The influence of cyclooxygenase pathway activation following thromboxane-endoperoxide (TP) receptor stimulation was studied in rat mesenteric resistance arteries (n=6 to 10 per group). We studied isolated, perfused, and pressurized mesenteric resistance arteries (mean internal diameter 214 μm) using an arteriograph, enabling us to study arteries in physiological conditions of flow and pressure. Changes in diameter were continuously recorded, and contractions measured as internal diameter reduction. Release of cyclooxygenase pathway metabolites was also assessed by enzyme immunoassay (EIA) analysis of mesenteric bed perfusions. The thromboxane A2 (TxA 2 ) analog U-46619 (1 μmol/L) induced a significant contraction (108 μm maximal diameter reduction). Inhibition by 3 chemically different cyclooxygenase inhibitors (ie, flurbiprofen, indomethacin, and aspirin) potently reduced the contraction to 27%, 25%, and 6% of control, respectively. The selective cyclooxygenase-1 inhibitor SC-58560 inhibited U-46619 contraction, whereas selective cyclooxygenase-2 inhibition (SC-58236) had no effect. Thromboxane synthase inhibition (furegrelate) did not affect U-46619-induced contraction, but it was reduced by cytosolic phospholipase A2 inhibition. Measurement of cyclooxygenase derivatives produced by the isolated mesenteric bed showed that PGE 2 was produced after TxA 2 -receptor stimulation with U-46619. Exogenous prostaglandin E 2 (in the presence of the TxA 2 receptor antagonist SQ 29 548) and U-46619 contracted mesenteric arteries with a similar potency (EC 50 : 0.30 and 0.48 μmol/L, respectively). This study provides the first evidence that TxA 2 -receptor-dependent contraction in a resistant artery involved cyclooxygenase stimulation and, at least in part, a PGE 2 formation. This mechanism of TxA 2 -dependent contraction in resistant arteries might be of importance in the understanding of diseases affecting resistant arteries and involving TxA 2 , such as hypertension.

UNLABELLED:The action of clopidogrel on platelet receptors was analysed using platelets obtained from 11 healthy volunteers given 75 mg of clopidogrel daily for 8 d. Samples of blood were taken before treatment and after 8 d of medication. Determination of 2-methylthioadenosine diphosphate trisodium (2MesADP)-induced platelet aggregation, serine/threonine and tyrosine phosphorylations were performed in the absence or presence of the P2Y1-receptor-specific antagonist: adenosine 3'-phosphate 5'-phosphate (A3P5P) or the strong inhibitor of GPIIb/IIIa activation: SR121566.MAJOR CONCLUSIONS:1). Serine and threonine phosphorylations of the myosin light chain (P20) and pleckstrin (P47) do not behave similarly, although they are both recognized as the result of phospholipase C pathway stimulation triggered by the P2Y1 receptor. P47 is strongly affected by the A3P5P, and this appears to be highly dependent on P2Y12. However, P20 phosphorylation occurs in the presence of A3P5P, suggesting that the P2Y12 receptor signal contributes to P20 phosphorylation mediated by a calcium-independent pathway. The results suggest that P2Y1 and P2Y12 receptors interact to modulate the phosphorylation of P20 and P47. 2). The inside-out signalling dependent on both P2Y12 and P2Y1 is necessary for GPIIb/IIIa activation. 3). Clopidogrel and SR121566 inhibited the increase in tyrosine phosphorylation induced by 2MesADP and concomitantly inhibited platelet aggregation, indicating that most of the phosphorylations are GPIIb/IIIa dependent. However, neither clopidogrel nor SR121566 inhibited the first wave of 80 kDa substrate (cortactin) which is involved in the reorganization of the cytoskeleton necessary for shape change and which appeared to be essentially P2Y1 dependent.

