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Prevention of Platelet Glycoprotein IIb/IIIa Activation by 3,4-Methylenedioxy-β-Nitrostyrene, A Novel Tyrosine Kinase Inhibitor

Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan

Received March 2, 2006; accepted July 11, 2006

	Abstract

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Binding fibrinogen to activated glycoprotein (GP)IIb/IIIa is the final common pathway of platelet aggregation and has become a successful target for antiplatelet therapy. In the present study, we found that a small chemical compound, 3,4-methyl-enedioxy-β-nitrostyrene (MNS), exhibited potent and broad-spectrum inhibitory effects on human platelet aggregation caused by various stimulators. Moreover, addition of MNS to human platelets that had been aggregated by ADP caused a rapid disaggregation. We demonstrated that the antiaggregatory activity of MNS is due to inhibition of GPIIb/IIIa activation by measuring the binding amount of PAC-1 in platelets. In contrast, MNS is not a direct antagonist of GPIIb/IIIa, because MNS did not affect fibrinogen binding to fixed ADP-stimulated platelets. By investigating how MNS inhibits GPIIb/IIIa activation, we found that MNS potently inhibited the activity of tyrosine kinases (Src and Syk) and prevented protein tyrosine phosphorylation and cytoskeletal association of GPIIb/IIIa and talin, but it had no direct effects on protein kinase C, Ca²⁺ mobilization, Ca²⁺-dependent enzymes (myosin light chain kinase and calpain), and arachidonic acid metabolism, and it did not affect the cellular levels of cyclic nucleotides. Therefore, MNS represents a new class of tyrosine kinase inhibitor that potently prevents GPIIb/IIIa activation and platelet aggregation without directly affecting other signaling pathways required for platelet activation. Because MNS inhibits GPIIb/IIIa functions in a manner different from GPIIb/IIIa antagonists, this feature may provide a new strategy for treatment of platelet-dependent thrombosis.

The cyclooxygenase inhibitor aspirin and the ADP receptor antagonists, such as clopidogrel, are widely used antiplatelet drugs and have been shown to be beneficial in the treatment of arterial thrombotic diseases (Antiplatelet Trialists' Collaboration, 1994a,b; Antithrombotic Trialists' Collaboration, 2002). However, the efficacy of aspirin and ADP receptor antagonists is limited because there are other pathways of platelet aggregation. Several GPIIb/IIIa antagonists have been developed based on the fact that the binding of fibrinogen to GPIIb/IIIa is the final common pathway for platelet aggregation. Although intravenous GPIIb/IIIa antagonists have had a significant impact on outcomes of percutaneous coronary intervention by reducing the rate of postprocedure ischemic complications, their efficacy in the treatment of acute coronary syndromes is restricted to high-risk patients (Boersma et al., 2002). Moreover, the oral GPIIb/IIIa antagonists have been discontinued because of a lack of efficacy and increased mortality (Leclerc, 2002; Quinn et al., 2003). It has been suggested that the deleterious effects of GPIIb/IIIa antagonists may be due to paradoxical GPIIb/IIIa activation and increased fibrinogen binding to blood platelets (Honda et al., 1998; Peter et al., 1998). Therefore, there remains an unmet need for the development of more effective and safe antiplatelet agents.

In our continuing search for new antiplatelet agents, more than 1000 synthetic compounds, natural products, and plant extracts were randomly screened for their ability to inhibit platelet aggregation. Among them, 3,4-methylenedioxy-β-nitrostyrene (MNS), a synthetic small-molecule compound, was found to possess potent inhibitory effect on platelet aggregation caused by various stimulators. In this study, we demonstrate that MNS inhibits platelet aggregation by preventing GPIIb/IIIa activation and that this effect is due to the inhibition of platelet tyrosine kinases.

	Materials and Methods

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Materials. MNS (Fig. 1A), bovine -thrombin, U46619 [GenBank] , collagen (type I, bovine Achilles tendon), phorbol 12,13-dibutyrate (PDBu), 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxyl]-2-[2-amino-5-methylphenoxy]ethane-N,N,N',N'-tetraacetic acid/ace-toxymethyl ester (fluo-3/acetoxymethyl ester) were obtained from Sigma-Aldrich (St. Louis, MO). The thromboxane B₂ immunoassay kit and the cAMP/cGMP immunoassay kit were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). FITC-labeled PAC-1 was from BD Biosciences (San Jose, CA). FITC-conjugated anti-CD62P (P-selectin) was from Serotec (Oxford, UK). Monoclonal anti-talin antibody was from Sigma-Aldrich. Anti-integrin β₃, anti-phospho-myosin light chain, anti-phosphotyrosine monoclonal anti-body PY20, phospho-myristoylated alanine-rich C kinase substrate (MARCKS)-specific polyclonal antibody, anti-Syk, and anti-phospholipase C (PLC)2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-Src (Tyr416) antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA). Baculo-virus-produced human Src, Syk, and JAK2 were purchased from Upstate Biotechnology (Charlottesville, VA). Recombinant human FAK was purchased from BioSource International (Camarillo, VA). The tyrosine kinase activity assay kit was from Chemicon International (Temecula, CA). All other chemicals were purchased from Sigma-Aldrich.

