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Biomedical subjects

C Bachelot

Publications and source records attributed to C Bachelot.

At least 19 recordsLinked to original sources
PubMed1996

Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c-Src in human platelets.

Protein tyrosine phosphatase 1C (PTP1C), highly expressed in hematopoietic cells, is a soluble protein tyrosine phosphatase containing two Src homology 2 (SH2) domains at the N-terminus and two putative sites of tyrosine phosphorylation at the C-terminus. This paper reports that PTP1C and c-Src could be coimmunoprecipitated during thrombin-induced platelet activation. Moreover, association between the two signalling proteins occurred only after PTP1C had been tyrosine phosphorylated. In in vitro experiments, PTP1C bound to the SH2 domain of c-Src, suggesting that association between tyrosine phosphorylated PTP1C and c-Src was mediated by the SH2 domain of c-Src. Finally, in resting platelets, PTP1C was mainly found in the Nonidet P-40 soluble fraction whereas following thrombin-induced activation, around 17% of PTP1C was associated with the insoluble fraction.

Blood Platelets↗
PubMed1996

Association of phosphatidylinositol 3-kinase, via the SH2 domains of p85, with focal adhesion kinase in polyoma middle t-transformed fibroblasts.

Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, becomes activated and phosphorylated on tyrosine in cells transformed with v-src. By cytoimmunofluorescence a sub-fraction of the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) localized in focal adhesion plaques. We examined the possibility that FAK associates with PI 3-kinase. In fibroblasts transformed with polyoma middle t, PI 3-kinase activity co-immunoprecipitated with pp125FAK using two different antibodies against this protein. PP125FAK from middle t-transformed cells associated with a glutathione-S-transferase fusion protein containing the 85-kDa subunit of phosphatidylinositol 3-kinase. Both of the SH2 domains and the SH3 domain of p85 also formed complexes with pp125FAK in vitro. Phosphopeptides that bind to the SH2 domains completely blocked the binding of full-length p85 to pp125FAK, while a peptide that binds to the SH3 domain was ineffective, indicating that the association between p85 and pp125FAK is mediated by the SH2 domains of p85.

3T3 Cells↗
PubMed1995

Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.

Androstadienes↗
PubMed1995

Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation.

Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.

Actins↗
PubMed1995

[Antiplatelet glycoprotein monoclonal antibodies: properties and practical value].

The aim of this review is (i) to classify the different monoclonal antibodies against platelet glycoproteins according to their properties and (ii) to take stock of their many diagnostic and therapeutic uses. Most of these antibodies recognize antigens on resting platelets without inducing activation, sometimes however they inhibit platelet function. Some of these antibodies, especially those against specific antigens (GPIIb/IIIa, CD9, CD36), have the capacity to activate platelets in vitro, either by direct binding of antibodies on the antigen or through the Fc domain binding to its platelet receptor Fc gamma RII. Other antibodies are directed against activation-dependent antigens that are expressed as a result of (i) a modification of a glycoprotein structure during platelet activation (as for GPIIb/IIIa), (ii) platelet release of granular antigens (GMP140, CD63, granulophysin...) or (iii) binding of soluble antigens on the activated platelet surface. Monoclonal antibodies find practical applications for both in vitro and in vivo diagnosis of bleeding or thrombotic pathology with some of them, notably anti-GPIIb/IIIa, having a promising future for antithrombotic therapy.

Antibodies, Monoclonal↗
PubMed1995

Role of Fc gamma RIIA gene polymorphism in human platelet activation by monoclonal antibodies.

