Biomedical subjects

C Thys

Publications and source records attributed to C Thys.

12 recordsLinked to original sources

PubMed2001

Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1 mutation.

A new mutation is described in the X-linked gene GATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for beta-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbbeta and GPIX but also a significantly lower expression of the nondirectly GATA1-regulated Gsalpha gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbalpha, GPIbbeta, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion, GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.

Adult↗

PubMed2001

Anti-vWf antibodies induce GPIbalpha and FcgammaRII mediated platelet aggregation only at low shear forces.

BACKGROUND/METHODS: Anti-von Willebrand factor (vWf) antibody mediated platelet activation was studied using 2 monoclonal anti-vWf antibodies promoting the binding of vWf to GPIbalpha: 1C1E7 (IgG2a) reacting with the vWf N-terminus and 75H4B12 (IgM), characterized in this paper and studied in association with 1C1E7. RESULTS: 75H4B12 binds to an N-terminal epitope in vWf, different from that reacting with 1C1E7. When com-bined, 1C1E7 and 75H4B12 promoted vWf binding to isolated GPIb under static conditions, even in the absence of ristocetin or botrocetin, and induced platelet aggregation synergistically in the presence of zero to subthreshold ristocetin concentrations. Specific inhibitors of GPIbalpha-vWf interactions prevented vWf binding to GPIbalpha in ELISA and during platelet aggregation. In addition, the 1C1E7 dependent platelet aggregation involved Fc receptor mediated platelet activation, a phenomenon even more pronounced when 1C1E7 and 75H4B12 were combined. A 75H4B12 binding phage expressing a peptide homologous with vWf sequence 88-95 neutralized the antibody induced platelet activation. However, at arterial shear rates, both 1C1E7 and 75H4B12 potently prolonged cartridge closure times in the PFA-100, compatible with inhibition of platelets by vWf, unfolded by the combined action of shear stress and antibodies. CONCLUSIONS: We conclude that antibodies directed against different epitopes in the N-terminus of vWf modify the folded vWf structure synergistically and enhance A1 domain mediated vWf binding to platelet GPIb at low shear forces. In addition, once platelet-bound, IgG antibodies potently activate platelets via the FcgammaII receptor. Thus, such antibodies may promote immune mediated thrombosis at low shear rates, typical of the venous circulation. In contrast, at arterial shear rates, anti-vWf antibodies may rather compromise platelet function following enhanced binding of the unfolded vWf multimers to platelets, shielding platelets from interacting with subendothelial and soluble ligands.

Antibodies, Monoclonal↗

PubMed2001

The ATP-gated P2X1 ion channel acts as a positive regulator of platelet responses to collagen.

ATP is a potent agonist of the P2X1 ion channel, mediating a rapid, quickly desensitized influx of Ca2+. In hirudinized PRP, containing apyrase, the two stable selective P2X1 agonists, alpha,beta-methylene ATP, and L-beta,gamma-methylene ATP induced extracellular Ca2+-dependent fast and reversible platelet shape change, leading to desensitization of the P2X1 ion channel. Preincubation with HPLC-purified ADP potently antagonized the subsequent alpha,beta-methylene ATP- and L-beta,gamma-methylene ATP-evoked platelet shape change. Accordingly, upon heterologous expression of P2X1 in Xenopus oocytes. HPLC-purified ADP acted as an antagonist of the ATP-induced current, but was inactive itself. Since ATP and ADP are co-released from dense granules during platelet activation, we investigated whether the P2X1 ion channel is involved in the response of platelets to collagen. We found that platelet shape change and aggregation induced by low concentrations of collagen were strongly inhibited after selective desensitization of P2X1 with its agonists or by pretreating the platelets with a low concentration of ADP (0.5 microM), that antagonizes the P2X1 channel without desensitizing the P2Y1 receptor. Our data suggest that, during collagen-initiated platelet activation, the early secretion of ATP results in the activation of the P2X1 ion channel, which plays a role as a positive regulator of further platelet responses.

Adenosine Diphosphate↗

PubMed2000

A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue.

The P2X1 receptor belongs to a family of oligomeric ATP-gated ion channels with intracellular N and C termini and two transmembrane segments separating a large extracellular domain. Here, we describe a naturally occurring dominant negative P2X1 mutant. This mutant lacks one leucine within a stretch of four leucine residues in its second transmembrane domain (TM2) (amino acids 351-354). Confocal microscopy revealed proper plasma membrane localization of the mutant in stably transfected HEK293 cells. Nevertheless, voltage-clamped HEK293 cells expressing mutated P2X1 channels failed to develop an ATP or ADP-induced current. Furthermore, when co-expressed with the wild type receptor in Xenopus oocytes, the mutated protein exhibited a dose-dependent dominant negative effect on the normal ATP or ADP-induced P2X1 channel activity. These data indicate that deletion of a single apolar amino acid residue at the inner border of the P2X1 TM2 generates a nonfunctional channel. The inactive and dominant negative form of the P2X1 receptor may constitute a new tool for the study of the physiological role of this channel in native cells.