View larger version (20K): [in this window] [in a new window]   Fig. 1. A, chemical structure of MNS. B, concentration-inhibition curves for MNS on human platelet aggregation. Washed human platelets were incubated with DMSO (vehicle control) or MNS at 37°C for 3 min, and then 100 µM AA, 10 µg/ml collagen, 0.1 U/ml thrombin, 2 µM U46619, or 5 µM ADP was added to trigger platelet aggregation. Values are presented as means ± S.E.M. (n = 4). C, disaggregation of ADP-aggregated platelets by MNS and PGE1. Washed platelets were challenged by 10 µM ADP to induce platelet aggregation. DMSO (control), 10 µM PGE1,or5, 10, and 20 µM MNS was added to aggregated platelets at times indicated by downward arrowheads. Representative tracings of three independent experiments are shown.

Measurement of Platelet Aggregation. Platelet aggregation was measured turbidimetrically with a light-transmission aggregometer (Chrono-Log Co., Havertown, PA). The platelet suspension was incubated with dimethyl sulfoxide (DMSO, vehicle) or MNS at 37°C for 3 min under a stirring condition (1200 rpm) before the addition of the platelet activators. The extent of platelet aggregation was measured as the maximal increase of light transmission within 5 min after the addition of inducers.

Measurement of Platelet Cyclic Nucleotide Contents. The contents of cyclic nucleotides in platelets were measured by the method described previously (Wu et al., 2004). In brief, the platelet suspension was incubated with MNS at 37°C for 3 min under a stirring condition (1200 rpm) in a light-transmission aggregometer. The reaction was stopped by adding 10 mM EDTA followed immediately by boiling for 2 min. Upon cooling to 4°C, cell debris was removed by centrifugation at 13,000g for 5 min. The supernatant was then used to assay for cAMP and cGMP using enzyme immunoassay kits.

Measurement of the Formation of Thromboxane B₂. Because thromboxane A₂ is very unstable and rapidly converted to more stable metabolite thromboxane B₂, we thus measured the latter instead of thromboxane A₂. After challenge of platelets with the aggregation inducer for 5 min, 2 mM EDTA and 50 µM indomethacin were added. The platelet suspensions were centrifuged for 3 min at 13,000 rpm, and the contents of thromboxane B₂ in the supernatants were assayed using an enzyme immunoassay kit according to the procedure described by the manufacturer.

Measurement of P-Selectin Expression and PAC-1 Binding by Flow Cytometry. Washed human platelets (3 x 10⁷ platelets/ml) were preincubated with DMSO or test compounds for 5 min and then treated with or without 0.2 U/ml thrombin in the presence of excessive amounts of FITC-conjugated monoclonal antibody (anti-CD62P or PAC-1) for 15 min at room temperature. The samples were then fixed at 4°C with 1% paraformaldehyde. Flow cytometric analysis was performed on an EPICS XL flow cytometer (Beckman Coulter, Inc., Fullerton, CA) with EXPO32 ADC software. Platelets were identified by logarithmic signal amplification for forward and side scatter. The levels of P-selectin expression and PAC-1 binding were expressed as the percentages of cells positive for anti-CD62P and PAC-1, respectively. The negative cut-off for each antibody was set using resting platelets that gave less than 4% of cells positive for binding of anti-CD62P or PAC-1.

Measurement of Intracellular Ca²⁺ Mobilization. Platelets pelleted from platelet-rich plasma were resuspended in Ca²⁺-free Tyrode's solution and then incubated with 2 µM fluo-3/acetoxymethyl ester at 37°C for 30 min. To prevent leakage of dye, 2.5 mM probenecid was added to the buffers throughout the experiments (Merritt et al., 1990). After washing twice, the fluo-3-loaded platelets were finally suspended in Ca²⁺-free Tyrode's solution at a concentration of 5 x 10⁷ platelets/ml. The fluo-3-loaded platelets were preincubated with MNS in the presence of 1 mM extracellular calcium at 37°C for 3 min before the addition of the platelet activators. Fluorescence (excitation 505 nm, emission 530 nm) was measured with a fluorescence spectrophotometer (model F4000; Hitachi, Tokyo, Japan).