The aim of this study was to determine if there is a correlation between the activity of a MoAb as an agonist and its ability to bind to the Fc platelet receptor, Fc gamma RIIa. A polymorphism at amino acid 131 [arginine (Arg) or histidine (His)] of Fc gamma RIIa was first shown to be determinant for MoAb-IgG1 binding on monocytes. To clarify the role of this polymorphism in platelet activation by MoAb-IgG1 we (i) established the Fc gamma RIIa polymorphism at the gene level by adapting the denaturating gradient gel electrophoresis method, (ii) analyzed the binding affinity of the MoAbs to Fc gamma RIIa on platelets from homozygous Arg, homozygous His, and heterozygous Arg/His donors, and (iii) characterized the different reactivities of platelets according to the Fc gamma RIIA polymorphism. Among 167 caucasian donors we found 46% heterozygous Arg/His, 36% homozygous His and 18% homozygous Arg. ALB6, and anti CD9, P256 an anti GPIIb-IIIa, and AP3 an anti-GPIIIa were chosen according to their ability (ALB6, P256) or not (AP3) to activate platelets. These 3 MoAbs-IgG1 bind to Fc gamma RIIa with a stronger affinity for the Arg-form of Fc gamma RIIa, a result which was confirmed with the use of diverse MoAbs directed against various antigens. The different abilities of MoAbs to bind to the two Fc gamma RIIa forms were well correlated to the different platelet responses induced by ALB6 and P256. However, low concentrations of ALB6, which allow full activation of platelets from homozygous Arg donors, as did P256, did not induce any activation of platelets from homozygous His donors, whereas P256 is able to induce a low aggregation. The results further define the respective roles of the antigen and the Fc receptor, depending on the MoAb, and the role of the Fc gamma RIIa polymorphism in platelet activation induced by MoAbs. In addition, the results obtained with MoAbs unable to induce platelet activation provided evidence that the binding of a MoAb on Fc gamma RIIa does not predict its ability to activate platelets.

Amino Acid Sequence↗
PubMed1994

Leukemic ophthalmopathy: a report of 21 pediatric cases.

A multicentric retrospective study on leukemic ophthalmopathy (LO) is reported. It includes 21 patients, 16 males and 5 females, with acute leukemia (AL) observed in 10 SIOP centers. LO developed in three patients at the time of diagnosis of AL; five patients were in first complete remission (three off therapy); four patients were in second or third remission; and nine were in combined relapse. Most frequent symptoms were blurred vision, photophobia, and ocular pain. Two patients with acute nonlymphoblastic leukemia died before treatment; another underwent bone marrow transplantation; one patient with B-cell acute lymphoblastic leukemia (B-ALL) treated with chemotherapy and radiotherapy died 4 months after LO; the remaining 17 children were treated according to different schedules with (10) or without (7) radiotherapy on the affected eye. Twelve patients achieved ocular remission and four of these had a second ocular relapse. Complete remission after LO treatment lasting for more than 3, 7, 24, 29 months was observed in four patients. The authors conclude that cure is possible in patients who had LO in first complete remission treated with chemotherapy and radiotherapy at high dose on the affected eye.

Acute Disease↗
PubMed1993

Indapamide inhibits human platelet aggregation in vitro: comparison with hydrochlorothiazide.

Among antihypertensive drugs with diuretic properties, indapamide was shown to inhibit platelet growth factors production in diabetic hypertensive patients, suggesting an antiplatelet activity. The present study aimed to demonstrate the antiaggregating properties of indapamide. The effect of indapamide on platelet function was compared in vitro to that of hydrochlorothiazide. Indapamide (100 microM) inhibited the second wave of adenosine diphosphate-induced aggregation and inhibited collagen-induced aggregation of platelet rich plasma by 50%. Using isolated platelets, indapamide also inhibited aggregation induced by low doses of thrombin (70% inhibition with 0.035 U/ml). This inhibition was dose-dependent and was still observed in presence of high thrombin concentrations, although the inhibition was moderate. Inhibitory effect of indapamide was more pronounced on the release reaction. Indapamide inhibited the thrombin-induced release of serotonin from dense granules by up to 80%. Hydrochlorothiazide at the same concentrations had no effect on platelet aggregation, and the inhibitory effect on the secretion was inconsistent and never exceeded 30%. By contrast, when the aggregation inducer was arachidonic acid, indapamide had no effect either on aggregation or on thromboxane formation, indicating that it was not acting on arachidonic catabolism. Calcium mobilization evoked by thrombin stimulation and measured with the fluorescent dye Indo 1 was also reduced in presence of indapamide by 30%. Myosin light chain and pleckstrin phosphorylation induced by thrombin were also reduced. These results demonstrate that indapamide inhibits platelet responses by inhibiting calcium mobilization. The anti-aggregating properties of indapamide could contribute to normalize the hyperresponsiveness of platelets from hypertensive patients.