The signalling pathways that link G-protein-coupled receptors to mitogen-activated protein kinases involve receptor and non-receptor tyrosine kinases and protein kinase C (PKC). We explored the pathways that are implicated in the thromboxane (TX) A(2)-dependent activation of extracellular-signal-regulated protein kinase (ERK) and the role of the two TX receptor (TP) isoforms, TP alpha and TP beta. ERK activation by IBOP, a TX analogue, was dependent on epidermal-growth-factor receptor (EGFR) in TP alpha- or TP beta-transfected cells and in human aortic smooth muscle cells (hASMCs), since AG1478, a selective inhibitor of tyrosine phosphorylation of the EGFR, strongly blocked ERK and EGFR phosphorylation. In addition, EGFR transactivation leading to ERK activation involved matrix metalloproteinases (MMPs), since BB2516, an inhibitor of MMP, decreased ERK and EGFR phosphorylation in TP alpha- or TP beta-transfected cells. Moreover, we showed that both isoforms activate ERK phosphorylation in an Src-kinase-dependent manner, whereas PKC was mainly implicated in ERK activation and EGFR phosphorylation by TP beta. In hASMCs, we showed that ERK activation depended on both pertussis-sensitive and -insensitive G alpha-proteins. We demonstrated further that EGFRs, PKC, Src kinase and MMPs are involved in ERK activation by TX. The results of the present study highlight a role for MMPs and PKC in EGFR transactivation triggered by the TPs and demonstrate this mechanism for the first time in primary cells, i.e. hASMCs.

Thrombospondin-1 (TSPI) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSPI, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with alphaIIb/beta3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The alphaIIb/beta3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of alphaIIb/beta3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cgamma2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new alphaIIb/beta3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSPI.

The serine protease thrombin present at the site of vascular injury triggers fibrin formation, platelet activation and different cellular responses including angiogenesis. We report a role for thrombin in the human monolayer cultured endothelial cell growth and angiogenesis in 3D collagen gel angiogenesis assay. The angiogenic activity of thrombin is, in part, related to the expression of the vascular endothelial growth factor (VEGF)165 mRNA, assessed by reverse transcriptase-polymerase chain reaction, either in monolayer cultured endothelial cells or in endothelial cells forming capillary-like structures in the 3D collagen gel assay. This expression of VEGF mRNA is associated with a VEGF secretion in the supernatant of thrombin-treated human umbilical vein endothelial cells. The thrombin-induced VEGF(165) mRNA expression is associated with the regulation of hypoxia-inducible factor 1alpha, analyzed by Western Blot, in endothelial cells.

GPCRs (G-protein coupled receptor) activation of extracellular signal- regulated kinases (ERK) has been extensively explored the last decade and showed a multitude of intracellular pathways involved including the activation of PKC, Ras, raf-1, etc []. Recently, it has been demonstrated that some GPCRs such as the lysophosphatidic acid, thrombin, bombesin and angiotensin receptor can activate ERK through the phosphorylation of the epidermal growth factor (EGF) receptor [[]]. This was mainly done using selective inhibitors of the tyrosine kinase activity of the EGF receptor such as the tyrphostin AG1478. Also, direct demonstration of the phosphorylation of the receptor of EGF in response to these different ligands has been demonstrated [[]].

Thromboxane A2 (TxA2) is a potent proaggregating, vasoconstrictor agent produced in many physiological and pathological situations. Although mitogen-activated protein (MAP) kinases [MAPK (ERK1/2 and p38)] have been shown to be activated after endoperoxide/thromboxane receptor (TP) stimulation, no study has investigated their potential role in resistance arteries, especially in physiological conditions of pressure and flow in which the arteries can contract. Thus, responses to TP stimulation by the stable agonist U46619 were studied in isolated rat mesenteric resistance arteries (inner diameter 262 ± 5 µm) mounted in an arteriograph. Changes in diameter were recorded under physiological levels of flow (90 µl/min) and pressure (50 mm Hg). TP stimulation induced a concentration-dependent contraction (EC50 value of 1.94 ± 0.22 × 10–7M), without desensitization. U46619-induced contraction was inhibited by calcium entry blockade (nifedipine) and protein kinase C inhibition (GF109203X), but it was not affected by tyrosine kinase inhibition (tyrphostin A25). MAPKK (MEK) inhibition (PD98059) did not alter U46619-dependent contraction, although ERK1/2 MAPK were activated. By contrast, p38 MAPK inhibition (SB203580) dose-dependently inhibited the contraction, and Western blot analysis showed activation of p38 MAPK in arteries contracted with U46619. Activation of p38 MAPK by U46619 was inhibited by nifedipine and in the absence of extracellular calcium. This study brings new insights in the transduction pathway involved in the contractile response of resistance arteries to TxA2/endoperoxide receptor stimulation. This contraction requires p38 MAPK activation, but did not involve ERK1/2 MAPK activation although both were activated.