Platelet Lysis and Immunoprecipitation. To prepare whole platelet lysates, the reaction was terminated at the indicated time points by adding an equal volume of 2x SDS sample buffer (1x, 2% SDS, 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, and 5% β-mercaptoethanol). The samples were boiled for 5 min and subjected to immunoblotting analysis. For preparation of immunoprecipitated proteins, platelet suspensions (containing 1 x 10⁹ cells) were lysed by adding a half-volume of ice-cold 3x radioimmunoprecipitation assay buffer (1x, 1% Triton X-100, 75 mM NaCl, 50 mM Tris-HCl, pH 7.4, 4 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM sodium orthovanadate). Lysates were incubated for 30 min on ice, clarified by centrifugation at 12,000g for 30 min, precleared with protein A-Sepharose beads, and incubated for 2 h at 4°C with anti-Syk or anti-PLC2. Immune complexes were precipitated with protein A-Sepharose beads for an additional 3 h, sedimented by brief centrifugation, and washed three times in ice-cold 1x radioimmunoprecipitation assay buffer. The anti-Syk and anti-PLC2 immunoprecipitates were subjected to immunoblotting analysis.

Fractionation of Platelets. After the indicated time of activation, platelets were lysed with an equal volume of 2x ice-cold lysis buffer (1x, 1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM sodium orthovanadate) and kept on ice for 30 min. The lysates were centrifuged at 15,000g for 5 min at 4°C. The resulting pellet (the Triton-insoluble fraction) was washed with 1x lysis buffer, solubilized in 1x SDS sample buffer, and subjected to immunoprecipitation analysis as described above.

Immunoblotting. Whole platelet lysates, subcellular fractions, and immunoprecipitates from the same cell equivalents were electrophoresed on a SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Trans-Blot; Bio-Rad, Hercules, CA). The membranes were blocked overnight in 5% nonfat dry milk in Tris-buffered saline supplemented with 0.1% Tween 20 (TBST), washed three times in TBST, and incubated for 1 h in the antibody solution of interest in TBST with 1% bovine serum albumin. The membrane was immunoblotted with primary antibodies against phosphotyrosine, Syk, phospho-Src, PLC2, integrin β3, talin, phospho-MARCKS, or phospho-myosin light chain. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. After washing with TBST, protein bands on the membrane were visualized by an enhanced chemiluminescence Western blotting detection system (Western Lightning; PerkinElmer Life and Analytical Sciences, Boston, MA).

Tyrosine Kinase Assay. For immune complex kinase assay, platelets treated with MNS, PP1, or piceatannol were lysed and immunoprecipitated with either anti-Src or anti-Syk antibody. In some experiments, recombinant human Src, Syk, FAK, or JAK2 was used and treated with MNS or tyrosine kinase inhibitors for 5 min. The activity of the immunoprecipitated kinases or the recombinant enzymes was determined by using a tyrosine kinase assay kit (Chemicon International). Samples were incubated with biotinylated poly[Glu:Tyr] (4:1), an exogenous substrate, in kinase assay buffer (20 mM HEPES, 5 mM MgCl₂, 5 mM MnCl₂, 0.1 mM sodium orthovanadate, and 1 mM dithiothreitol) for 37°C for 30 to 45 min. After stopping the enzyme reaction with EDTA (final concentration of 20 mM), both the phosphorylated and dephosphorylated substrates were immobilized by binding to the streptavidin-coated plate. The fraction of phosphorylated substrate is determined using a phosphotyrosine monoclonal antibody conjugated to HRP and an ensuing chromogenic substrate reaction.

Fibrin Clot Retraction. Washed human platelets (3 x 10⁸ platelets/ml) were suspended in Tyrode's solution containing 250 µg/ml fibrinogen and incubated with DMSO or MNS for 3 min without stirring in a glass tube. Fibrin clot formation was induced by adding 1 U/ml thrombin. Clot retraction was allowed to develop at 37°C for 4 h, and pictures were taken at 0.5, 1, 2, 3, and 4 h using a digital camera. The images were analyzed, and the areas occupied by clots were calculated using Scion Image software (Scion Corporation, Frederick, MD). Data were expressed as follows: percentage of clot retraction = [1 - (area t/area t₀)] x 100.

Statistics. Results are expressed as the mean ± S.E.M. and comparisons were made using Student's t test. A probability of 0.05 or less was considered significant.

	Results

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Effect of MNS on the Aggregation of Washed Human Platelets. In washed human platelets, MNS completely inhibited 2 µM U46619 [GenBank] -(a thromboxane A₂ mimic), 5 µM ADP-, 100 µM arachidonic acid-(AA), 10 µg/ml collagen-, and 0.1 U/ml thrombin-induced platelet aggregation in a concentration-dependent manner with IC₅₀ values of 2.1 ± 0.5, 4.1 ± 0.5, 5.8 ± 0.9, 7.0 ± 0.4, and 12.7 ± 1.1 µM, respectively (Fig. 1B). In addition, platelet aggregation caused by either the calcium ionophore A23187 [GenBank] (1 µM) or the protein kinase C (PKC) activator PDBu (200 nM) was also inhibited by MNS with IC₅₀ values of 25.9 ± 3.1 and 4.8 ± 0.6 µM, respectively.

Moreover, the addition of 5 to 20 µM MNS to human platelets that had been aggregated by ADP caused rapid disaggregations in a concentration-dependent manner (Fig. 1C). This ability of MNS to induce platelet disaggregation was even more potent than that of prostaglandin E₁ (PGE₁).

