Thrombin induces angiogenesis and vascular endothelial growth factor expression in human endothelial cells: possible relevance to HIF-1alpha.

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 VEGF165 mRNA expression is associated with the regulation of hypoxia-inducible factor 1alpha, analyzed by Western Blot, in endothelial cells.

Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 requires the docking protein LAT but is largely independent of alphaIIb/beta3.

Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, 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 TSP1.

Modulation of COX-2 expression by statins in human aortic smooth muscle cells. Involvement of geranylgeranylated proteins.

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 microm 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-1alpha-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.

Induction of cyclo-oxygenase-2 in human endothelial cells by SIN-1 in the absence of prostaglandin production.

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)-1alpha resulted in increased induction of COX-2 compared with IL-1alpha 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-1alpha. 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-1alpha, 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.

A comparison of early intensive methotrexate/mercaptopurine with early intensive alternating combination chemotherapy for high-risk B-precursor acute lymphoblastic leukemia: a Pediatric Oncology Group phase III randomized trial.

A prospective, randomized multicenter study was performed to evaluate the relative efficacy of two different concepts for early intensive therapy in a randomized trial of children with B-precursor acute lymphoblastic leukemia (ALL) at high risk (HR) for relapse. Four hundred and ninety eligible children with HR-ALL were randomized on the Pediatric Oncology Group (POG) 9006 phase III trial between 7 January 1991 and 12 January 1994. After prednisone (PDN), vincristine (VCR), asparaginase (ASP) and daunorubicin (DNR) induction, 470 patients received either 12 intensive parenteral treatments of intermediate dose (1 g/m2 each) methotrexate (MTX) and mercaptopurine (MP) over 24 weeks (regimen A) or 12 intensive course of alternating myelosuppressive drug combinations given over 30 weeks (regimen B). These drug combinations included MTX/MP, teniposide (VM-26)/cytosine arabinoside (AC) and VCR/PDN/DNR/AC/ASP. Central nervous system (CNS) prophylaxis was age-adjusted triple intrathecal chemotherapy. Patients with CNS disease at diagnosis were treated with craniospinal irradiation after the intensive phase. Continuation was standard doses of MTX and MP for 2 years. This trial was closed early because of an apparent early difference favoring regimen B. Results show that 470 patients achieved remission (97%). Two hundred and thirty two were randomized to regimen A and 238 to regimen B. The estimated 4-year event-free survival (EFS) for patients treated with regimen A is 61.6 % (s.e. = 3.3%) and with regimen B is 69.4% (s.e. = 3.1%), P = 0.091. Toxicities were more frequent on regimen B. In conclusion, for children with B-precursor ALL at high risk to relapse, early intensification with myelosuppressive combination chemotherapy was more toxic but produced no significant difference in EFS when compared to those treated with parenteral methotrexate and mercaptopurine.

Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation.

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.

Heme oxygenase-1 induction by nitric oxide in RAW 264.7 macrophages is upregulated by a cyclo-oxygenase-2 inhibitor.

Unstimulated RAW 264.7 macrophages express negligible heme oxygenase-1 (HO-1) protein but incubation with the nitric oxide (NO) donor spermine nonoate (SPNO) induced HO-1 and weakly cyclo-oxygenase-2 (COX-2) protein. This effect was potentiated by coincubation with the COX-2 selective inhibitor, SC58125. Cells incubated with SPNO showed a strong increase in HO-1 mRNA levels after 4 h with a significant potentiation in the presence of SC58125, which did not modify HO-1 mRNA stability. The induction of HO-1 by NO and its potentiation by anti-inflammatory agents may play a role in inflammatory and immune responses.

Rhegmatogenous retinal detachment in retinoblastoma patients undergoing chemoreduction and cryotherapy.

PURPOSE: To report the development of rhegmatogenous retinal detachment, with the retinal break adjacent to a cryotherapy scar, in three patients undergoing systemic chemotherapy for intraocular retinoblastoma. METHOD: Case series. RESULTS: Three patients with intraocular retinoblastoma were referred after poor response to systemic chemotherapy and local cryotherapy; three eyes of these three patients were noted to have rhegmatogenous retinal detachment and active retinoblastoma, with retinal breaks adjacent to cryotherapy scars. CONCLUSION: Rhegmatogenous retinal detachment may result from retinal necrosis associated with cryotherapy in the setting of intraocular retinoblastoma treated with systemic chemotherapy and local cryotherapy.

Enhanced expression of haem oxygenase-1 by nitric oxide and antiinflammatory drugs in NIH 3T3 fibroblasts.

1. 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. 2. NO donors increased basal PGE(2) release although they reduced PGE(2) 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. 3. 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. 4. The selective COX-2 inhibitors SC58125 and NS398 as well as the non-selective COX inhibitor, indomethacin, strongly reduced PGE(2) synthesis and showed a synergy with NO donors in HO-1 and COX-2 induction. Addition of PGE(2) had no effect, suggesting a mechanism independent of PGs formation. 5. In inflammatory conditions a number of factors could cooperate to induce HO-1 and COX-2, with a positive regulation by COX inhibitors.

Regulation of c-jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets.

