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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 assessment of platelet activation levels may be useful for identifying patients who would benefit from antiplatelet therapy and prediction of ischemic events. Laboratory markers of platelet activation include activation-dependent changes in glycoprotein (GP) IIb/IIIa complex, exposure of granule membrane proteins, binding of secreted platelet proteins, and development of procoagulant surfaces. Whole blood flow cytometry is a popular and useful method for the detection of these markers of platelet activation. Monoclonal antibodies against platelet surface glycoproteins are used to identify activated platelets.

Bisphenol A (BPA) is an environmental endocrine disrupter. Excess exposure to BPA may increase susceptibility to many metabolic disorders, but it is unclear whether BPA exposure has any adverse effects on the development of atherosclerosis. To determine whether there are such effects, we investigated the response of Watanabe heritable hyperlipidemic (WHHL) rabbits to 400-µg/kg BPA per day, administered orally by gavage, over the course of 12 weeks and compared aortic and coronary atherosclerosis in these rabbits to the vehicle group using histological and morphometric methods. In addition, serum BPA, cytokines levels and plasma lipids as well as pathologic changes in liver, adipose and heart were analyzed. Moreover, we treated human umbilical cord vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (SMCs) with different doses of BPA to investigate the underlying molecular mechanisms involved in BPA action(s). BPA treatment did not change the plasma lipids and body weights of the WHHL rabbits; however, the gross atherosclerotic lesion area in the aortic arch was increased by 57% compared to the vehicle group. Histological and immunohistochemical analyses revealed marked increases in advanced lesions (37%) accompanied by smooth muscle cells (60%) but no significant changes in the numbers of macrophages. With regard to coronary atherosclerosis, incidents of coronary stenosis increased by 11% and smooth muscle cells increased by 73% compared to the vehicle group. Furthermore, BPA-treated WHHL rabbits showed increased adipose accumulation and hepatic and myocardial injuries accompanied by up-regulation of endoplasmic reticulum (ER) stress and inflammatory and lipid metabolism markers in livers. Treatment with BPA also induced the expression of ER stress and inflammation related genes in cultured HUVECs. These results demonstrate for the first time that BPA exposure may increase susceptibility to atherosclerosis in WHHL rabbits.

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [32P]Sph-1-P, but not [32P]LPA, was observed in [32P]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca++ mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

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.

Because sphingosine (Sph) is actively incorporated into platelets and rapidly converted to sphingosine 1-phosphate (Sph-1-P), which is then released extracellularly, it is important to study the effects of Sph and Sph-1-P on endothelial cells from the viewpoint of platelet-endothelial cell interaction. In this study, we found that Sph, as well as ceramide, induces apoptosis in human umbilical vein endothelial cells (HUVECs). In contrast, Sph-1-P acts as a HUVEC survival factor; this bioactive lipid was shown to protect HUVECs from apoptosis induced by the withdrawal of growth factors and to stimulate HUVEC DNA synthesis. In metabolic studies, [3H]Sph, incorporated into HUVECs, was converted to [3H]Cer and further to [3H]sphingomyelin in a time-dependent manner, whereas [3H]Sph-1-P formation from [3H]Sph was weak and transient. These findings in HUVECs are very different from those of platelets, which possess a highly active Sph kinase but lack Sph-1-P lyase. As a result, platelets abundantly store Sph-1-P, whereas HUVECs contain much less Sph-1-P. Finally, HUVECs, in contrast to platelets, failed to release Sph-1-P extracellularly, indicating that HUVECs themselves are not able to supply the survival factor Sph-1-P, but receive it from activated platelets. Our results suggest that platelets may maintain the integrity of endothelial cells by incorporating Sph and releasing Sph-1-P.