Adenosine Diphosphate↗
PubMed1993

[Microangiopathic anemia following thrombopenic purpura].

BACKGROUND: Chronic relapsing microangiopathic hemolytic anemia is rare in children. This report describes a case associated with thrombocytopenia following idiopathic thrombocytopenic purpura. CASE REPORT: A 4 year-old girl was admitted for acute idiopathic thrombocytopenic purpura (platelet count: 12,000/mm3) without anemia or fragmented red cells. The patient was given intravenous gammaglobulins without success, followed by prednisone (2 mg/kg/day). The platelet count was normalized, but decreased when the treatment was discontinued. The patient developed acute intracranial hypertension at the age of 5 yr 8 mo, following two cerebral hematomas. The platelet count was 9,000/mm3. A second course of intravenous gammaglobulins and prednisone was unsuccessful, so a splenectomy was performed. One year later, the patient was admitted because of diffuse purpura, anemia and jaundice. Hematologic findings were: Hb 8.4 g/dl, reticulocytes 448,200/mm3, fragmented red cells 16%, platelets 15,000/mm3, WBC 22,400/mm3. Seroimmunologic investigation showed a high titer of antinuclear antibodies. Examination for viral etiology was negative. Intravenous gammaglobulins had a transient effect on platelets, reticulocytes and fragmented red cells. The patient was then given vincristine plus prednisone; they were only effective when high doses were used. A second intracerebral hemorrhage occurred when the patient was given low doses of drugs. After 3 other hematologic relapses, the vincristine was stopped without further complication. CONCLUSION: The criteria for systemic lupus erythematous were not satisfied, despite the presence of antinuclear antibodies. A congenital deficiency of an unidentified plasma factor that reverses microangiopathic hemolysis and thrombocytopenia was not demonstrated in this patient, who could not be given fresh frozen plasma.

Acute Disease↗
PubMed1992

Inhibition of platelet activation by tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers (tyrphostins) inhibit in a dose-dependent fashion thrombin-induced aggregation and serotonin release with IC50 values in the 10-35 microM concentration range. The inhibition of thrombin-induced aggregation correlates with their potency in inhibiting phosphorylation of proteins on tyrosine residues. Using metabolically 32P-labelled human platelets, it was found that the tyrphostins have no effect on the decrease in [32P]phosphatidylinositol bisphosphate but prevent the replenishment of [32P]polyphosphoinositide. Tyrphostins decreased [32P]phosphatidic acid production induced by thrombin, although never by more than 50%, and only delayed the peak of diacylglycerol, suggesting that phospholipase C was still activated. Tyrphostins inhibited the thrombin-elicited early phosphorylation of p43 and p20, substrates for protein kinase C (PKC) and myosin light chain kinase, respectively, at short times of activation. This inhibition, however, was overcome after 1 min of stimulation with thrombin. Tyrphostin AG213 also inhibited platelet aggregation and tyrosine protein phosphorylation induced by phorbol myristate acetate (PMA), but did not inhibit pleckstrin phosphorylation. These results suggest that thrombin induces the phosphorylation of proteins on tyrosine residues which most probably results in the activation of phosphoinositide kinases. The ability of tyrphostins to inhibit phosphorylation of p43 and p20 when induced by thrombin but not when induced by PMA confirms that PTKs may be involved subsequent to PKC activation.

Catechols↗
PubMed1992

Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors.