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 alpha(IIb)beta(3) integrin 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 alpha(IIb)beta(3) integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab')(2) fragments of a monoclonal antibody specific for alpha(IIb)beta(3), demonstrating that, like ERK2, alpha(IIb)beta(3) 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 alpha(IIb)beta(3) integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet alpha(IIb)beta(3). 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 alpha(IIb)beta(3) engagement and positively by mechanical forces in platelets.

Platelet signal transduction pathways: could we organize them into a 'hierarchy'?

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 alphaIIbbeta(3). The numerous biological processes consecutive to thrombin binding to platelet membrane are mainly controlled by phosphorylation mechanisms organized into signalling pathways. Schematically, the phospholipase Cbeta 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 beta(3). 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 alphaIIbbeta(3), 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.

Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change.

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)alpha. 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.

The Miami pediatric quality of life questionnaire: parent scale.

Because there were limited measures available to assess health-related quality of life (HRQL) in children with chronic illnesses, this study was initiated to develop an empirically derived questionnaire for use in evaluating HRQL issues in children treated for cancer. Extensive interviews were conducted with 30 families of children with cancer, 10 of pre-school age, 10 of school age and 10 of adolescent age. Responses were videotaped and transcribed, then categorized to develop a pool of 56 items, which were administered to 132 children with cancer and to their parents. This report focuses on parental responses to objective items and ratings of importance of each of these items. Three primary categories, Self-Competence, Emotional Stability and Social Competence, were identified, each of which had solid internal consistency, sensitivity and reliability across 1-month intervals. The measure demonstrated the ability to discriminate between children with different types of cancer, offers an alternative to measures relying on expert judgment to assess HRQL and may lead to greater inclusion of psychological and social concerns as primary factors in determining HRQL in children participating in clinical trials.

Abnormal tyrosine phosphorylation linked to a defective interaction between ADP and its receptor on platelets.

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 alpha-subunits, and with rabbit antibodies specific for Gialpha1-3, and Galpha12 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 alpha-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+ signalling in platelets from healthy volunteers: effects of aspirin and relationship with expression of endomembrane Ca2+-ATPases.

Increased Ca2+ signal generation may lead to hyperactivity of platelets and contribute to thrombotic complications. Using fura-2-loaded platelets from 51 healthy volunteers, high variability was detected in the Ca2+ responses evoked by the receptor agonists, thrombin and collagen, and the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), thapsigargin (Tg). Oral intake of 500mg aspirin reduced the magnitude of the Ca2+ responses, and lowered the intra-individual coefficients of variance of the responses by 50%. However, the corresponding inter-individual variance coefficients were only a little influenced by aspirin intake, pointing to subject-dependent factors in Ca2+ handling that are unrelated to thromboxane formation. With each agonist, 6-9% of the subjects had platelets with relatively high Ca2+ responses (> mean + SD) both before and after aspirin intake. In 90% (9/10) of these cases the high responsiveness was confirmed in platelets obtained 6-12 months later. The Tg- but not thrombin-induced Ca2+ responses correlated inversely with the expression levels of SERCA PL/IM 430 (SERCA-3b) in platelets. After aspirin intake, the Ca2+ responses with collagen but not thrombin correlated inversely with SERCA-2b expression. These results suggest that, in the absence of potentiating effects of thromboxane, (i) the amount of PL/IM 430-recognizable SERCA may control the Ca2+ signal when SERCA-2b is specifically inhibited (with Tg), and (ii) the expression of SERCA-2b determine the collagen- but not the thrombin-evoked Ca2+ signal. Accordingly, limited Ca2+-pumping activity by low expression of one of the SERCA isoforms is likely to be one of the factors resulting in increased platelet activity towards collagen or thapsigargin but not thrombin.

Thrombopoietin activates human platelets and induces tyrosine phosphorylation of p80/85 cortactin.

The mechanism of human platelet activation by thrombopoietin (TPO) was investigated in vitro. We found that rHuTPO stimulated thromboxane A2 formation and serotonin secretion, despite the absence of shape change and aggregation. Blockade of the arachidonic acid pathway did not inhibit rHuTPO-induced platelet secretion. rHuTPO stimulated the tyrosine phosphorylation of 64, 80/85, 95, 130 and 140 kDa proteins, but phosphoproteins of 100-105 and 125 kDa obtained when platelets aggregated in the presence of thrombin were absent. rHuTPO stimulated and potentiated the thrombin-induced tyrosine phosphorylation of a 80 kDa protein identified as the cortical actin-associated protein, p80/85 cortactin. When platelets were aggregated in the presence of rHuTPO and fibrinogen, cortactin phosphorylation was enhanced as compared to rHuTPO alone. Treatment with RGDS or cytochalasin D respectively reduced or abolished cortactin tyrosine phosphorylation. This confirms the existence of fibrinogen binding-dependent and independent pools of phosphorylated cortactin, both requiring intact actin polymerization. Cytoskeleton-binding proteins may be implicated in in vitro platelet activation by rHuTPO.

Contribution of thromboxane and endomembrane Ca2+-ATPases to variability in Ca2+ signalling of platelets from healthy volunteers.

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.

Clopidogrel: a review of its mechanism of action.

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.