Human platelets were activated either by glycoprotein (GP) Ia/IIa agonist (rhodocytin) or by a GPVI agonist (collagen-related peptide, CRP), and the intracellular signal transduction pathways were compared in the presence of various inhibitors. Rhodocytin isolated from Calloselasma rhodostoma venom was verified as a GPIa/IIa agonist, based on the inhibitory effects of three mAbs directed against GPIa. Platelet activation mediated by GPIa/IIa led to overt platelet aggregation, elevation of intracellular Ca 2+ , and tyrosine phosphorylation of several proteins, similar to that of GPVI. p72 syk and phospholipase Cγ2 (PLCγ2) tyrosine phosphorylation were also observed with GPIa/IIa-mediated platelet aggregation, although they peaked slightly later than that of GPVI. In contrast to GPVI-mediated platelet activation, most of these phenomena induced by GPIa/IIa activation were markedly suppressed by acetylsalicylic acid (ASA) or cytochalasin D. These findings suggest that the requirements for thromboxane A 2 (TXA 2 ) production and actin polymerization, which are the characteristics of collagen-induced platelet activation, are derived from the GPIa/IIa-mediated signal transduction, but not from that of GPVI.

To clarify the relationship between platelet function and diabetic complications, we investigated spontaneous platelet aggregation (SPA) and agonist-induced platelet aggregation by a particle counting method using light scattering (LS) and by a conventional light transmission method (LT) in 23 age- and sex-matched control subjects and 74 patients with type II diabetes mellitus. We also observed platelets using the FIC-2 (TOA Medical Electronics, Kobe, Japan) flow cytometer and imaging device. Observation by the FIC-2 device showed microaggregates of platelets in samples with increased SPA-LS. SPA-LS was significantly elevated in patients with type II diabetes mellitus as a whole compared with control subjects. SPA-LS also showed significant differences between control subjects and three diabetic patient subgroups with a varying severity of retinopathy, nephropathy, or neuropathy, and the mean values increased along with the increasing severity of complications. On the other hand, although SPA-LT also showed significant differences between these groups, the absolute values were all less than 10%, which we believe does not warrant quantitative analysis. Adenosine-5'-diphosphate (ADP)-induced platelet aggregation failed to show significant differences between controls and subjects with a varying severity of retinopathy by either LS or LT, which indicates that SPA is more sensitive than agonist-induced platelet aggregation in relation to diabetic complications. We observed significant correlations between SPA-LS and the patients' age, hemoglobin A1c (HbA1c) level, plasma fibrinogen level, or 6-keto-PGF1alpha (6KF) to 11-dehydro-thromboxane B2 (TXB2) ratio. Our study demonstrated a close relationship between platelet hyperaggregability and diabetic complications, and a longitudinal prospective study of SPA-LS in diabetic patients is warranted to clarify cause-and-effect relationships.

von Willebrand factor (vWF ) in the presence of botrocetin induces p72syk activation, assessed as its autophosphorylated level and in vitro kinase assays, the transient association of p72syk with p60c-src, and the translocation of p60c-src and p54/58lyn 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.

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.

Platelet function in patients with NIDDM is enhanced. We have found that spontaneous aggregation (i.e., the formation of small-sized aggregates in the absence of agonist stimulation) occurs at a high rate in platelets from NIDDM patients. We then investigated basal myosin light chain 20 (MLC) phosphorylation, which plays a key role in platelet shape change and aggregation, using a monoclonal antibody against a phosphorylation site (serine 19 residue) in the MLC molecule in platelets from these patients. Standard calibration curves obtained from purified MLC or the phosphorylated form of myosin light chain 20 (MLC-P) were linear within the range of 0–150 ng for MLC and 0–3 ng for MLC-P. The amount of MLC or MLC-P in platelets was estimated, and basal MLC phosphorylation was calculated. Platelets were obtained from 9 young healthy control subjects, 13 age- and sex-matched nondiabetic control subjects, and 13 patients with NIDDM. The basal MLC phosphorylation in platelets was significantly higher in the NIDDM patients than in the control subjects, irrespective of age. These findings suggest that platelets from NIDDM patients are activated in vivo. Platelets obtained from NIDDM patients generated spontaneous aggregation, the degree of which was significantly higher than that in control subjects. Platelet spontaneous aggregation correlated well with basal MLC phosphorylation. These findings suggest that increases in basal MLC in platelets may be one factor leading to hyperaggregability of platelets in these patients.