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.

Blood Platelets↗
PubMed1992

[Parameningeal cervical rhabdomyosarcoma in the neonatal period].

A case of parameningeal cervical rhabdomyosarcoma with severe bone destruction is reported in a 3 month-old infant; symptoms were present at birth. The treatment consisted of exclusive intensive chemotherapy. The outcome was favourable with complete tumor regression and vertebral bone reconstruction. The child was on complete remission without sequellae two years later.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

Involvement of platelet glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin) in thrombin-induced synthesis of phosphatidylinositol 3',4'-bisphosphate.

32P-Labeled human platelets were incubated with thrombin (1 unit/ml) for 5 min at 37 degrees C under conditions allowing maximal synthesis of [32P]phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2). Incorporation of 32P into the latter phosphoinositide was dose-dependently reduced (to a maximal level averaging 60%) by the tetrapeptide RGDS, an inhibitor of fibrinogen binding to activated glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin). Identical results were obtained with the fibrinogen gamma-chain dodecapeptide HHLGGAKQAGDV, whereas the tripeptide RGD and the tetrapeptide RGES displayed reduced or undetectable effects on 32P labeling of PtdIns(3,4)P2, respectively, in good correlation with their ability to inhibit platelet aggregation and fibrinogen binding to activated alpha IIb-beta 3 integrin. In addition, pathological platelets from three patients suffering thrombasthenia, which lack alpha IIb-beta 3 integrin and fail to aggregate in response to thrombin, displayed hardly detectable increases in the 32P labeling of PtdIns(3,4)P2. In contrast, thrombin-stimulated synthesis of PtdIns(3,4)P2 was unaltered in other deficient platelets lacking the glycoprotein Ib-IX complex (Bernard-Soulier syndrome). Although additional pathways seem to be involved in the regulation of phosphatidylinositol-3-kinase, these data indicate a strong relationship between platelet aggregation involving fibrinogen binding to activated alpha IIb-beta 3 integrin and the synthesis of the novel phosphoinositides phosphorylated at position D-3 of the inositol ring.

Amino Acid Sequence↗
PubMed1991

Mechanisms involved in platelet activation induced by a monoclonal antibody anti glycoprotein IIb-IIIa: inositol phosphate production is not the primary event.

The mechanisms involved in platelet aggregation by a monoclonal antibody (mAb) P256 specific for the GPIIb-IIIa complex was investigated following metabolic 32P labelling of platelets. When compared with thrombin, inositol phosphates (InsP) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [32P]-Ins(1,4,5)P3 and [32P]-Ins(1,3,4,5)P4, was observed between 20 and 90 s. [32P]-Ins(1,3,4)P3 was also produced with a maximum after 90 s. Addition of the ADP scavenger creatinine phosphate/creatine phosphokinase (CP/CPK) and of the cyclooxygenase inhibitor aspirin together with P256 almost totally abolished InsP formation, whereas platelet aggregation and protein phosphorylation were partially inhibited. F(ab')2 fragments of P256 also aggregated platelets but to a smaller extent than IgG, and without any measurable InsPs. To characterize further P256-induced activation, the phosphorylation of p43, the main substrate of protein kinase C (PKC) and the phosphorylation of tyrosine protein (P-Tyr) was also studied. PKC activation was smaller with P256-IgG than with thrombin but both thrombin and P265-IgG induced a similar profile of P-Tyr involving seven major bands, whereas P256-F(ab')2 only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular weight P-Tyr. The data indicate that the binding of P256 to GPIIb-IIIa, in contrast with thrombin, does not initially lead directly to the activation of the phosphoinositide phospholipase C to produce InsP's but rather involves the activation of protein kinases and also both fragments F(ab')2 and Fc play a specific role in the platelet responses to the mAb. Only the crosstalk between the two pathways evoked by F(ab')2 and Fc respectively allows the activation of all platelet activation systems.

Antibodies, Monoclonal↗