Inhibitory signaling of 17β-estradiol in platelet activation: the pivotal role of cyclic AMP-mediated nitric oxide synthase activation

Arterial thromboses are mostly composed of platelets adherent to ruptured endothelial surfaces. Platelets are anucleated cells; therefore, they represent an excellent and unique model to selectively investigate the signaling pathways mediating the nongenomic effects of estrogen. The aim of this study was to examine the signal transduction pathways of 17β-estradiol in preventing platelet activation. In this study, 17β-estradiol (5~10 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin). 17β-Estradiol-inhibited collagen-stimulated platelet activation accompanied by [Ca(2+)]i mobilization, thromboxane A? (TxA?) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. 17β-Estradiol markedly increased cyclic AMP and cyclic GMP levels, nitric oxide (NO) release, vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and endothelial nitric oxide synthase (eNOS) expression. SQ 22536, an inhibitor of adenylate cyclase, markedly reversed the 17β-estradiol-mediated effects (i.e., platelet aggregation, and PLCγ2, VASP, and eNOS phosphorylation). Furthermore, ICI 182,780, a pure estrogen receptor antagonist, also reversed the 17β-estradiol-mediated effects on platelet aggregation and eNOS activation. In conclusion, the most important findings of this study demonstrate for the first time that the inhibitory effect of 17β-estradiol in platelet activation involves activation of the cyclic AMP-eNOS/NO-cyclic GMP pathway, resulting in inhibition of PLCγ2 and p38 MAPK activation, which may lower the incidence of cardiovascular events in postmenopausal women.     

The mechanism of anti-platelet activity of davallialactone: involvement of intracellular calcium ions, extracellular signal-regulated kinase 2 and p38 mitogen-activated protein kinase

This study was designed to investigate the effect of davallialactone, which was isolated from the mushroom Inonotus xeranticus, on platelet aggregation induced by collagen, thrombin and ADP. We found that davallialactone dose-dependently inhibited platelet aggregation that was stimulated either by collagen (2.5 microg/ml), a potent ligand of integrin alpha2beta1 and glycoprotein VI, or by thrombin (0.1U/ml), a potent agonist of the protease-activated receptors (PARs) PAR1 and PAR3. In addition, davallialactone inhibited platelet aggregation induced by ADP, an agonist of P2Y receptor. To understand the mechanism of anti-platelet activity, we determined whether davallialactone affected the downstream signaling in collagen-activated platelets. Using the fura-2/AM fluorometric assay, we found that davallialactone dose-dependently inhibited intracellular calcium concentration levels ([Ca2+]i). Moreover, davallialactone inhibited the phosphorylation of extracellular signal-regulated protein kinase (ERK)-2 and p38 mitogen-activated protein kinase (MAPK), in a dose-dependent manner. The tyrosine phosphorylation of 60 and 85kDa proteins, which were activated by collagen, were differentially inhibited by davallialactone. Taken together, these data suggest that davallialactone may have potential anti-platelet aggregation activity via suppression of intracellular downstream signaling pathways.     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

PAR1, but not PAR4, activates human platelets through a Gi/o/phosphoinositide-3 kinase signaling axis

Thrombin-mediated activation of platelets is critical for hemostasis, but the signaling pathways responsible for this process are not completely understood. In addition, signaling within this cascade can also lead to thrombosis. In this study, we have defined a new signaling pathway for the thrombin receptor protease activated receptor-1 (PAR1) in human platelets. We show that PAR1 couples to G(i/o) in human platelets and activates phosphoinositide-3 kinase (PI3K). PI3K activation regulates platelet integrin alphaIIbbeta3 activation and platelet aggregation and potentiates the PAR1-mediated increase in intraplatelet calcium concentration. PI3K inhibitors eliminated these effects downstream of PAR1, but they had no effect on PAR4 signaling. This study has identified an important role for the direct activation of G(i/o) by PAR1 in human platelets. Given the efficacy of clopidogrel, which blocks the G(i/o)-coupled P2Y purinoceptor 12, as an antiplatelet/antithrombotic drug, our data suggest that specifically blocking only PAR1-mediated G(i/o) signaling could also be an effective therapeutic approach with the possibility of less unwanted bleeding.     