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.

(2004). Letter to the EditorClear visual detection of circulating platelet aggregates in acute myocardial infarction using a flow cytometer equipped with an imaging device. Platelets: Vol. 15, No. 1, pp. 61-62.

NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced protein tyrosine phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa proteins in human platelets, whereas F(ab')2 fragments of NNKY 1-19 did not, suggesting that the stimulation of Fc gamma II receptors is required for the induction of protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa were associated with aggregation, while NNKY 1-19-induced protein tyrosine phosphorylation was completely inhibited by prostaglandin I2 (PGI2). The activity of p72syk was assessed in immunoprecipitation kinase assays to determine at which step the signal transduction pathway leading to protein tyrosine phosphorylation was suspended. NNKY 1-19 induced a rapid and transient increase in the p72syk-associated tyrosine kinase activity that peaked at 10 s and subsided to the original level 2 min after stimulation. Coinciding with this time course, p60c-src transiently associated with p72syk. In platelets preexposed to GRGDS peptides or PGI2, NNKY 1-19 also increased the p72syk-associated tyrosine kinase activity and led to the association of p60c-src with p72syk. However, in contrast to the control without any inhibitor, the elevated tyrosine kinase activity and the associated state of the two tyrosine kinases persisted as long as 5 min after stimulation. F(ab')2 fragments of NNKY 1-19 induced changes similar to those observed with the effects of GRGDS peptides or PGI2 treatment on intact IgG NNKY 1-19 stimulation. F(ab')2 fragments of another CD9 MoAb, PMA2, had effects on p72syk essentially similar to those of NNKY 1-19. These findings suggest that the binding of anti-CD9 MoAb to CD9 on the platelet membrane per se induces an increase in the p72syk-associated tyrosine kinase activity but that Fc gamma II receptor-mediated signal(s) is required for the full activation of platelets and the appearance of tyrosine-phosphorylated proteins. The elevated intracellular cAMP level induced by PGI2 acts at a step distal to the activation of p72syk and inhibited the signal transduction pathway leading to protein tyrosine phosphorylation and aggregation. p72syk activation occurs in the absence of aggregation, but aggregation appears to reduce the elevated p72syk activity induced by anti-CD9 MoAb.

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.

The interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) induced by ristocetin or botrocetin resulted in associated platelet aggregation, protein tyrosine phosphorylation (PTP) of a 64 kDa protein, as detected by a monoclonal antibody against phosphotyrosine (PY-20), and intracellular Ca 2+ elevation that is largely dependent upon Ca 2+ influx in human platelets. It is of interest that 75–80, 97 and 125 kDa proteins which are strongly tyrosine-phosphorylated in platelet activation induced by thrombin and other agonists were not detected. Neither vWF nor a coaggregating agent (ristocetin or botrocetin) alone induced aggregation, [Ca 2+ ] i elevation or the 64 kDa PTP. NMC-4, an antibody which inhibits both ristocetin- or botrocetin-induced vWF binding to GPIb, abolished the appearance of the 64 kDa PTP as well as other responses, suggesting that it is specifically induced by the GPIb-vWF interaction. Aspirin, or ONO-3708, a competitive inhibitor of thromboxane A 2 , did not modify the 64 kDa PTP, while [Ca 2+ ] i elevation was moderately suppressed. Depletion of extracellular Ca 2+ or RGD peptides suppressed neither the 64 kDa PTP nor aggregation. H-7, a protein kinase C inhibitor, did not inhibit the 64 kDa PTP, while staurosporine, a potent protein kinase inhibitor, inhibited the 64 kDa PTP and Ca 2+ influx, but not aggregation, in a dose-dependent manner. It is suggested that the 64 kDa PTP is associated with platelet aggregation induced by the interaction between GPIb and vWF.

