A large number of similarities have previously been noted between the blood and milk clotting phenomena [Jollès, P. (1975) Mol. Cell. Biochem. 7, 73–85; Jollès, P. & Henschen, A. (1982) Trends Biochem. Sci. 7, 325–328]: some analogous features have also been found between fibrinogen and k‐casein. In this connection, the effect of a natural and a synthetic peptide derived from k‐casein on platelet function was studied: the undecapeptide Met‐Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp‐Lys (residues 106 → 116 of cow k‐casein) inhibited both aggregation of ADP‐treated platelets and binding of 125I‐fibrinogen to ADP‐treated platelets: its behaviour was similar to that of the structurally related C‐terminal dodecapeptide of human fibrinogen γ‐chain.

The presence of prostaglandin (PG) H 2 in the supernatant of human umbilical vein endothelial cells (HU-VEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B 2 . Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1␣ or a phorbol ester increases this formation. HU-VEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3-or 6-h induction of Cox-2 with phorbol ester or IL-1␣. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50 -60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6 -8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF 1␣ , PGE 2 , PGF 2␣ ) versus transcellular (TXB 2 ) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acidtreated cells, after 6-h stimulation with IL-1␣, newly synthesized Cox-2 produced less TXB 2 than 6-keto-PGF 1␣ compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.

SummaryIn a group of 6 patients with lupus anticoagulant (LA) and antiphospholipid (aPL) antibodies detected by ELISA ovemight urine and blood were simultaneously collected. A significantly increased urinary excretion of the platelet-derived thromboxane (TX) metabolite 11-dehydro-TXB2 was found in this group, as compared to 12 healthy individuals. In contrast, a small but significant reduction of the vascular prostacyclin (PGI2) metabolite 2,3-dinor-6-keto-prostaglandin F1α was observed.To further elucidate the effect of these antibodies on platelet activation we isolated the F(ab’)2 fragments from IgG of the 6 patients and 5 Controls, and we evaluated the effect of these fragments on the responses of isolated normal platelets to thrombin.Patients’ F(ab’)2 increased platelet aggregation and serotonin release of platelets stimulated by low dose thrombin (0.01 U/ml). At threshold thrombin concentration (0.05 U/ml) an enhanced TXB2 production was also observed.In summary, our results show, in addition to the altered TXA2/PGI2 balance observed in vivo, a direct stimulatory effect of aPL antibodies on platelet activation in vitro. This effect is related to recognition of phospholipid epitopes on platelets as shown by its neutralization upon preincubation with phospholipids. This phenomenon may be relevant for the thrombotic tendency of these patients.

Extracellular signal-regulated kinase (ERK) activation pathways have been well characterized in a number of cell types but very few data are available for platelets. The thrombin-induced signaling pathway leading to ERK2 activation in platelets is largely uncharacterized. In this study, we investigated the kinases involved in thrombin-induced ERK2 activation in conditions of maximal ERK2 activation. We found that thrombin-induced mitogen-activated protein kinase/ERK kinase (MEK)1/2 activation was necessary for ERK2 phosphorylation. We obtained strong evidence that conventional protein kinase Cs (PKCs) and calcium are involved in thrombin-induced ERK2 activation. First, ERK2 and MEK1/2 phosphorylation was totally inhibited by low concentrations (1 W WM) of RO318425, a speci¢c inhibitor of conventional PKCs. Second, Ca 2+ , from either intracellular pools or the extracellular medium, was necessary for ERK2 activation and conventional PKC activation, excluding the involvement of a new class of calcium-insensitive PKCs. Third, LY294002 and wortmannin had no signi¢cant e¡ect on ERK2 activation, even at concentrations that inhibit phosphatidylinositol (PI)3-kinase (5 W WM to 25 W WM and 50 nM, respectively). This suggests that PI3-kinase was not necessary for ERK2 activation and therefore, that PI3-kinase-dependent atypical PKCs were not involved. Surprisingly, in contrast to proliferative cells, we found that the serine/threonine kinases Raf-1 and B-Raf were not an intermediate kinase between conventional PKCs and MEK1/2. After immunoprecipitation of Raf-1 and B-Raf, the basal glutathione S-transferase^MEK1 phosphorylation observed in resting platelets was not upregulated by thrombin and was still observed in the absence of anti-Raf-1 or anti-B-Raf antibodies. In these conditions, the in vitro cascade kinase assay did not detect any MEK activity. Thus in platelets, thrombin-induced ERK2 activation is activated by conventional PKCs independently of Raf-1 and B-Raf activation. ß

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.

Cyclooxygenases are involved in the metabolism of arachidonic acid to prostaglandins (PGs) and thromboxane (TX) A 2 (6). In vascular biology, the two major products of COX are TXA 2 , which is mainly formed by the constitutive form of COX, COX-1 in activated platelets, and prostacyclin or PGI 2 , which is mainly produced in vascular cells by COX-1 and the inducible form of COX, COX-2 (7, 8). TXA 2 participates in platelet aggregation and vascular contraction, whereas PGI 2 acts as an antiaggregant for platelets and a vasodilator. PGI 2 plays an important role in vascular physiology as illustrated by the therapeutic effect of stable analogs of PGI 2 such as iloprost (9). Platelets from patients suffering from hypercholesterolemia are characterized by hypersensitivity to various aggregating agents. Notarbartolo et al. (10) have shown that simvastatin decreased platelet aggregation in hypercholesterolemic subjects and supported a decrease in the thromboxane platelet production, although the underlying mechanism of the statin effect on platelet function remains unclear.In this study, we demonstrated in human aortic smooth muscle cells (hASMC) that two different statins, mevastatin and lovastatin, increased COX-2 expression and PGI 2 formation. We further demonstrated using selective inhibitors of geranylgeranyltransferases and modulators of Rho GTPases that geranylgeranylated proteins such as Rho seem to be responsible for COX-2 down-regulation, which is prevented by statins.

Thromboxane A 2 (TxA 2 ) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7-3-(3-hydroxy-4-(4-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2.1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F 2 ␣. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal.Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA 2 -induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA 2 high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.