Expression of amyloid beta peptide in human platelets: pivotal role of the phospholipase Cgamma2-protein kinase C pathway in platelet activation.

The amyloid beta peptide (Abeta), a mediator of neuronal and vascular degeneration in the pathogenesis of Alzheimer's disease and cerebral amyloid angiopathy may have peripheral actions. Platelets are enriched with Abeta and have been shown to enhance platelet actions. However, the detailed signaling pathways through which Abeta activates platelets have not been previously explored. In this study, we examined the intra-platelet Abeta distribution using a gold labeling technique and noted that Abeta was predominantly localized in the cytoplasm of resting platelets. A marked increase in Abeta-gold labeling in an open canalicular system was observed in collagen-activated platelets. Exogenous Abeta (2-10 microM) stimulated platelet aggregation accompanied by phospholipase Cgamma2 (PLCgamma2) phosphorylation, phosphoinositide breakdown, and [Ca(2+)]i mobilization as well as protein kinase C (PKC) activation. Ro318220, an inhibitor of PKC, suppressed Abeta-induced platelet aggregation, PKC activation, and [Ca(2+)]i mobilization in platelets, suggesting that the PLCgamma2-PKC pathway is involved in Abeta-induced platelet aggregation. In the electron spin resonance study, Abeta (2 and 10 microM) markedly triggered hydroxyl radical formation in platelets. In an in vivo study, Abeta (2mg/kg) significantly shortened the latency for inducing platelet plug formation in the mesenteric venules of mice. In conclusion, we are the first to demonstrate (1) the distribution of Abeta in human platelets; and that (2) Abeta activation of platelets is mediated, at least partially, by the PLCgamma2-PKC pathway; and (3) Abeta triggers thrombus formation in vivo.     

The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation

BACKGROUND AND PURPOSE

Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated.

EXPERIMENTAL APPROACH

The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca²⁺ mobilization respectively.

KEY RESULTS

When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca²⁺ rises. Blockade of ADP P2Y₁₂ receptor with 2-methylthioadenosine 5′-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5′-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

These results suggest that PAR4 acts in parallel with the P2Y₁₂/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.     

Inhibition of agonist-induced p42 and p38 mitogen-activated protein kinase phosphorylation and CD40 ligand/P-selectin expression by cyclic nucleotide-regulated pathways in human platelets

Platelet activation and adhesion to endothelial cells and extracellular matrix proteins are crucial events in the development of arterial cardiovascular diseases. Platelet activation is initiated by stimulation of intracellular signaling cascades, including the p42 mitogen-activated protein kinase (MAPK) and p38 MAPK pathways, followed by major changes in the platelet cytoskeleton and expression and activation of platelet surface receptors, such as P-selectin (CD62P) and CD40 ligand (CD40L). Activated platelets directly bind to vascular endothelial cells via CD40L/CD40 interactions and induce inflammatory reactions that initiate or aggravate atherosclerotic lesions. The aim of this study was to investigate effects of two known platelet inhibitors-the cAMP-elevating prostaglandin E(1) (PG-E(1)) and the cGMP-elevating sodium nitroprusside (SNP)-on platelet p42 MAPK and p38 MAPK activation as well as on surface expression of CD62P and CD40L. MAPK activation was analyzed by Western blot experiments using phosphorylation-specific antibodies, and surface CD40L and CD62P expression was determined by flow cytometry analysis. PG-E(1) and SNP strongly inhibited p42 and p38 MAPK phosphorylation as well as CD40L and CD62P expression in response to thrombin, a thromboxane A(2) analog, and ADP. These data indicate that adenosine and guanosine 3',5'-cyclic monophosphate-dependent protein kinases not only inhibit platelet pathways leading to activation and aggregation, but also those resulting in enhanced surface expression of protein ligands involved in inflammation. Expression of CD40L and CD62P was found to be independent of MAPK activation, since it was not inhibited by specific MAPK inhibitors. Inhibition of platelet-induced inflammatory responses including CD62P- and CD40L-mediated interaction of platelets with leukocytes and endothelial cells, respectively, is suggested to be an important component of the long-term vasoprotective effects of cyclic nucleotide-elevating prostaglandins and NO donors.     