Amino Acid Sequence↗

PubMed1998

Recurrent arterial thrombosis linked to autoimmune antibodies enhancing von Willebrand factor binding to platelets and inducing Fc gamma RII receptor-mediated platelet activation.

A patient with a history of recurrent late fetal loss associated with multiple placental infarcts and cerebrovascular ischemia at the age of 36, followed a year later by a myocardial infarction, was referred for further investigation. Coronary angiography was normal. Antinuclear factor, lupus anticoagulant, anticardiolipin antibodies, and other thrombophilia parameters were negative, but there was moderate hyperthyroidism with positive thyroid peroxidase antibodies. Platelet numbers and von Willebrand factor (vWF) were normal. Her platelets showed spontaneous aggregation that disappeared with aspirin intake. However, aggregation still was induced by low levels of ristocetin (0.3 to 0.5 mg/mL). The low-dose ristocetin aggregation in patient platelet-rich plasma (PRP) was completely blocked by neutralizing antiglycoprotein Ib (GPIb) and anti-vWF antibodies. The monoclonal anti-Fc gamma RII receptor antibody IV.3 inhibited partly, which suggests that PRP aggregation by low-dose ristocetin was elicited by vWF-immunoglobulin (Ig) complexes. Upon addition to washed human platelets, with vWF (10 micrograms/mL), purified patient Igs dose-dependently enhanced ristocetin (0.15 mg/mL)-induced aggregation between 0 and 500 micrograms/mL, an effect that disappeared again above 1 mg/mL. Aggregation was dependent on the vWF concentration and was blocked by IV.3 or neutralizing anti-GPIb or anti-vWF antibodies. The spontaneous aggregation of normal platelets resuspended in patient plasma could be inhibited totally by IV.3 and partially by neutralizing anti-GPIb or anti-vWF antibodies. Perfusion with normal anticoagulated blood, enriched with 10% of control or patient plasma, over surfaces coated with vWF showed increased platelet adhesion and activation in the presence of patient antibodies. Treatment of the patient with the antithyroid drug thiamazol and temporary corticosteroids, aspirin, and ticlopidine did not correct the platelet hypersensitivity to ristocetin. These observations suggest that some autoantibodies to vWF may both enhance vWF binding to platelets and cause platelet activation through binding to the Fc gamma RII receptor, and thereby may be responsible for a new form of antibody-mediated thrombosis.

Adult↗

PubMed1996

Dose-responses to inducers and inhibitors of platelet aggregation analysed via a micro-method.

The ex vivo study of platelet function in small experimental animals treated with anti-platelet drugs is restricted by the limited availability of platelet-rich plasma (PRP). To circumvent this obstacle, a micro-method for platelet aggregation was developed that enables ED50 measurements for various inducers of platelet aggregation. Fifty microliters of human, hamster, or rabbit PRP was mixed with agonist in each well of a microtiter plate, and shaken at 900 rpm at 37 degrees C for intervals up to 5 min. After stopping the aggregation with formaldehyde in PBS, light scattering was measured vs platelet-poor plasma (PPP) at 620 nm. Thus, aggregation in human PRP by ADP (t = 2 min) occurred with an ED50 = 1.8 microM, whereas the collagen (t = 3 min; ED50 = 2 micrograms/ml) and AA (t = 1 min; ED50 = 0.3 microM) induced aggregation occurred at those concentrations that induce aggregation in classical aggregometry. Likewise, aggregation was inhibited by the anti-GPIIb/IIIa antibody MA-16N7C2 and by the GPIIb/IIIa antagonists G4120 or MK-852. In comparison with human PRP, hamster (ED50 = 0.8 microM at t = 2 min) and rabbit (ED50 = 5 microM at t = 4 min) platelet aggregation by ADP occurred with comparable sensitivities, whereas the aggregation of rabbit platelets by collagen (ED50 = 15 micrograms/ml at t = 3 min) appeared to be slightly less sensitive and subject to large interindividual variations. The method was applied to measure plasma levels of a GPIIb/IIIa antagonist following injection into hamsters.

Adenosine Diphosphate↗

PubMed1995