Effect of MNS on P-Selectin Expression and Thromboxane Formation in Platelets. We next examined whether MNS could inhibit the processes of platelet activation, such as -granule secretion and thromboxane formation. In the present study, the -granule secretion was monitored by the surface expression of P-selectin, a major platelet -granule protein that is crucial in promoting platelet-leukocyte interactions and fibrin accumulation (Shebuski and Kilgore, 2002; Andre, 2004). As shown in Fig. 2, thrombin-induced P-selectin expression on platelets was decreased by MNS to levels comparable to those observed in PGE₁-treated platelets.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effect of MNS on P-selectin expression. Washed human platelets were preincubated with DMSO or test compounds for 5 min and then treated with or without 0.2 U/ml thrombin in the presence of FITC-conjugated anti-CD62P for 15 min at room temperature. The samples were then fixed at 4°C with 1% paraformaldehyde. P-selectin expression was measured by flow cytometry as described under Materials and Methods. Results are presented as mean ± S.E.M. (n = 3). #, P < 0.001 compared with the resting value. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with the control.

Effect of MNS on Platelet GPIIb/IIIa. Platelet GPIIb/IIIa activation was monitored by the binding of FITC-labeled PAC-1, because this monoclonal antibody only binds to the activated form of GPIIb/IIIa. Figure 3A shows that 1 to 20 µM MNS inhibited thrombin-induced PAC-1 binding to platelets in a concentration range similar to that observed with the inhibition of platelet aggregation.

View larger version (14K): [in this window] [in a new window]   Fig. 3. A, effect of MNS on GPIIb/IIIa activation. Washed human platelets were preincubated with DMSO or test compounds for 5 min and then treated with or without 0.2 U/ml thrombin in the presence of FITC-conjugated PAC-1 for 15 min at room temperature. The samples were then fixed at 4°C with 1% paraformaldehyde. The percentage of PAC-1-positive platelets was analyzed by flow cytometry as described under Materials and Methods. Results are presented as mean ± S.E.M. (n = 3). #, P < 0.001 compared with the resting value. ***, P < 0.001 compared with the control. B, effects of MNS, PGE1, and RGDS on fibrinogen-induced aggregation in ADP-stimulated fixed platelets. Washed human platelets were treated with 20 µM ADP and then fixed with 0.8% para-formaldehyde for 30 min at room temperature. The fixed platelets were washed and resuspended in Tyrode's solution. The fixed platelets were treated with DMSO (control), 20 µM MNS, 10 µM PGE1, or 100 µM RGDS before (left) or after (right, at times indicated by downward arrow-heads) addition of 200 µg/ml fibrinogen. Representative tracings of three independent experiments are shown.

Effect of MNS on Cyclic Nucleotide Levels in Platelets. To assess whether the action of MNS was due to elevation of intracellular levels of cAMP and/or cGMP, which are two major inhibitory messengers in regulating platelet aggregation, the effect of MNS on cyclic nucleotide levels in platelets was examined. In washed human platelets, we found that MNS affected neither cAMP levels (picomoles/8 x 10⁸ platelets, 4.77 ± 0.18 versus basal 4.61 ± 0.98; n = 4) nor cGMP levels (picomoles/8 x 10⁸ platelets, 0.65 ± 0.09 versus basal 0.88 ± 0.14; n = 4). These results indicate that the antiplatelet effect of MNS is not related to cyclic nucleotides.

Effect of MNS on the Intracellular Free Calcium of Platelets. As shown in Fig. 4, in the presence of 1 mM extracellular calcium, pretreatment of fluo-3-loaded platelets with 20 µM MNS did not inhibit the calcium signal elicited by thrombin, ADP, U46619 [GenBank] , or AA. In contrast, collagen-induced intracellular calcium mobilization was almost abolished by MNS treatment.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of MNS on intracellular calcium mobilization in human platelets. Fluo-3-loaded human platelets were incubated with DMSO or 20 µM MNS at 37°C for 3 min in the presence of 1 mM extracellular Ca2+, and 100 µM AA, 10 µg/ml collagen, 0.1 U/ml thrombin, 5 µM ADP, or 2 µM U46619 was then added to trigger the increase of [Ca2+]i (fluorescence, F). Similar results were obtained in three separate experiments.

As shown in Fig. 5A, MNS had no apparent inhibitory effect on MLC phosphorylation induced by either thrombin or A23187. [GenBank] In contrast, the MLC kinase inhibitor ML-7 (50 µM) markedly inhibited MLC phosphorylation elicited by these two inducers.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Effects of MNS on thrombin- or A23187-induced myosin light chain (MLC) phosphorylation (A) and talin proteolysis (B). Human platelets were preincubated with 20 µM MNS, 50 µM ML-7, or 100 µM calpeptin at 37°C for 3 min and then stimulated with either 1 µM A23187 or 0.1 U/ml thrombin for another 30 s. After stimulation, platelets were lysed and electrophoresed as described under Materials and Methods. Western blot analysis was performed on platelet lysates using anti-phospho-MLC antibody and anti-talin antibody, respectively. Similar results were obtained in three separate experiments.