Involvement of p38 MAPK phosphorylation and nitrate formation in aristolochic acid-mediated antiplatelet activity

Aristolochic acid (AsA) is produced from Aristolochia fangchi, and has been used as a Chinese herbal medicine. AsA possesses various biological activities including antiplatelet, antifungal, and anti-inflammatory properties. The aim of this study was to examine the mechanisms of AsA in inhibiting platelet aggregation. AsA (75 - 150 microM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen (1 microg/mL) than other agonists. AsA (115 and 150 microM) inhibited collagen-induced platelet activation accompanied by [Ca+2)]i mobilization, thromboxane A2 (TxA2) formation and phosphoinositide breakdown. On the other hand, AsA also markedly increased levels of NO/cyclic GMP, and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. AsA inhibited p38 MAPK but not ERK1/2 phosphorylation in washed platelets. In conclusion, the most important findings of this study suggest that the inhibitory effects of AsA possibly involve the (1) inhibition of the p38 MAPK-cytosolic phospholipase A2-arachidonic acid-TxA2-[Ca+2)]i cascade, and (2) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C. These results imply that Aristolochia fangchi treatment alone or in combination with other antiplatelet drugs, may result in alteration of hemostasis in vivo.     

Induction of central signalling pathways and select functional effects in human platelets by beta-boswellic acid

We have recently shown that in polymorphonuclear leukocytes, 11-keto boswellic acids (KBAs) induce Ca2+ mobilisation and activation of mitogen-activated protein kinases (MAPK). Here we addressed the effects of BAs on central signalling pathways in human platelets and on various platelet functions. We found that beta-BA (10 microM), the 11-methylene analogue of KBA, caused a pronounced mobilisation of Ca2+ from internal stores and induced the phosphorylation of p38 MAPK, extracellular signal-regulated kinase (ERK)2, and Akt. These effects of beta-BA were concentration dependent, and the magnitude of the responses was comparable to those obtained after platelet stimulation with thrombin or collagen. Based on inhibitor studies, beta-BA triggers Ca2+ mobilisation via the phospholipase (PL)C/inositol-1,4,5-trisphosphate pathway, and involves Src family kinase signalling.Investigation of platelet functions revealed that beta-BA (> or =10 microM) strongly stimulates the platelet-induced generation of thrombin in an ex-vivo in-vitro model, the liberation of arachidonic acid (AA), and induces platelet aggregation in a Ca2+-dependent manner. In contrast to beta-BA, the 11-keto-BAs (KBA or AKBA) evoke only moderate Ca2+ mobilisation and activate p38 MAPK, but fail to induce phosphorylation of ERK2 or Akt, and do not cause aggregation or significant generation of thrombin.In summary, beta-BA potently induces Ca2+ mobilisation as well as the activation of pivotal protein kinases, and elicits functional platelet responses such as thrombin generation, liberation of AA, and aggregation.     

Inhibition of the MEK/ERK pathway has no effect on agonist-induced aggregation of human platelets

The activation of human platelets by a variety of agonists is accompanied by the phosphorylation of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein (MAP) kinases. However, the role(s) of, and the substrate(s) for, these enzymes in platelet function remain unclear. Studies on ERKs in platelets have relied on pharmacological tools, including an inhibitor of ERK activation, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene]. In the present study, the effects of U0126 and its "inactive" analogue, U0125 [1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene], on human platelet aggregation and MAP kinase activity were examined. Several agonists with a variety of signaling pathways were studied including thrombin, a thromboxane analogue, arachidonic acid, collagen, calcium ionophores, and the phorbol ester phorbol myristate acetate (PMA). U0126, at concentrations consistent with inhibition of the isolated enzyme, inhibited ERK phosphorylation, and therefore MEK activation, in response to each agonist. Under such conditions, U0126 did not affect the phosphorylation of a second MAP kinase, p38(MAPK); however, platelet aggregation was also unaffected. Higher concentrations of U0126, and of U0125, inhibited platelet aggregation in response to collagen and PMA with no effect on that induced by the other agonists. These results dissociate ERK activation from platelet aggregation, suggesting an alternative role for ERKs in platelet function. In addition, the effects of higher concentrations of U0126 are likely due to an action on protein kinase C, likely unrelated to ERK inhibition, suggesting that the inhibitor concentration is crucial to the interpretation of such studies.     

