Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa.
Ontology highlight
ABSTRACT:
SUBMITTER: Pietu G
PROVIDER: S-EPMC1132596 | biostudies-other | 1992 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:BackgroundWe previously described a mouse model in which platelet immunization between selected strains leads to production of alloantibodies and severe autoimmune thrombocytopenia and mimics the human condition posttransfusion purpura (PTP). This report describes studies defining epitopes recognized by these alloantibodies.Study designHybridomas were produced from spleen cells of immunized mice. Glycoprotein (GP) targets of resulting monoclonal antibodies were characterized by immunoprecipitation using platelets from the immunizing strains. Antigens defined by single amino acid (AA) polymorphisms recognized by monoclonal antibodies were identified by mutagenizing target glycoproteins expressed in Chinese hamster ovary cells and observing the effects on antibody binding.ResultsThree monoclonal antibodies (417.1, 417.3, 425.1) were produced that recognized GPIIb on immunizing platelets. Monoclonal antibodies 417.1 and 417.3 both required G111 and 425.1 required V37, located on the beta propeller domain of GPIIb, for binding to platelets from the immunizing strains C57 and PWK, respectively. Injection of 417.3 and 425.1 into mice caused platelet destruction only in mice with GPIIb containing the targeted AAs.ConclusionsFindings made provide evidence that alloantibodies produced by mice experiencing thrombocytopenia in a mouse model of PTP are specific for single AA polymorphisms that differ in GPIIb/IIIa integrin of the immunizing and immunized strains and therefore closely resemble the potent alloantibodies found in patients with PTP. The observations show that naturally occurring single AA differences in GPIIb/IIIa integrin of various mouse strains are highly immunogenic in the mouse strains studied and readily induce antibodies comparable to human platelet antigen-specific antibodies found in transfused and pregnant humans.
| S-EPMC8035281 | biostudies-literature
Project description:The von Willebrand factor (VWF) A1-glycoprotein (GP) Ib? interaction is of major importance during thrombosis mainly at sites of high shear stress. Inhibitors of this interaction prevent platelet-dependent thrombus formation in vivo, without major bleeding complications. However, the size and/or protein nature of the inhibitors currently in development limit oral bioavailability and clinical development. We therefore aimed to search for a small molecule protein-protein interaction inhibitor interfering with the VWF-GPIb? binding. After determination of putative small molecule binding pockets on the surface of VWF-A1 and GPIb? using site-finding algorithms and molecular dynamics, high throughput molecular docking was performed on both binding partners. A selection of compounds showing good in silico docking scores into the predicted pockets was retained for testing their in vitro effect on VWF-GPIb? complex formation, by which we identified a compound that surprisingly stimulated the VWF-GPIb? binding in a ristocetin cofactor ELISA and increased platelet adhesion in whole blood to collagen under arterial shear rate but in contrast inhibited ristocetin-induced platelet aggregation. The selected compound adhering to the predicted binding partner GPIb? could be confirmed by saturation transfer difference NMR spectroscopy. We thus clearly identified a small molecule that modulates VWF-GPIb? binding and that will now serve as a starting point for further studies and chemical modifications to fully characterize the interaction and to manipulate specific activity of the compound.
| S-EPMC3308782 | biostudies-literature
Project description:Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.
| S-EPMC1135775 | biostudies-other
Project description:Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY:Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.
| S-EPMC5949882 | biostudies-literature
Project description:The major platelet integrin, glycoprotein IIb-IIIa, binds soluble fibrinogen only after platelet activation. To investigate the mechanism by which platelets convert glycoprotein IIb-IIIa into a functional fibrinogen receptor, we characterized the opening and closing of fibrinogen-binding sites in isolated platelet membranes and compared the regulatory properties of membrane-bound glycoprotein IIb-IIIa with those of the detergent-solubilized receptor. Basal fibrinogen binding to the membranes possessed many of the properties of fibrinogen binding to activated platelets; however, less than 10% of glycoprotein IIb-IIIa in the membranes was capable of binding fibrinogen. Preincubating the membranes with either an activating glycoprotein IIb-IIIa antibody or alpha-chymotrypsin increased fibrinogen binding. In contrast, agents that require intracellular mediators, such as platelet agonists, guanine-nucleotide-binding-protein activators and purified protein kinase C, did not stimulate fibrinogen binding to the membranes, suggesting that cytosolic factor(s) may be required for activation of the receptor in platelets. Occupancy of glycoprotein IIb-IIIa in the membranes with RGD (Arg-Gly-Asp)-containing peptides reversibly exposed neoantigenic epitopes and fibrinogen-binding sites in the receptor. These conformational changes required membrane fixation to be maintained following peptide removal. Similar results were obtained with purified glycoprotein IIb-IIIa incorporated into phospholipid vesicles, indicating that the resting state of the receptor is favoured in these environments. In contrast, when the conformation of detergent-solubilized glycoprotein IIb-IIIa was altered by exposure to RGD-containing peptides, the receptor remained active even after incorporation into phospholipid vesicles. These results demonstrate that platelet membranes are a useful model in which to study the regulation of glycoprotein IIb-IIIa and suggest that the environment surrounding the receptor may have a profound influence on this process.
