ml) was inhibitable by an anti-alphaIIbbeta3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to alphaIIbbeta3-dependent aggregation. Additional evidence suggested that, like convulxin, alborhagin activated platelets by a mechanism involving GPVI. First, alborhagin- and convulxin-treated platelets showed a similar tyrosine phosphorylation pattern, including a similar level of phospholipase Cgamma2 phosphorylation. Second, alborhagin induced GPVI-dependent responses in GPVI-transfected K562 and Jurkat cells. Third, alborhagin-dependent aggregation of mouse platelets was inhibited by the anti-GPVI monoclonal antibody JAQ1. Alborhagin had minimal effect on convulxin binding to GPVI-expressing cells, indicating that these venom proteins may recognize distinct binding sites. Characterization of alborhagin as a GPVI agonist that is structurally distinct from convulxin demonstrates the versatility of snake venom toxins and provides a novel probe for GPVI-dependent platelet activation.

Activation of protein-tyrosine kinase pathways in human platelets stimulated with the A1 domain of von Willebrand factor.

The binding of multimeric von Willebrand Factor (vWF) to its specific receptor on platelets, glycoprotein (GP)Ib, is a critical event, allowing platelet activation and subsequent thrombus formation in the vessels. In this study, the effects of the monomeric A1 domain, which contains the GPIb-binding site of the vWF molecule, on platelet activation were examined. The binding of the A1 domain to GPIb resulted in Syk activation and association with Src, as is the case with intact vWF. However, the A1 domain, in contrast to vWF, did not induce platelet cytoskeletal association of tyrosine kinases, Src and Lyn. When platelet functional responses, such as aggregation and intracellular Ca2+ mobilization, were monitored, the A1 domain failed to induce the responses by itself and blocked the responses induced by the multimeric vWF molecule. These results suggested that the A1 domain triggers at least some of tyrosine kinase-related signals via GPIb and may be a partial agonist as well as a competitive antagonist for the vWF-GPIb interaction.

Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin.

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.

Platelet activation mediated through membrane glycoproteins: involvement of tyrosine kinases.

Fc gamma RII cross-linking and anti-CD9 mAbs included tyrosine phosphorylation of Fc gamma RII, Syk, and Lyn associated with Fc gamma RII in Fc gamma RII cross-linking but not in anti-CD9 mAb-induced platelet activation. We prepared various GST fusion proteins expressing one or two SH2 domains of Syk and evaluated the association between these GST fusion proteins with Fc gamma RII. Based on the results obtained from these experiments, we suggest that only one tyrosine residue in ITAM of Fc gamma RII is phosphorylated with anti-CD9 mAb and that both are phosphorylated with Fc gamma RII cross-linking. Platelet activation mediated by GPIb, the receptor for vWF, is also related with tyrosine phosphorylation. Botrocetin and vWF induced Syk activation. Shc was also rapidly and heavily tyrosine phosphorylated. Sre and Lyn, a 54-kDa tyrosine kinase, was associated with cytoskeletal proteins. When GPIb was immunoprecipitated with nonfunctional anti-GPIb mAbs after platelets were activated with vWF and botrocetin, an in vitro kinase assay revealed the transient association of a kinase activity with GPIb after platelet activation. Phosphoamino acid analysis of phosphorylated proteins in this assay demonstrated that only tyrosine residues but not serine or threonine were phosphorylated, suggesting that the kinase was indeed a tyrosine kinase.

Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alphaIIbbeta3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2.

Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKbeta or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on alphaIIbbeta3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for alphaIIbbeta3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N-terminal SH3 domain of Grb2 and that the phosphotyrosine residue of Shc binds to the SH2 domain of Grb2. Although Pyk2 and FAK have been reported to be differentially regulated in many cell types, our results suggest that, in human platelets, the mode of Pyk2 activation is mostly similar to that of FAK, in terms of alphaIIbbeta3 integrin-dependent and PKC-dependent tyrosine phosphorylation. Furthermore, Pyk2, as well as FAK, might have one or more important roles in post-aggregation tyrosine phosphorylation events, in association with the cytoskeleton and through interaction with adapter proteins including Grb2 and Shc.

LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI.

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.

FcgammaRII tyrosine phosphorylation differs between FcgammaRII cross-linking and platelet-activating anti-platelet monoclonal antibodies.

Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.

Suppression of protein kinase C is associated with inhibition of PYK2 tyrosine phosphorylation and enhancement of PYK2 interaction with Src in thrombin-activated platelets.

Blood platelets have recently been shown to express PYK2, a nonreceptor tyrosine kinase belonging to the FAK gene family. In this study, we examined the involvement of protein kinase C (PKC) in PYK2-related responses in human platelets. While PYK2 tyrosine phosphorylation induced by thrombin was inhibited by preincubation of platelets with PKC inhibitors, staurosporine and Ro31-8220, PYK2 association with Src was markedly enhanced under the same conditions. Platelet intracellular Ca2+ mobilization induced by thrombin was hardly inhibited by these PKC inhibitors. p130Cas is a docking protein that associates with FAK or PYK2 through the SH3 domain. Although we identified p130Cas in platelets for the first time, this docking protein failed to interact with PYK2. These results suggest that PKC activation (but not Ca2+ mobilization) is involved in PYK2 tyrosine phosphorylation and that PYK2 associates with Src without PYK2 tyrosine phosphorylation or p130Cas involvement in platelets.

Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum.

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.

Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody.

NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ib alpha, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-Fc gamma II receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab' fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab' fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the Fc gamma II receptor. Cross-linking of the receptor-bound Fab' fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5-induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.

Glycoprotein Ib-von Willebrand factor interactions activate tyrosine kinases in human platelets.

von Willebrand factor (vWF ) in the presence of botrocetin induces p72(syk) activation, assessed as its autophosphorylated level and in vitro kinase assays, the transient association of p72(syk) with p60(c-src), and the translocation of p60(c-src) and p54/58(lyn) to cytoskeletal fractions. Jararaca glycoprotein Ib-binding protein (GPIb-BP), which specifically binds to GPIb, abolished these phenomena, suggesting that they are mediated by the vWF-GPIb interaction. These tyrosine kinase-related events were not inhibited by GRGDS peptide (plus EGTA), indicating that GPIIb/IIIa is not involved in the observed responses. Shc, an adaptor protein, was also tyrosine phosphorylated by the botrocetin-vWF activation. When GPIb was immunoprecipitated with nonfunctional monoclonal antibodies (MoAbs) directed against GPIb, a kinase activity was found to associate with GPIb upon botrocetin-vWF activation. On the other hand, anti-GPIb MoAbs that inhibit the vWF-GPIb interaction did not coprecipitate a kinase activity. Because the recovery of GPIb did not differ significantly, it is suggested that the excessive presence of inhibitory anti-GPIb MoAb dissociated a kinase activity from GPIb. Phosphoamino acid analysis showed that the kinase activity was that of a tyrosine kinase. The identity of the tyrosine kinase and the mode of interaction with the cytoplasmic region of GPIb await to be determined. Our findings suggest that the tyrosine kinase associated with GPIb serves at a most proximal step in the signal transduction pathway involved in the vWF-GPIb-induced platelet activation, which leads to other tyrosine kinase-related intracellular signals.

Activation of protein-tyrosine kinase Syk in human platelets stimulated with lysophosphatidic acid or sphingosine 1-phosphate.

It has been reported that not only lysophosphatidic acid (LPA) but also its sphingolipid counterpart, sphingosine 1-phosphate (Sph-1-P), induce platelet functional responses. We report here Syk activation in human platelets stimulated with these lysophospholipids. LPA rapidly induced platelet protein-tyrosine phosphorylation, including that of Syk, and Syk activation, assessed by immunoprecipitation kinase assay. Sph-1-P, although rather weaker, mimicked LPA in inducing these tyrosine kinase-related events. Pretreatment of platelets with staurosporine, a potent protein kinase inhibitor, diminished LPA-induced Syk phosphorylation and activation, but not intracellular Ca2+ mobilization. These results demonstrate that, in platelets, the bioactive lysophospholipids induce Syk activation, which, however, may not be related to Ca2+ mobilization.

Differential activation of human platelets induced by Fc gamma receptor II cross-linking and by anti-CD9 monoclonal antibody.

Platelet activation induced by anti-CD9 mAb, which depends upon Fc gammaRII, has been considered to be similar to that induced by Fc gammaRII cross-linking. In this work, we present several lines of evidence to suggest that the mode of platelet activation induced by anti-CD9 mAb is distinct from that induced by Fc gammaRII cross-linking. Ca2+ release from intracellular Ca2+ stores induced by anti-CD9 mAb depended almost totally upon thromboxane A2 production and released ADP, whereas that induced by Fc gammaRII was affected only minimally by these factors. Fc gammaRII cross-linking induced Ca2+ channel opening, which is dependent upon the depletion of intracellular Ca2+ stores. In contrast, anti-CD9 mAb appeared to directly open Ca2+ channels, irrespective of intracellular Ca2+ stores (Kuroda et al., 1995. J. Immunol. 155: 4427). The Ca2+ requirement for the Ca2+ channels opened by Fc gammaRII cross-linking was also distinct from that induced by anti-CD9 mAb. The early phase of Fc gammaRII tyrosine phosphorylation was dependent upon thromboxane A2 production with anti-CD9 mAb-induced activation, whereas that of Fc gammaRII cross-linking was not. p72(syk) and p53/56(lyn) appeared to associate with Fc gammaRII in platelet activation induced by Fc gammaRII cross-linking, whereas there was little, if any, association between Fc gammaRII and these tyrosine kinases in anti-CD9 mAb-induced activation. Piceatannol, a selective inhibitor of p72(syk), enhanced Fc gammaRII tyrosine phosphorylation induced by Fc gammaRII cross-linking, whereas it attenuated the process in anti-CD9 mAb-induced platelet activation. It is suggested that the regulatory mechanism of Fc gammaRII tyrosine phosphorylation differs between these two modes of platelet activation.