Extracellular signal-regulated kinase plays an essential role in endothelin-1-induced homotypic adhesion of human neutrophil granulocytes

1. Endothelin-1 (ET-1) stimulates integrin-dependent adhesion of neutrophil granulocytes to endothelial cells, one of the early key events in acute inflammation. However, the signalling pathway(s) of ET-1-stimulated neutrophil adhesive responses has not been elucidated. Previous studies indicated that extracellular signal-regulated kinase (ERK) activation could mediate rapid responses of neutrophil granulocytes to various stimuli. In this study, we investigated the role of ERK signalling in human neutrophil granulocytes challenged with ET-1. 2. ET-1 rapidly down-regulated the expression of L-selectin and up-regulated the expression of CD11b/CD18 on the neutrophil surface. Concomitantly, ET-1 induced homotypic adhesion (aggregation) of neutrophils, that was blocked by a monoclonal antibody to CD18. 3. ET-1, through ET(A) receptors, evoked activation of Ras and subsequent phosphorylation of Raf-1, mitogen-activated protein kinase kinase (MAPK/ERK kinase) and ERK 1/2. ERK activation by ET-1 was rapid, concordant with the kinetics of ET-1-stimulated neutrophil aggregation. 4. Neutrophil responses to ET-1 were markedly attenuated by the MAPK/ERK kinase inhibitor PD98059, whereas inhibitors of p38 MAPK, tyrosine kinases and phosphatidylinositol 3-kinase had no detectable effects. We have observed a tight correlation between neutrophil ERK activation and homotypic adhesion. 5. These data indicate an essential role for ERK in mediating ET-1-stimulated adhesive responses of human neutrophil granulocytes.     

Transforming growth factor-beta 1 impairs endothelin-1-mediated contraction of brain vessels by inducing mitogen-activated protein (MAP) kinase phosphatase-1 and inhibiting p38 MAP kinase

Brain levels of transforming growth factor-beta1 (TGF-beta1) are increased in Alzheimer's disease and have been implicated in the associated cerebrovascular pathology. We recently reported that transgenic mice that overexpress TGF-beta1 (TGF+ mice) display, with aging, selectively reduced endothelin-1 (ET-1)-mediated contractions. Because ET-1 is a key regulator of cerebrovascular tone and homeostasis, we investigated how increased levels of TGF-beta1 could selectively alter this contractile response. We found that ETA receptors, via activation of p38 mitogen-activated protein (MAP) kinase, mediate the ET-1-induced contraction in mouse cerebral arteries, a response significantly decreased in aged TGF+ mice (-39%; p < 0.01) despite unaltered ETA receptor levels or affinity. In cerebrovascular smooth muscle cell cultures, long-term treatment with TGF-beta1 significantly decreased (>50%; p < 0.05) the ET-1-induced activation of the p38 MAPK/27-kDa heat shock protein (HSP27) signaling pathway. This occurred with no effect upstream to p38 MAP kinase but with the concomitant induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. Inhibition of MKP-1 expression with Ro-31-8220 or suppression of MKP-1 expression by short interfering RNA restored the ET-1-mediated p38 MAP kinase response. These results disclose a new role for long-term increases of TGF-beta1 in modulating cerebrovascular tone by dampening ET-1-mediated activation of the p38 MAPK/HSP27 signaling pathway. Such changes in ET-1-mediated signaling may help maintain vascular wall homeostasis by compensating for the diminished dilatory function induced by TGF-beta1 and amyloid-beta; brain levels of these two molecules are increased in patients with Alzheimer's disease.     