Effect of MNS on Protein Kinase C Activation. In addition to calcium signaling, agonist-induced PKC activation may also contribute to the regulation of GPIIb/IIIa activation (van Willigen et al., 1996; Hers et al., 1998). We thus examined the effect of MNS on PKC activation by measuring the phosphorylation of MARCKS, which is a major substrate of PKC (Elzagallaai et al., 2000). As shown in Fig. 6, 20 µM MNS markedly inhibited thrombin-but not PDBu-induced MARCKS phosphorylation. In contrast, the PKC inhibitor GF109203X (10 µM) abolished MARCKS phosphorylation caused by these two inducers. Based on the fact that PDBu-induced platelet aggregation is completely prevented by MNS, these results indicate that MNS inhibits GPIIb/IIIa activation without directly inhibiting PKC.

View larger version (59K): [in this window] [in a new window]   Fig. 6. Effect of MNS on thrombin- or PDBu-induced MARCKS phosphorylation. Washed platelets were preincubated with 20 µM MNS or 10 µM GF109203X for 3 min at 37°C and then stimulated with either 0.1 U/ml thrombin or 200 nM PDBu for another 30 s. Western blot analysis was performed on platelet lysates using anti-phospho-MARCKS antibody. Similar results were obtained in three separate experiments.

Effect of MNS on Protein Tyrosine Phosphorylation in Platelets. Figure 7A shows that thrombin stimulation of platelets leads to time-dependent tyrosine phosphorylation of a number of proteins with molecular masses of 130, 110, 90, 80, 75, and 60 kDa. A similar profile was observed after collagen-induced platelet activation. Pretreatment of platelets with 20 µM MNS markedly inhibited protein tyrosine phosphorylation at either 0.5 or 3 min after thrombin or collagen stimulation. The concentration of MNS required to inhibit protein tyrosine phosphorylation was comparable to that required to inhibit platelet aggregation.

View larger version (53K): [in this window] [in a new window]   Fig. 7. Effects of MNS and genistein on thrombin- and collagen-induced protein tyrosine phosphorylation in human platelets. A, washed platelets were preincubated with DMSO, 20 µM MNS, or genistein (600 µM for thrombin treatment, but 20 µM for collagen treatment) at 37°C for 3 min. Then, platelets were treated with or without 0.1 U/ml thrombin or 10 µg/ml collagen for the indicated periods. Western blot analysis was performed on whole platelet lysates using a monoclonal anti-phosphotyrosine antibody (PY20). B, washed platelets were preincubated with DMSO, 20 µM MNS, or 600 µM genistein at 37°C for 3 min. After treatment with or without 0.1 U/ml thrombin, platelets were lysed with Triton X-100 lysis buffer. The Triton X-100-insoluble (cytoskeletal) fraction was separated with centrifugation. Western blot analysis was performed on cytoskeletal fractions using PY20. Similar results were obtained in three separate experiments.

The tyrosine kinase inhibitor genistein at 20 µM inhibited collagen-induced platelet aggregation by 81% and markedly prevented protein tyrosine phosphorylation (Fig. 7A). However, a 30-fold higher concentration (600 µM) of genistein was required to significantly prevent thrombin-induced protein tyrosine phosphorylation. At 600 µM, genistein partially inhibited thrombin-induced platelet aggregation by 56%.

We also examined the effect of MNS on tyrosine phosphorylated proteins in the cytoskeleton. Upon thrombin stimulation, there was a time-dependent increase in the levels of phosphotyrosine of multiple proteins in the cytoskeletal fraction (Fig. 7B). Pretreatment of platelets with 20 µM MNS almost completely reduced the quantity of tyrosine-phosphorylated proteins in the cytoskeleton at either 0.5 or 3 min after thrombin stimulation. Genistein (600 µM) also decreased tyrosine-phosphorylated proteins in the cytoskeleton but to a lesser extent than that observed with MNS (Fig. 7B).

Effect of MNS on Thrombin-Induced Cytoskeletal Association of GPIIb/IIIa and Talin. Upon stimulation of platelet with thrombin, GPIIb/IIIa and talin were detectable in Triton X-100-insoluble cytoskeletal fraction within 0.5 min and became more pronounced at 3 min (Fig. 8A). Both 20 µM MNS and 600 µM genistein markedly inhibited talin and GPIIb/IIIa redistribution to cytoskeletal fractions.