Effects of three sulfonylurea agents on arachidonic acid metabolism of platelet homogenates were evaluated using HPLC. Gliclazide had no significant inhibitory effects on arachidonic acid metabolism. Glibenclamide and glimepiride both inhibited the production of cyclooxygenase-related metabolites, thromboxane B2 (TXB2) and 12-hydroxy 5,8,10-heptadecatrienoic acid (HHT), whereas 12-hydroxy 5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase-related product, was unaffected. These findings confirmed part of our previous report using intact platelets, except that we found in the present study that glibenclamide had no inhibitory effect on 12-lipoxygenase.

To elucidate the mechanism involving synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), which is the main species of 3-phosphorylated phosphoinositides in activated blood platelets, we observed a correlation among protein-tyrosine phosphorylation, protein kinase C (PKC) activation, and PtdIns(3,4)P2 synthesis in these anucleate cells. Thrombin (1 U/ml) elicited marked protein-tyrosine phosphorylation, PKC activation, and PtdIns(3,4)P2 synthesis. In contrast, 1 microM 12-O-tetrade-canoylphorbol 13-acetate barely induced tyrosine phosphorylation and PtdIns(3,4)P2 synthesis although it strongly activated PKC. A variety of kinase inhibitors were tested for their ability to inhibit the thrombin effects. Both staurosporine and tyrphostin inhibited thrombin-stimulated tyrosine phosphorylation and PtdIns(3,4)P2 synthesis. H-7, which specifically, although weakly, inhibited PKC activation, had no effect on tyrosine phosphorylation and PtdIns(3,4)P2 production. Among the various kinase inhibitors tested, staurosporine was the most potent inhibitor of protein tyrosine phosphorylation and PtdIns(3,4)P2 synthesis, and there was a good correlation of the inhibition between these two parameters, although it also inhibited PKC activation. To examine the involvement of PtdIns 3-kinase, which is believed to play an important role in 3-phosphorylated phosphoinositide synthesis, we studied tyrosine phosphorylation and the association with tyrosine-phosphorylated proteins of the p85 alpha subunit of PtdIns 3-kinase in thrombin-stimulated platelets. We did not detect tyrosine-phosphorylated protein by Western blotting where p85 alpha was located. Similarly, when platelet lysates were precipitated with anti-p85 alpha antibodies and then blotted with anti-phosphotyrosine antibodies, tyrosine-phosphorylated p85 alpha was undetectable. Furthermore, when the cell lysates were precipitated with anti-phosphotyrosine antibodies, no p85 alpha was found in the immunoprecipitates. These results show that PtdIns(3,4)P2 synthesis in stimulated platelets is mediated by tyrosine phosphorylation, as it is in proliferating cells, but the p85 alpha subunit of PtdIns 3-kinase may not be a target for tyrosine kinases and that staurosporine, though non-specific, would be a useful tool for elucidating signal transduction involving D-3-phosphorylated phosphoinositide generation and protein-tyrosine phosphorylation in blood platelets.

A novel method to detect platelet aggregation by means of the particle counting technique using light scattering has been developed. An optical device designed to focus on a limited area of platelet-rich plasma measured the intensity of light scattered by particles passing through the area, minimizing multiple light scattering. The use of polystyrene spheres of different diameters confirmed that the light scattering intensity increases in proportion to the particle size in a suspension. Platelet activation induced by various agonists resulted in light scattering of higher intensities, which correlated well with the number and size of aggregates as observed under a microscope. These findings confirmed that the intensity of light scattering detected by the new device provides information on the number and size of aggregates in a suspension. The new method was compared with conventional platelet aggregometry using overall light scattering or changes in light transmission (optical density). The new device appeared to be particularly sensitive to small aggregates such as those formed in platelet activation induced by low concentrations of agonists. Furthermore, the new method has an advantage over the conventional aggregometry, in that it allows the aggregate size distribution and the extent of aggregation to be estimated.