Activation of p38 mitogen-activated protein kinase by formyl-methionyl-leucyl-phenylalanine in rat neutrophils

The signaling pathways leading to p38 mitogen-activated protein kinase (MAPK) activation in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils were examined. Immunoblot analysis with antibodies against a phosphorylated form of p38 MAPK showed that fMLP-stimulated p38 MAPK activation was dependent on a pertussis toxin-sensitive G protein. Two phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), did not affect the p38 MAPK activation. Phosphorylation of p38 MAPK was concentration dependently attenuated by a tyrosine kinase inhibitor, genistein, and by a Ca(2+)-dependent protein kinase C inhibitor, 13-cyanoethyl-12-methyl-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3 , 4-c]carbazole-7-one (G?6976). However, the protein kinase C inhibitors with a broader spectrum, 2-[1-(3-dimethylaminopropyl)-5-methoxy-1H-indol-3-yl]-3-(1H-indol-3-y l)-maleimide (G?6983) and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimi de (GF109203X), had no inhibitory effect. fMLP-stimulated p38 MAPK phosphorylation was also reduced in cells pretreated with a phospholipase C inhibitor, 1-[6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), or preloaded with an intracellular Ca(2+) chelator, 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). We conclude that phosphorylation of p38 MAPK by fMLP stimulation in rat neutrophils is dependent on G(i/o) protein, nonreceptor tyrosine kinase, phospholipase C/Ca(2+), and probably Ca(2+)-dependent protein kinase C pathways.     

Characterization of a novel protein from Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist.

Many toxins from viperid venoms have been characterised as powerful activators of platelets. Here, the venom from the East African Lowland viper, Proatheris superciliaris, was investigated for its effect on platelets and the coagulation system. Whole P. superciliaris venom stimulated platelet shape change and aggregation; however, the stimulation of platelet activation was unaffected by the structurally distinct Src family kinase inhibitors PP1 and PD0173952, suggesting that platelet activation was mediated by a G protein-coupled receptor. A platelet reactive 34-kDa protein was isolated from P. superciliaris venom which we have designated proatherocytin. This protein induced Src kinase-independent aggregation of both human and mouse platelets that was inhibited by the serine protease inhibitor AEBSF. Proatherocytin did not clot bovine or human fibrinogen, degrade fibrinogen or hydrolyse the serine protease substrate benzoyl-FVR-pNA. It activated human PAR1 on stably transfected rat kidney epithelial cells, whereas no activation of the trypsin receptor PAR2 was shown. Surprisingly, Edman degradation of proatherocytin revealed sequence identity with existing disintegrin-like domains of snake venom metalloproteinases. These results suggest that proatherocytin is a highly selective PAR1 agonist that also causes mouse platelet aggregation, probably through cleavage of PAR4.     

Synthesis of novel peptide inhibitors of thrombin-induced platelet activation

Inhibitors of the activation of platelet aggregation have promise as important therapeutic agents for the management of acute coronary syndrome (ACS). Platelet activation by thrombin, a serine protease, occurs by binding to and cleavage of the extracellular N-terminal domains of protease-activated receptors 1 and 4 (PAR1 and PAR4). The proteolysis of the PARs exposes new tethered ligands that then signal through transmembrane domains to initiate platelet activation as a downstream effect. A pentapeptide cleavage product of bradykinin with the sequence Arg-Pro-Pro-Gly-Phe serves as a thrombin inhibitor by blocking alpha- and gamma-thrombin-induced platelet aggregation. Analogs of RPPGF have been prepared that result in improved inhibition of thrombin activation of platelets. Specific amino acid residues required for activity against platelet aggregation have been identified, and a lead compound, rOicPaPhe(p-Me)-NH(2) (FM19), has been developed. FM19, which completely inhibits threshold gamma-thrombin-induced platelet aggregation at a concentration of 16 +/- 4 microm, represents an important lead compound in the development of inhibitors of thrombin-mediated platelet aggregation for treatment of ACS.     