Nitric oxide (NO) regulates cyclo‐oxygenase (COX) activity in various cell systems and reports conflict in regard to its stimulatory versus inhibitory role. Incubation of human umbilical vein endothelial cells (HUVEC) with SIN‐1 (3‐morpholinosydnonimine), a donor of NO, resulted in a rapid and dose‐dependent increase in the expression of COX‐2 as analysed by Western and Northern blotting. Incubation of HUVEC with SIN‐1 and interleukine (IL)‐1α resulted in increased induction of COX‐2 compared with IL‐1α alone and corresponded to an additive effect. The COX‐2 induction was dependent on a de novo synthesis since cycloheximide, an inhibitor of protein synthesis, blocked the enzyme expression. The increase in COX‐2 expression was not accompanied by a corresponding change in prostaglandin (PG) production. However, the COX activity was partially recovered when immunoprecipitated COX‐2 was incubated with arachidonic acid and haematin. Peroxynitrite, a highly reactive nitrogen molecule derived from the interaction of NO and superoxide anion, significantly increased COX‐2 expression. Under these conditions and within the limit of detection of the antibody, selective antibody for nitrotyrosine failed to detect nitrated COX‐2 in immunoprecipitated COX‐2 when cells where incubated with SIN‐1 or SIN‐1+IL‐1α. Ro 31‐8220, a specific inhibitor of protein kinase (PK) C, blocked the induction of COX‐2. Also, SB203580, the selective inhibitor of p38 MAP kinase, strongly blocked the induction of COX‐2 by SIN‐1 in the presence or absence of IL‐1α, whereas the MEK‐1 inhibitor, PD 98059, affected it to a lesser extent. These data demonstrate that SIN‐1 induces COX‐2 in HUVEC in the absence of PG formation and suggest a complex regulation of COX‐2 expression and PG formation by NO in endothelial cells. British Journal of Pharmacology (2001) 133, 1163–1171; doi:10.1038/sj.bjp.0704163

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.

Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis. Recently, cholesterol-independent effects of statins have been described. In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells. Treatment of cells with 25 muM mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production. Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect. GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2. Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1 alpha -dependent COX-2 induction. These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.

Haem oxygenase‐1 (HO‐1) can exert protective effects against oxidative stress and inflammation. Fibroblasts participate in inflammatory responses where they produce high levels of prostaglandins (PGs) and nitric oxide (NO). However, little is known of the presence of HO‐1 in these cells and the possible interactions among these pathways. Incubation of cells with NO donors, spermine nonoate (SPNO) and S‐nitroso‐N‐acetylpenicillamine (SNAP), induced a dose‐ and time‐dependent expression of HO‐1 protein. NO donors increased basal PGE2 release although they reduced PGE2 accumulated in the medium and cyclo‐oxygenase (COX) activity when cells were stimulated with lipopolysaccharide (LPS). COX‐2 protein was weakly induced by SPNO in basal conditions and in the presence of LPS a synergy for HO‐1 and COX‐2 protein expression was observed. Our results indicate that reactive oxygen species participate in the inductive effect of NO donors or LPS on HO‐1 expression, whereas endogenous NO production may play a role in the mechanism of the synergy exhibited by SPNO and LPS on HO‐1 and COX‐2 expression. In this system, zinc protoporphyrin IX did not affect nitrite levels but reduced COX activity. The selective COX‐2 inhibitors SC58125 and NS398 as well as the non‐selective COX inhibitor, indomethacin, strongly reduced PGE2 synthesis and showed a synergy with NO donors in HO‐1 and COX‐2 induction. Addition of PGE2 had no effect, suggesting a mechanism independent of PGs formation. In inflammatory conditions a number of factors could cooperate to induce HO‐1 and COX‐2, with a positive regulation by COX inhibitors. British Journal of Pharmacology (2000) 130, 57–64; doi:10.1038/sj.bjp.0703281