Platelets exposed to shear stress aggregate in the absence of exogenously added agonists, utilizing distinct platelet membrane receptors and ligands depending upon the level of shear stress applied. Using a modified cone and plate type viscometer, we previously demonstrated that, under low shear stress (18 dyn/cm2), aggregation is mediated by platelet membrane glycoprotein (GP) IIb-IIIa and fibrinogen, whereas aggregation induced by high shear stress (108 dyn/cm2) requires the binding of von Willebrand factor (vWF) to both GPIb-IX and GPIIb-IIIa (Ikeda, Y., M. Handa, K. Kawano, T. Kamata, M. Murata, Y. Araki, H. Anbo, Y. Kawai, K. Watanabe, I. Itagaki, et al. 1991. J. Clin. Invest. 87:1234-1240). Here we report that vWF-dependent aggregation occurs under low shear stress in citrated platelet-rich plasma (PRP) from two types of congenital bleeding disorders, platelet-type von Willebrand disease (vWD) and type IIB vWD, in both of which ristocetin-induced aggregation is known to be heightened. Aggregation induced by low shear stress was enhanced in both types of disorders compared to normal controls, and the enhancement was completely abolished by anti-vWF monoclonal antibody NMC-4, which blocks the GPIb-binding site on vWF. Under high shear stress, the extent of maximal aggregation was not different between controls and the patient groups although maximal aggregation was reached much more quickly in the latter. When citrated PRP was exposed to a gradient of shear stress (6 to 108 dyn/cm2 over a 5-min period), vWF-dependent aggregation, as judged from the inhibitory effect of NMC-4, first occurred at 14 dyn/cm2 in platelet-type vWD and at 10-12 dyn/cm2 in type IIB vWD, as compared with more than 81 +/- 20.1 dyn/cm2 in control platelets. These results suggest that an abnormality in either vWF or GPIb-IX triggers the aggregation-inducing interaction of the two molecules under low shear stress, which might explain the intravascular platelet clumping, that presumably underlies the thrombocytopenia observed in these bleeding disorders.

Phosphoinositide metabolism elicited by anti-CD9 monoclonal antibody, a well-characterized platelet activator, was studied using acetylsalicylic acid-treated human platelets. TP82, which is an anti-CD9 monoclonal antibody, induced classical phosphatidylinositol 4,5-bisphosphate hydrolysis, as monitored by intracellular Ca2+ mobilization and phosphatidic acid production, and synthesis of phosphatidylinositol 3,4-bisphosphate, which is a major component of newly-described 3-phosphorylated inositol phospholipids produced during platelet activation. These changes were severely inhibited by 1 μM staurosporine, a potent, though non-selective, protein kinase inhibitor, which also abolished TP82 induction of tyrosine phosphorylation of multiple platelet proteins. Protein-tyrosine phosphorylation appears necessary to initiate both the classical phosphoinositide turnover and synthesis of the newly-described 3-phosphorylated inositol phospholipids in anti-CD9 monoclonal antibody-induced platelet activation.

Sodium fluoride (NaF) alone below the concentration of 10 mM had no effect on platelet intracellular Ca2+ ([Ca2+]i). When platelets were incubated with low concentrations of NaF (10 mM) prior to thrombin stimulation, the second phase of [Ca2+]i elevation which is attributable to Ca2+ influx was suppressed, while the initial rapid peak of [Ca2+]i which is attributable to internal Ca2+ release was unaffected. Ca2+ influx assessed by the addition of extracellular Ca2+ to cells preactivated by thrombin in the absence of extracellular Ca2+ was also inhibited by NaF in a dose-dependent manner. NaF was also effective in inhibiting thrombin- or U-46619-induced Mn2+ entry. This inhibitory effect of NaF on Ca2+ influx occurred after a lag of at least 30 s. However, Ca2+ influx induced by ionomycin-induced Ca2+ depletion or by thapsigargin, a Ca(2+)-ATPase inhibitor, was only partially suppressed by NaF treatment. It is suggested that Ca2+ entry induced by receptor-mediated activation is NaF-sensitive and that the depletion of Ca2+ storage sites by artificial procedures facilitates the opening of Ca2+ channels via NaF-insensitive pathways.