View larger version (40K): [in this window] [in a new window]   Fig. 8. A, effect of MNS on thrombin-induced cytoskeletal association of talin and GPIIb/IIIa. Washed platelets were preincubated with DMSO, 20 µM MNS, or 600 µM genistein at 37°C for 3 min. After treatment with or without 0.1 U/ml thrombin, platelets were lysed with Triton X-100 lysis buffer, and the cytoskeletal fraction was isolated. Western blot analysis was performed on cytoskeletal fractions using anti-talin anti-body and anti-β3 integrin antibody, respectively. Similar results were obtained in three separate experiments. B, effect of MNS on fibrin clot retraction. Washed platelets were suspended in Tyrode's solution containing 250 µg/ml fibrinogen and incubated with DMSO or MNS for 3 min without stirring in a glass tube. Fibrin clot formation was induced by adding 1 U/ml thrombin. Clot retraction was allowed to develop at 37°C for 4 h. Top, time frames of a retracting clot at the indicated time points. Bottom, kinetic curves of retraction in the presence of different concentrations of MNS were generated by plotting clot areas versus time. Results represent the means from two independent experiments.

Effect of MNS on Activation of Src, Syk, and PLC2. To investigate the mechanism of inhibition of agonist-induced protein tyrosine phosphorylation by MNS, the activation of two major tyrosine kinases, Src and Syk, in platelets was examined. We assessed the autophosphorylation of Src in thrombin- or collagen-stimulated platelets by Western blotting using a specific anti-phospho-Src (Tyr416) antibody, which specifically detects Src phosphorylated at Tyr416, a process indicative of Src activation (Osusky et al., 1995). Figure 9A shows that stimulation of platelets with thrombin or collagen induced Tyr416 phosphorylation of Src within 1 min. MNS pretreatment of platelets led to marked inhibition of Src activation by both these two agonists. To further determine the effect of MNS on endogenous Src activity, the immune complex kinase assay was performed. As shown in Fig. 9B, thrombin treatment of human platelets led to a marked increase in tyrosine phosphorylation and kinase activity of Src. As expected, both MNS and the Src inhibitor PP1 significantly inhibited thrombin-induced Src phosphorylation and kinase activity.

View larger version (34K): [in this window] [in a new window]   Fig. 9. A, effect of MNS on the activation of Src. Washed platelets were preincubated with DMSO, 20 µM MNS, or 20 µM PP1 at 37°C for 3 min and then stimulated with 0.1 U/ml thrombin or 30 µg/ml collagen for the indicated periods. Western blot analysis was performed on platelet lysates using a specific anti-phospho-Src antibody (Tyr416). B, platelet lysates were immunoprecipitated with anti-Src antibody, and the immunoprecipitates were subjected to Western blot analysis for PY20 or in vitro kinase assay. Results of the in vitro kinase assay are presented as mean ± S.E.M. (n = 3). C, effect of MNS on Syk phosphorylation and kinase activity. Washed platelets were preincubated with DMSO, 20 µM MNS, or 100 µM piceatannol at 37°C for 3 min and then stimulated with 0.1 U/ml thrombin or 30 µg/ml collagen for the indicated periods. Platelet lysates were immunoprecipitated with anti-Syk antibody, and the immunoprecipitates were subjected to Western blot analysis for PY20 or in vitro kinase assay. Results of the in vitro kinase assay are presented as mean ± S.E.M. (n = 3). D, effect of MNS on PLC2 phosphorylation. PLC2 was immunoprecipitated from lysates made from platelets stimulated with 0.1 U/ml thrombin or 30 µg/ml collagen in the presence of DMSO or 20 µM MNS. The immunoprecipitates were subjected to Western blot analysis for PY20.

In human platelets, PLC2 is downstream of Syk and is essential for both collagen-elicited inositol 1,4,5-trisphosphate formation and intracellular Ca²⁺ mobilization. As shown in Fig. 9D, both thrombin and collagen induced tyrosine phosphorylation of PLC2 in human platelets. Treatment of platelets with MNS resulted in attenuation of PLC2 phosphorylation induced by these two agonists.

Direct Effect of MNS on Purified Tyrosine Kinases. We then examined the direct inhibition of MNS on tyrosine kinase activity by using recombinant human Src and Syk. The results in Table 1 demonstrate that MNS at 20 µM profoundly inhibited Syk activity by more than 90% with an IC₅₀ value of 2.5 µM, which was approximately 15-fold more potent than piceatannol, a well known inhibitor of Syk. At higher concentrations, MNS also inhibited Src activity, but the potency was less pronounced (IC₅₀ = 29.3 µM). To further examine the selectivity of MNS for other tyrosine kinases, recombinant human FAK and JAK2 were used. Table 1 shows that even at the highest concentration (50 µM) tested, MNS only inhibited FAK and JAK2 by 36.6 ± 17.7 and 5.2 ± 2.2%, respectively. These data indicate that MNS selectively inhibits the kinase activity of Syk and, to a lesser extent, Src, but not FAK and JAK2. In contrast, genistein inhibited Src, Syk, and FAK with similar IC₅₀ values, indicating it is a nonselective inhibitor of tyrosine kinases.

