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Gab2, a recently identified docking protein, contains a pleckstrin homology domain and potential binding sites for SH2 and SH3 domain-containing proteins. Gab2 has been shown to support growth, differentiation, and function in a number of haematopoietic cells, although its role in platelets remains to be determined. Here we report that cross-linking of the collagen receptor GPVI by the snake venom toxin convulxin stimulates tyrosine phosphorylation of Gab2. Furthermore, platelet aggregation induced by submaximal concentrations of convulxin is attenuated in the absence of Gab2, although recovery is seen with higher concentrations of the toxin. Consistent with this, tyrosine phosphorylation of Fc receptor γ-chain, Syk, Btk, and phospholipase Cγ2 by convulxin is reduced in the absence of Gab2. In comparison, the G protein-coupled receptor agonist, thrombin, does not induce phosphorylation of Gab2 and aggregation is unaltered in the absence of the toxin. These findings provide evidence for a functional role of Gab2 in supporting platelet activation by GPVI.

The binding of von Willebrand factor (VWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3– kinase (PI 3–kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3–kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3–kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)–fusion protein coprecipitated the p85 subunit of PI 3–kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3–kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3– kinase.

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.

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) γ chain, Syk, SLP-76, and phospholipase C γ2 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 γ 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 αIIbβ3-independent aggregation induced by 4N1-1 was also observed in fixed platelets and platelets from patients with Bernard-Soulier syndrome, which are deficient in GPIbα. 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 γ-chain signaling pathway and through agglutination. The latter pathway is independent of signaling events and does not use GPIbα or αIIbβ3. Neither of these pathways is mediated by IAP.

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 platelet collagen receptor glycoprotein VI (GPVI) signals through a similar pathway to that used by immunoreceptors with pivotal roles for the Fc receptor (FcR) γ-chain and the tyrosine kinase Syk (28). Cross-linking of GPVI leads to tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif in the FcR γ-chain by a Src-like kinase, enabling the binding of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif via its tandem SH2 domains (8, 13, 16). This leads to autophosphorylation and activation of Syk and regulation of downstream events that culminate in activation of phospholipase Cγ2 (PLCγ2). The activation of PLCγ2 is critical for the majority of functional responses to collagen, including aggregation and dense-granule secretion (38). Several proteins play important roles in the activation of PLCγ2 downstream of Syk in platelets. These include the adapter SLP-76, which is a substrate for Syk and is essential for tyrosine phosphorylation of PLCγ2 (12, 19); the tyrosine kinase Btk, which is implicated in the tyrosine phosphorylation of PLCγ2 (29); and the p85/110-kDa heterodimeric form of phosphatidylinositol 3-kinase (PI 3-kinase), which is required for regulation of PLCγ2 activity but not tyrosine phosphorylation (27). Cross-linking of GPVI also leads to the tyrosine phosphorylation of a 36- to 38-kDa protein in platelets independent of PLCγ2, which is proposed to play a role in the regulation of the phospholipase (5). The 36- to 38-kDa protein has many of the characteristics of a protein with a similar molecular mass in T cells, including the ability to bind to the SH2 domains of PLCγ1 and Grb2 (31). The cloning of the 36- to 38-kDa protein from T cells, the linker for activation of T cells (LAT) (41), enabled the identification of the platelet protein as LAT (17). LAT was shown to associate with the p85α subunit of PI 3-kinase in platelets following cross-linking of GPVI by collagen or the snake venom convulxin, suggesting that it mediates recruitment to the membrane and activation of its associated p110 catalytic subunit (17). LAT has also been shown to be phosphorylated in thrombin-stimulated platelets (32). LAT is a transmembrane protein which is found in glycolipid-enriched membrane domains (43). There are nine conserved tyrosine residues in the cytosolic domain in the murine and human sequences, five of which contain the optimal binding sequence for binding to the SH2 domain of Grb2. LAT is heavily phosphorylated on tyrosine residues upon T-cell receptor stimulation, most probably by ZAP-70 (41). Studies with a LAT-deficient Jurkat T-cell line, J.Cam2, have shown that LAT plays a critical role in the regulation of PLCγ1 and the mitogen-activated protein kinase pathway downstream of the T-cell antigen receptor (15, 42). LAT-deficient mice have no mature peripheral T cells, but they have a normal B-cell population, demonstrating that LAT is critical for T-cell but not B-cell development (42). The present study was undertaken to investigate whether LAT is required for the activation of PLCγ2 and the onset of functional responses in platelets stimulated by cross-linking of GPVI by using platelets from mice engineered to lack the adapter (42). A collagen-related peptide (CRP) which does not bind to integrin α2β1 (25) was used rather than collagen to activate GPVI because it gives a more powerful and reproducible response between experiments and, unlike collagen, can be used in flow cytometry. Several studies have shown that activation of platelets by CRP mimics that by collagen (1, 2, 16). The results show that LAT plays a critical role in PLCγ2 tyrosine phosphorylation and activation and in subsequent functional events in response to GPVI activation. In contrast, the response to the G-protein-receptor-coupled agonist thrombin, which mediates activation through PLCβ isoforms (7) and induces minimal tyrosine phosphorylation of PLCγ2 (6), is not altered.