Comparison of the effects of PAR1 antagonists, PAR4 antagonists, and their combinations on thrombin-induced human platelet activation

Thrombin activates human platelets through proteolytic activation of two protease-activated receptors (PARs), PAR1 and PAR4. In the present study, we show that, RWJ-56110, a potent synthetic PAR1 antagonist, inhibited platelet aggregation caused by a low concentration (0.05 U/ml) of thrombin, but lost its effectiveness when higher concentrations of thrombin were used as stimulators. YD-3, a non-peptide PAR4 antagonist, alone had little or no effect on thrombin-induced platelet aggregation, significantly enhanced the anti-aggregatory activity of PAR1 antagonist. In addition, we demonstrate for the first time that P-selectin expression in thrombin-stimulated platelets can be synergistically prevented by combined treatment of PAR1 antagonist and PAR4 antagonist. These results indicate that thrombin-induced platelet activation cannot be effectively inhibited by just blocking either single thrombin receptor pathway, and suggest a rationale for potential combination therapy in arterial thrombosis.     

Activation of the p38 and p42/p44 mitogen-activated protein kinase families by the histamine H(1) receptor in DDT(1)MF-2 cells

1. The mitogen-activated protein kinases (MAPKs) consist of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. In this study we have examined the effect of histamine H(1) receptor activation on MAPK pathway activation in the smooth muscle cell line DDT(1)MF-2. 2. Histamine stimulated time and concentration-dependent increases in p42/p44 MAPK activation in DDT(1)MF-2 cells. Responses to histamine were inhibited by the histamine H(1) receptor antagonist mepyramine (K(D) 3.5 nM) and following pre-treatment with pertussis toxin (PTX; 57% inhibition). 3. Histamine-induced increases in p42/p44 MAPK activation were blocked by inhibitors of MAPK kinase 1 (PD 98059), tyrosine kinase (genistein and tyrphostin A47), phosphatidylinositol 3-kinase (wortmannin and LY 294002) and protein kinase C (Ro 31-8220; 10 microM; 41% inhibition). Inhibitors of Src tyrosine kinase (PP2) and the epidermal growth factor tyrosine kinase (AG1478) were without effect. Removal of extracellular Ca(2+), chelation of intracellular Ca(2+) with BAPTA and inhibition of focal adhesion assembly (cytochalasin D) had no significant effect on histamine-induced p42/p44 MAPK activation. 4. Histamine stimulated time and concentration-dependent increases in p38 MAPK activation in DDT(1)MF-2 cells but had no effect on JNK activation. Histamine-induced p38 MAPK activation was inhibited by pertussis toxin (74% inhibition) and the p38 MAPK inhibitor SB 203580 (95% inhibition). 5. In summary, we have shown the histamine H(1) receptor activates p42/p44 MAPK and p38 MAPK signalling pathways in DDT(1)MF-2 smooth muscle cells. Interestingly, signalling to both pathways appears to involve histamine H(1) receptor coupling to G(i)/G(o)-proteins.     

Involvement of p38 mitogen-activated protein kinase in PLL-AGE-induced cyclooxgenase-2 expression

In the present study, murine RAW 264.7 macrophages were incubated with poly-L-lysine-derived advanced glycosylation end products (PLL-AGEs) to examine cyclooxygenase-2 protein expression. Treatment of RAW 264.7 cells with PLL-AGEs caused the dose-dependent expression of cylooxygenase-2 but not cylooxygenase-1 and an increase in cylooxygenase activity. Increased cylooxygenase-2 expression was seen at 6 h and reached a maximum at 24 h. The tyrosine kinase inhibitor, genistein, and the p38 mitogen-activated protein kinase (MAPK) inhibitor, [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (SB 203580), inhibited PLL-AGE-induced cylooxygenase-2 expression, while the Ras inhibitor, FPT inhibitor II, and the MAP kinase kinase inhibitor, (2'-amino-3'-methoxyflavone) (PD 98059), had no effect on PLL-AGE-induced cylooxygenase-2 expression. Incubation of RAW 264.7 cells with PLL-AGEs resulted in activation of p38 MAPK, and this activation was suppressed by genistein and SB 203580. Taken together, our results suggest that activation of protein tyrosine kinase and p38 MAPK is involved in AGE-induced cyclooxygenase-2 expression in RAW 264.7 macrophages.