	Discussion

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In the present study, we show that MNS, a β-nitrostyrene derivative, exhibited potent and broad-spectrum inhibitory effects on human platelet aggregation caused by various receptor agonists and chemical reagents (a PKC activator and aCa²⁺ ionophore) that bypass receptor-mediated processes. In addition to preventing platelet aggregation, MNS also induced rapid disaggregation of aggregated platelets. These results imply that MNS may act on GPIIb/IIIa, the final common pathway of platelet aggregation. In the following experiment, we have demonstrated that the anti-aggregatory effect of MNS is due to inhibition of GPIIb/IIIa activation by measuring the binding amount of PAC-1 in platelets. On the other hand, MNS is apparently not a direct antagonist of GPIIb/IIIa, because MNS did not affect fibrinogen binding to fixed ADP-stimulated platelets.

The activation of GPIIb/IIIa is controlled in a complex manner by positive and negative signaling elements in blood platelets. Elevation of intracellular cyclic nucleotide levels, either by stimulation of cyclases or by inhibition of phosphodiesterases, is the most potent inhibitory mechanism of GP IIb/IIIa activation (Haslam et al., 1999; Schwarz et al., 2001). However, the failure of MNS to increase cAMP and cGMP levels in platelets excludes the possibility that cyclic nucleotides are involved in the action of MNS. Several positive signaling molecules have been implicated in the regulation of GPIIb/IIIa function; among them, phosphatidylinositide-specific PLC plays a central role (Nozawa et al., 1993). Two major forms of PLC, β and , have been identified in human platelets. The β forms are regulated by G_q protein-coupled receptors for soluble agonists (thrombin, ADP, and thromboxane A₂), whereas the forms (predominantly PLC2) are activated by tyrosine phosphorylation in response to adhesion receptors for subendothelial matrix (collagen and von Willebrand factor) (Levy-Toledano, 1999; Gibbins, 2004). Once activated, PLC hydrolyzed phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol-1,4,5-triphosphate, which promote activation of PKC and the increase of cytosolic Ca²⁺, respectively. We show here that collagen-induced tyrosine phosphorylation of PLC2 and Ca²⁺ signaling were both abolished by MNS, indicating that the activity of PLC2 had been inhibited. In contrast, MNS did not inhibit the increase in intracellular Ca²⁺ caused by soluble agonists, indicating that MNS has no inhibitory effect on PLCβ activation. Furthermore, calcium-induced activation of both MLCK and calpain were not affected by MNS, even though platelet aggregation was inhibited. These results suggest that MNS is able to prevent GPIIb/IIIa activation and platelet aggregation by acting at sites distal to the PLC/Ca²⁺ pathway. It is noteworthy that we found that the calpain inhibitor calpeptin did not inhibit either thrombin- or A23187 [GenBank] -induced platelet aggregation, even though calpain activation was completely prevented. This result is inconsistent with that of Croce et al. (1999), and the reason for this discrepancy is not clear. To examine the effect of MNS on the PKC pathway, we found that although MNS was able to prevent thrombin-induced MCRCKS phosphorylation, it did not inhibit PDBu-induced MCRCKS phosphorylation despite the fact that platelet aggregation was abolished. The indirect inhibition of thrombin-induced PKC activation by MNS might result from its interfering with tyrosine kinases, because PKC and PKC are known to be functionally regulated by tyrosine phosphorylation (Murugappan et al., 2005; Pula et al., 2005). Moreover, we also found that the Src inhibitor PP1 decreased thrombin-induced MARCKS phosphorylation in platelets (data not shown).

Protein tyrosine phosphorylation is another important stimulation pathway involved in the regulation of GPIIb/IIIa activation and platelet aggregation (Jackson et al., 1996; Shattil et al., 1998). Platelets contain high amounts of different nonreceptor tyrosine kinases, including Src family kinases (Src, Fyn, Lyn, Hck, and Yes), Syk, FAK family kinases (FAK and PYK), and JAK family kinases (JAK and TYK). Upon stimulation of platelets with agonists, there is a dramatic increase in the levels of phosphotyrosine of multiple platelet proteins. A role for protein tyrosine phosphorylation in inside-out GPIIb/IIIa signaling is suggested by the observation that tyrosine kinase inhibitors decrease GPIIb/IIIa activation and platelet aggregation (Asahi et al., 1992; Rendu et al., 1992; Furman et al., 1994). Furthermore, Syk null murine platelets have been shown to cause a reduction in fibrinogen binding in response to ADP and epinephrine (Law et al., 1999). Protein tyrosine phosphorylation also plays a critical role in outside-in GPIIb/IIIa signaling events, such as cytoskeletal recruitment of various proteins (talin, GPIIb/IIIa, and PLC), cytoskeleton reorganization, and clot retraction (Schoenwaelder et al., 1994; Banno et al., 1996; Santos et al., 2000). In our experiments, MNS effects on agonist-induced total protein tyrosine phosphorylation in platelets and activation of Src and Syk paralleled a reduction in the activation of GPIIb/IIIa and platelet aggregation, indicating that the inhibition of inside-out GPIIb/IIIa signaling by MNS is due to the inhibition of tyrosine kinases and protein tyrosine phosphorylation. Furthermore, platelets treated with MNS resulted in a complete absence of thrombin-induced tyrosine-phosphorylated proteins in the cytoskeletal fraction and largely prevented the cytoskeletal association of talin and β₃ integrin, suggesting that MNS disrupted cytoskeletal reorganization and the interaction between GPIIb/IIIa and cytoskeleton. This would contribute to the significant inhibition of clot retraction, which is dependent on the anchorage of GPIIb/IIIa to the contractile cytoskeleton (Tuszynski et al., 1984).