We investigated the effects on human platelet aggregation of several agents that increase either intracellular cyclic AMP or cyclic GMP, using a platelet aggregometer that allows quantification of the size and number of platelet aggregates. During the initial phase of aggregation induced by epinephrine and ADP, small aggregates consisting of < 100 cells predominated; large aggregates formed later. Prostaglandin I2 (PGI2), which increases intracellular cyclic AMP, suppressed the formation of small as well as large aggregates induced by epinephrine, with ID50 values of 10.7 +/- 2.8 and 3.8 +/- 0.5 nM, respectively. ADP-induced formation of small and large aggregates was also inhibited by PGI2, with similar ID50 values. Dibutyryl cyclic AMP (db cyclic AMP), a cell-permeant form of cyclic AMP, also inhibited small and large aggregate formation induced by epinephrine or ADP, with ID50 values of 420-560 microM for small aggregates and 139-166 microM for large aggregates, respectively. On the other hand, nitroprusside, which increases intracellular cyclic GMP, inhibited only the formation of large aggregates, with an ID50 value of 454 +/- 191 nM for epinephrine-induced activation and of 2.1 +/- 0.6 microM for ADP-induced activation. Nitroprusside at 1 mM did not affect the formation of small aggregates induced by epinephrine, whereas that of large aggregates was completely blocked at 10 microM. 8-Bromo cyclic GMP (8-br cyclic GMP) also inhibited only the formation of large aggregates, with ID50 values of 140-170 microM, but not that of small aggregates induced by epinephrine and ADP. Milrinone, which increases the intracellular level of both cyclic AMP and cyclic GMP, suppressed the formation of small and large aggregates induced by epinephrine and ADP. These findings suggest that cyclic AMP and cyclic GMP differentially modify the size of aggregates formed during epinephrine or ADP activation.

The effects of S2-serotonergic receptor antagonists, ketanserin, MCI-9042, and one of its major metabolite, M-1, were evaluated on human platelet activation induced by serotonin. A newly developed method for detecting particles in suspensions was used to assess serotonin-induced platelet aggregation. Serotonin added to platelets in plasma induced transient formation of small aggregates but not that of large ones. All the three antagonists in a dose-dependent manner suppressed serotonin-induced platelet aggregation. The ID50 values for ketanserin, MCI-9042, and M-1 are 10 nM, 0.6 microM, and 40 nM, respectively. The effects of these antagonists were also evaluated on [Ca+2]i elevation and shape change, the measurement of which does not require the presence of plasma proteins. These antagonists effectively inhibited [Ca+2]i elevation and shape change induced by serotonin. The ID50 value for MCI-9042 was approximately 1/10 for platelet aggregation. These findings suggest that MCI-9042 tightly binds to plasma proteins with resultant reduction in overall potency. The ID50 values obtained in this study are essentially equivalent to those reported for S2-serotonergic receptor binding in rabbit platelets, suggesting that these agents are also potent antagonists serotonin-induced activation of human platelets.

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)

We have evaluated the effects of different anti-coagulants or agonists on the generation of platelet-derived microparticles (PMPs) using flow cytometry. Twenty microg/ml of collagen induced significantly greater PMP formation in whole blood anti-coagulated with argatroban, a selective thrombin inhibitor, as compared with platelet-rich plasma, or whole blood anti-coagulated with citrate. Thus, whole blood kept at the physiological Ca2+ concentration provides an optimal condition for the formation of PMP. Convulxin, a GPVI-selective agonist, also induced PMP formation at the magnitude which far exceeds those of other agonists, such as thrombin receptor-activating peptide, ADP or epinephrine. These findings suggest that GPVI-mediated platelet activation plays a key role in the formation of PMP in the presence of physiological Ca2+ in whole blood. The addition of red blood cells to PRP potentiated PMP formation induced by collagen. Pretreatment of whole blood with the combination of creatine phosphate and creatine phosphokinase reduced PMP formation induced by collagen. Blockade of ADP receptors, P2Y12 with AR-C69931MX and P2Y1 with A3P5P, respectively, further suppressed collagen-induced PMP formation. We conclude that ADP released from red blood cells enhances PMP formation induced by collagen, and that both P2Y12 and P2Y1 contribute to ADP-potentiation of PMP generation induced by collagen.

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.

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.

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.

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.

NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ibα, 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-FcγII 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 FcγII 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.

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.