| S-EPMC1134177 | biostudies-other
Project description:The seven-amino-acid peptide LSARLAF has been reported to activate platelets via the integrin GPIIb-IIIa (glycoprotein IIb-IIIa). Activation by LSARLAF is reinforced by release of ADP and thromboxanes, but the initiating event in the signalling cascade is not known. In the present study, we demonstrate that LSARLAF stimulates Src kinase-dependent tyrosine phosphorylation of many of the proteins in the GPIIb-IIIa cascade, including the tyrosine kinase Syk, the adapter SLP-76 (SH2-containing leucocyte phosphoprotein of 76 kDa) and PLCgamma2 (phospholipase Cgamma2). A critical role for PLCgamma2 in signalling by LSARLAF was demonstrated by abolition of aggregation in PLCgamma2-/- murine platelets to low concentrations of the peptide, although a partial recovery was seen with higher concentrations. In sharp contrast with the GPIIb-IIIa-regulated signalling cascade, aggregation was inhibited in murine platelets deficient in the adapter LAT (linker for activation of T-cells) and the Fc receptor gamma-chain. Aggregation was also partially inhibited by the cholesterol-lowering reagent, beta-methyl-cyclodextrin, at concentrations that disrupt membrane rafts, but do not interfere with signalling by GPIIb-IIIa. Furthermore, LSARLAF also stimulated tyrosine phosphorylation in GPIIb-deficient murine platelets, confirming that the integrin is not critical for activation of intracellular signalling pathways. LSARLAF also stimulated Ca2+ elevation in RBL-2H3 cells, which lack the platelet glycoproteins GPIIb, GPVI and GPIb. These results demonstrate that LSARLAF activates platelets through a PLCgamma2-dependent pathway that lies downstream of Src kinases and which is partially dependent on the Fc receptor gamma-chain, LAT and lipid rafts. The mechanism of cell activation by LSARLAF remains to be established, although the present results indicate that more than one surface glycoprotein may mediate this response.
| S-EPMC1223919 | biostudies-other
Project description:The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.
| S-EPMC8876638 | biostudies-literature
Project description:Circulating von Willebrand factor (VWF) adopts a closed conformation that shields the platelet glycoprotein Ibα (GPIbα) binding site in the VWF-A1 domain. Immobilized at sites of vascular injury, VWF is activated by its interaction with collagen and the exertion of increased hemodynamic forces. Studies on native VWF strings and isolated A1 domains suggest the existence of multiple A1 binding states in different biophysical contexts. In this single-molecule study, we have used a biomembrane force probe (BFP) and a flow chamber to identify and characterize a collagen binding induced conformation with a higher affinity to platelet GPIbα. As force increases, our results show that collagen binding increases the stability of GPIbα bond with both VWF and isolated A1 domain. However, the collagen 2D binding affinity for VWF-A3 domain is 10 times of that for A1 domain, suggesting the initial VWF capture is mediated by A3-collagen interaction while A1-collagen regulates the subsequent VWF activation. Our results reveal the molecular mechanism of collagen-regulated, A1-mediated platelet adhesion enhancement. Characterization of different A1 states provides insights into binding heterogeneity of VWF in different scenarios of inflammation and thrombosis.
| S-EPMC4553094 | biostudies-literature
Project description:It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s(-1)) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf (-/-) mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf (-/-) and Vwf (+/+) blood, exposed to VWF at 1800 s(-1) in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf(-/-) mice than from Vwf(+/+) mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf(-/-) mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s(-1) on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.
| S-EPMC3667798 | biostudies-literature
Project description:Platelet-type von Willebrand disease is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIbα-von Willebrand factor (vWF) interaction. GPIbα and vWF play a role in megakaryocytopoiesis, thus we aimed to investigate megakaryocyte differentiation and proplatelet-formation in platelet-type von Willebrand disease using megakaryocytes from a patient carrying the Met239Val variant and from mice carrying the Gly233Val variant. Platelet-type von Willebrand disease megakaryocytes bound vWF at an early differentiation stage and generated proplatelets with a decreased number of enlarged tips compared to control megakaryocytes. Moreover, they formed proplatelets upon contact with collagen, differently from normal megakaryocytes. Similarly, collagen triggered megakaryocytes showed defective activation of the RhoA-MLC2 axis, which prevents proplatelet formation, and increased phosphorylation of Lyn, which acts as a negative regulator of GPVI signaling, thus preventing ectopic proplatelet-formation on collagen. Consistently, human and murine bone marrow contained an increased number of extravascular platelets compared to controls. In addition, platelet survival of mutant mice was shortened compared to control mice, and the administration of desmopressin, raising circulating vWF, caused a marked drop in platelet count. Taken together, these results show for the first time that thrombocytopenia in platelet-type von Willebrand disease is due to the combination of different pathogenic mechanisms, i.e. the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/vWF complexes.
| S-EPMC6601082 | biostudies-literature