Several tyrosine kinase inhibitors have been used to investigate the role of protein tyrosine phosphorylation in platelet functions; however, these compounds usually suffer limitations, such as lack of potency, specificity, and/or cell permeability. For example, it has been demonstrated that besides inhibiting tyrosine kinases, genistein also antagonizes the thromboxane A₂ receptor on platelets (Nakashima et al., 1991; McNicol, 1993). In addition, erbstatin was found to directly inhibit protein kinase C besides tyrosine kinases (Bishop et al., 1990). In this study, we show that MNS is more potent than genistein in inhibiting agonist-induced protein tyrosine phosphorylation in platelets. In the pure enzyme system, MNS is also more potent than genistein and piceatannol in inhibiting the activities of tyrosine kinases, especially that of Syk. Moreover, MNS does not inhibit PLCβ/Ca²⁺ signaling and arachidonic acid metabolism and has no direct effect on PKC, MLCK, and calpain, suggesting its high specificity for tyrosine kinases. In addition, MNS can readily penetrate the plasma membrane, because it effectively inhibits tyrosine kinases both in vitro as well as in intact cells.

As noted in previous reports (Rendu et al., 1992; Ozaki et al., 1993) and in this study, although tyrosine kinase inhibitors, including genistein, PP1, and piceatannol, are effective in inhibiting collagen-induced platelet aggregation (IC₅₀ of 13.7 ± 0.6, 2.1 ± 0.4, and 17.2 ± 2.3 µM, respectively; n = 3), they are much less effective in inhibiting platelet aggregation caused by thrombin (IC₅₀ > 200 µM). In contrast, although MNS had distinct effect on Ca²⁺ signaling triggered by thrombin and collagen, it inhibited platelet aggregations by these two stimulators in a similar range of concentration. These data indicate that tyrosine kinases sensitive to MNS may be involved in the final common steps leading to the activation of GPIIb/IIIa. The discrepancy between MNS and other tyrosine kinase inhibitors in inhibition of thrombin-induced platelet aggregation can probably be attributed to their distinct specificity and efficacy toward diverse tyrosine kinases, which is supported by the observation that treatment with MNS caused a different pattern of inhibition of protein tyrosine phosphorylation in agonist-stimulated platelets compared with genistein. Further investigation of the effect of MNS for different tyrosine kinases in platelets may help to elucidate the precise mechanism for how and which tyrosine kinases are involved in the final steps of inside-out GPIIb/IIIa signaling pathway.

In conclusion, MNS represents a new class of tyrosine kinase inhibitor that potently prevents GPIIb/IIIa activation and platelet aggregation caused by various stimulators without directly affecting other signaling pathways required for platelet activation. In addition, MNS not only inhibits platelet aggregation but also promotes platelet disaggregation, indicating that MNS has the ability to reverse the processes of GPIIb/IIIa and thereby has the potential to accelerate thrombolysis. Because MNS inhibits GPIIb/IIIa functions in a manner different from GPIIb/IIIa antagonists, it might avoid adverse effects of the latter, such as paradoxical GPIIb/IIIa activation. These features of MNS may provide a new strategy for treatment of platelet-dependent thrombosis.

	Footnotes

 
This work was supported by National Science Council of Taiwan Grant NSC 94-2320-B-037-041.

ABBREVIATIONS: GP, glycoprotein; MNS, 3,4-methylenedioxy-β-nitrostyrene; U46619 [GenBank] , 9,11-dideoxy-9, 11-methanoepoxy PGF₂; PDBu, phorbol 12,13-dibutyrate; FITC, fluorescein isothiocyanate; JAK, Janus tyrosine kinase; MARCKS, myristoylated alanine-rich C kinase substrate; DMSO, dimethyl sulfoxide; PLC, phospholipase C; TBST, Tris-buffered saline supplemented with 0.1% Tween 20; AA, arachidonic acid; A23187 [GenBank] , calcimycin; PKC, protein kinase C; PGE, prostaglandin E₁; RGDS, Arg-Gly-Asp-Ser; MLCK, myosin light chain kinase; GF109203X, 3-[1-[3-(dimethyl-aminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)homopiperazine.

Address correspondence to: Dr. Chin-Chung Wu, Graduate Institute of Natural Products, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd., Kaohsiung, 807, Taiwan. E-mail: ccwu{at}kmu.edu.tw

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