Platelets are an interesting model for studying the relationship betwen adhesion and mitogen-activated protein (MAP) kinase activation. We have recently shown that in platelets, ERK2 was activated by thrombin and downregulated by IIbβ3integrin engagement. Here we focused our attention on the c-Jun NH2-terminal kinases (JNKs) and their activation in conditions of platelet aggregation. We found that JNK1 was present in human platelets and was activated after thrombin induction. JNK1 phosphorylation was detected with low concentrations of thrombin (0.02 U/mL) and after 1 minute of thrombin-induced platelet aggregation. JNK1 activation was increased (fivefold) when fibrinogen binding to IIbβ3 integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab′)2 fragments of a monoclonal antibody specific for IIbβ3, demonstrating that, like ERK2, IIbβ3 integrin engagement negatively regulates JNK1 activation. Comparison of JNK1 activation by thrombin in stirred and unstirred platelets in the presence of RGDS peptide showed a positive regulation by stirring itself, independently of IIbβ3 integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet IIbβ3. The same positive regulation by stirring was found for ERK2. These results suggest that MAP kinases (JNK1 and ERK2) are activated positively by thrombin and stirring. In conclusion, we found that JNK1 is present in platelets and can be activated after thrombin induction. Moreover, this is the first report showing that two different MAP kinases (ERK2 and JNK1) are regulated negatively by IIbβ3 engagement and positively by mechanical forces in platelets.

The search for active antiplatelet drugs within the original chemical class of the thienopyridines, led to the discovery of clopidogrel, a novel ADP-selective agent whose antiaggregating properties are several times higher than those of ticlopidine. The antiaggregating properties of this compound are well known and, very recently, new results have clarified its mechanism of action. Clopidogrel is active only after intravenous or oral administration, and no circulating activity has been found in the plasma of treated animals or human volunteers. Experiments in rats have demonstrated that the antiaggregating activity was caused by a shortlasting metabolite generated in the liver by a cytochrome P450-dependent pathway. The antiaggregating property of clopidogrel is caused by an inhibition of the binding of ADP to its platelet receptors, and more specifically to the low affinity receptors, the high affinity binding sites being unaffected by clopidogrel. Several events in the ADP activation process, including adenylyl cyclase down-regulation, protein tyrosine phosphorylation, activation of the GPIIb-IIIa complex, fibrinogen binding, aggregation and release, were inhibited by clopidogrel and indicate their close relationship with the activation of a low affinity receptor by ADP. In contrast, binding of ADP to its high affinity binding sites (clopidogrel-resistant receptors) induced shape change, cytosolic calcium increase and phosphorylations of several other proteins, some events which were clopidogrel-sensitive. Thus, clopidogrel not only constitutes a potent antithrombotic drug in humans but also a good tool to study the effect of ADP on platelets.

Summary ADP, a primary stimulus of platelets, binds to one or more populations of receptors on the platelet surface. These receptors are linked to discrete activation pathways. Both G proteins and tyrosine kinases have been implicated in the cellular responses to this agonist. We have studied a patient with a congenital abnormality of ADP-induced platelet aggregation in an effort to gain information on the signalling pathways used by ADP. Immunoblotting with a broadly reactive rabbit antibody recognizing the GTP-binding domain of G protein α-subunits, and with rabbit antibodies specific for Giαl-3, and Gα12 all showed normal reactivity when tested against the patient‘s platelets. The phosphorylation of proteins was studied using an anti-phosphotyrosine MoAb (4G10) and platelets stimulated in a platelet aggregometer with ADP, a thromboxane A2 mimetic (IBOP), TRAP-14-mer peptide and α-thrombin. With normal platelets, a time-dependent phosphorylation of several bands in the 60 to 130 kDa mol. wt. range was observed with all agonists. For the patient, minimal aggregation and little or no phosphorylation of proteins of 80-85 kDa (cortactin), 100-105 kDa and 125-130 kDa were seen in response to ADP. The aggregation and phosphorylation responses were slightly modified in the presence of low doses of thrombin but were normal with high doses. Aggregation and tyrosine phosphorylation were virtually absent with IBOP, a finding reproduced when normal platelets were incubated with IBOP and the CP/CPK ADP scavenging system, thereby underlining the role of ADP in the response to IBOP. Our results show that the ADP receptor pathway deficient in the patient is linked to a selective tyrosine phosphorylation response.