Both thrombin and thrombin receptor agonist peptide (TRAP) activated p72 syk and p60 c-src with similar magnitudes. Both thrombin and TRAP induced translocation of p60 c-src and p54/58 lyn to cytoskeleton in an aggregation-dependent manner. Thrombin also induced cytoskeletal association of p72 syk , but independent of platelet aggregation. Furthermore, p72 syk associated with cytoskeleton underwent marked proteolysis, which was partially dependent upon calpain activation. In contrast, TRAP, even at concentrations as high as 100 μ M, did not induce p72 syk translocation to cytoskeleton. Our findings suggest that cytoskeletal translocation of p72 syk induced by thrombin is governed by a mechanism distinct from those of p60 c-src and p54/58 lyn translocation. It is also suggested that p72 syk translocation induced by thrombin requires additional signal(s) other than that mediated by the recently-cloned thrombin receptor that couples with GTP-binding proteins and interacts with TRAP.

SummaryIn human platelets treated with acetylsalicylic acid, collagen induced protein-tyrosine-phosphorylation of several proteins. The major 75 kDa band included cortactin and autophosphorylated p72 syk . p72 syk activity rapidly increased upon collagen stimulation, whereas p60c-src activation was below detectable levels. A combination of inhibitors to remove the effects of extracellular and intracellular Ca2+, released ADP, and fibrinogen binding to GPIIb/IIIa delayed and attenuated the major 75 kDa band. By contrast, p72 syk activation was not inhibited by these treatments. Cytochalasin D completely inhibited protein tyrosine phosphorylation and p72 syk activation. It also potently inhibited aggregation and [Ca2+]i elevation. Anti-GPMIa/IIa MoAb in a concentration-dependent manner partially attenuated protein tyrosine phosphorylation and p72 syk activation. Its inhibitory effects on intracellular Ca2+ mobilization, release of intracellular granule contents, and aggregation also were partial. No tyrosine kinase activity was coprecipitated with GPIa/IIa. These results suggest that p72 syk activation lies upstream of protein tyrosine phosphorylation, Ca2+ mobilization, ADP release, thromboxane A2 production and aggregation. GPIa/IIa plays a key role in p72 syk activation induced by collagen, but other collagen receptors may work in synergy to fully activate p72 syk . Actin polymerization is a prerequisite for both p72 syk activation and other intracellular signal transduction pathways.

Platelet aggregate size was determined with a newly-developed platelet aggregometer, PA-100 (KOWA), which can quantitatively evaluate the size and number of platelet aggregates by means of the particle counting method based upon light scattering. Epinephrine-induced platelet aggregation consists of two phases, the former characterized by the formation of small-sized aggregates (less than 100 cells), which is followed by the phase of large aggregate formation with concomitant decrease in the number of small aggregates. These findings suggest that small aggregates fuse to form large aggregates. Effects of various inhibitors and antibodies directed against platelet membrane glycoproteins were evaluated on the size of platelet aggregates induced by epinephrine. Cyclooxygenase inhibitors, thromboxane A2 receptor antagonists, and a Na + H + exchanger inhibitor (ethylisopropylamiloride) inhibited the formation of large aggregates (more than 100 cells) but not that of small aggregates. Cytochalasin B, which interferes with microfilaments, suppressed large aggregate formation, whereas taxol, which reacts with microtubules, had no effects. Anti - GPIIb IIIa monoclonal antibody (MoAb) inhibited both the formation of small and large platelet aggregates, while antibodies directed against GPIb, thrombospondin, P-selectin, or PECAM-1 had no effects on platelet aggregate formation. These findings, taken together, suggest that intracellular alkalinization, thromboxane A2 formation and microfilament rearrangement are prerequisites for large platelet aggregate formation. GPIIb IIIa is involved in the formation of small as well as large aggregates, but a membrane glycoprotein(s) responsible for the transition of small aggregates into large aggregates awaits to be determined.

The effects of three hypoglycaemic agents—glimepiride, glibenclamide and gliclazide—were evaluated on platelet aggregation and intracellular Ca2+ elevation induced by arachidonic acid. Platelet aggregation was assessed both by the conventional method using changes in light transmission and by a newly-developed procedure using light scattering which allows the detection of small as well as large aggregates. Glimepiride and glibenclamide inhibited the formation of small and large aggregates induced by optimal concentrations of arachidonic acid in a dose-dependent manner. The id 50 values for the inhibition of platelet aggregation were approximately one third of those for arachidonic acid metabolism, suggesting that both agents have certain direct inhibitory effects on platelet aggregation unrelated to arachidonic acid metabolism. Gliclazide inhibited the formation of small aggregates induced by low concentrations of arachidonic acid to a limited extent. However, it inhibited the formation of large aggregates but not small aggregates when higher concentrations of arachidonic acid were used. Glimepiride and glibenclamide inhibited [Ca2+]i elevation induced by arachidonic acid in a dose-dependent manner, whereas gliclazide had no inhibitory effect. Taken together, these suggest that gliclazide does not inhibit arachidonic acid metabolism but does have certain direct inhibitory effects on platelet aggregation.