Inter-individual variability in Ca2+ signal generation was studied in platelets from 15 healthy volunteers. The possible involvement of variation in thromboxane A production and variation in sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) was investigated by using platelets isolated before and after intake of 500 mg aspirin, and by measuring the expression levels of two main SERCA isoforms (SERCA-2b and PL/IM 430-recognizable SERCA). Considerable difference in Ca2+ responses were detected after platelet stimulation with thrombin, collagen or the SERCA-2b inhibitor, thapsigargin (TG), with inter-individual coefficients of variance of 22-43% in the absence and 15-41% in the presence of aspirin. Differences in thromboxane A2 generation and SERCA expression contributed to this variability in various ways. In the absence of aspirin, the amount of formed thromboxane A2 partially explains the level of the Ca2+ response induced by TG. On the other hand, in the absence of thromboxane-dependent effects, the expression levels of SERCA-2b and SERCA PL/IM 430 were inversely related to the responses evoked by collagen and TG, respectively. None of these factors were related to the level of the thrombin-evoked Ca2+ signal.

Platelet activation results in shape change, release of granule contents, aggregation and clot retraction. An intense intracellular ‘machinery’ is engaged to achieve these functions. Thrombin is one of the most important agonists for platelet recruitment and aggregation which is mediated by the binding of fibrinogen to its adhesive receptor: the glycoprotein (GP) IIb/IIIa complex or integrin αIIbβ3. The numerous biological processes consecutive to thrombin binding to platelet membrane are mainly controlled by phosphorylation mechanisms organized into signalling pathways. Schematically, the phospholipase Cβ pathway activated by G protein coupled to the seven transmembrane thrombin receptors, provides the first intracellular relay and would generate regulators such as protein kinase C, phosphorylated pleckstrin but also modifications of the intracellular domain of β3. This inside-out signalling would lead to some changes in the extracellular domain of GPIIb/IIIa increasing access of fibrinogen to the receptor. Ligand interaction with GPIIb/IIIa induced reorganization of the cytoskeleton and would mediate the outside-in signals which involve a series of intracellular events including tyrosine kinases, phosphatidylinositol 3 kinases, MAP kinases and phosphatases. Some of these pathways and/or signalling metabolites could be associated to some well-characterized platelet functions: cortactin phosphorylation is involved in platelet shape change, phosphatidylinositol 3 kinase (p85) in the stabilisation of platelet aggregates and MAP kinase (p44) in postaggregation events. But in fact the sequence of events which has been described has to be viewed as integrated networks. At least three biochemical processes govern the highly integrated organization to send just the appropriate quanta of signal for a specific need: the reorganisation of the cytoskeleton following the binding of fibrinogen to αIIbβ3, the structure of the signal transducers that contain SH2, SH3, and PH domains leading to the formation of macromolecules of signalling and the crosstalk phenomena between the different pathways. Elucidating the mechanisms of such networks becomes an increasingly exciting project.

SHP-1 is a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells. Upon thrombin stimulation of human platelets, SHP-1 is rapidly phosphorylated on both serine and tyrosine residues, and becomes associated with the cytoskeleton, where it could participate in the formation of multiprotein signalling complexes. In order to discriminate between signalling events occurring downstream of G-protein-coupled thrombin receptor and those subsequent to integrin alpha IIb beta 3 engagement, SHP-1 behaviour was examined in platelets from two patients lacking integrin alpha IIb beta 3 (Glanzmann's thrombasthenia). Upon thrombin stimulation, phosphorylation of SHP-1 occurred normally in thrombasthenic platelets, whereas association with the cytoskeleton was abolished. Moreover, inhibition of normal platelet aggregation with the tetrapeptide arg-gly-asp-ser (RGDS) which impairs fibrinogen binding to integrin alpha IIb beta 3, did not alter significantly SHP-1 phosphorylation. It is concluded that SHP-1 phosphorylation is not a consequence of integrin signalling but might rather occur downstream of thrombin receptor and heterotrimeric G-proteins.