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Yoshihiro Fujimura - One of the best experts on this subject based on the ideXlab platform.
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Mutant Botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination.
Journal of thrombosis and haemostasis : JTH, 2017Co-Authors: Taei Matsui, Yoshihiko Sakurai, Masanori Matsumoto, Jiharu Hamako, Yasuhiro Ozeki, A. Hori, Fumio Matsushita, Masaki Hayakawa, Yoshihiro FujimuraAbstract:Essentials Botrocetin-2 (Bot2) binds to von Willebrand factor (VWF) and induces platelet agglutination. We identified Bot2 residues that are required for binding to VWF and glycoprotein (GP) Ib. We produced a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Mutant Bot2 could be used as a potential anti-thrombotic reagent to block VWF–GPIb interaction. SummaryBackground Botrocetin-2 (Bot2) is a Botrocetin-like protein composed of α and β subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-Botrocetin column. The mutant rBot2 with either Ala at Asp70 in the β subunit (Aspβ70Ala), or Argβ115Ala and Lysβ117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspβ70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argβ115Ala/Lysβ117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argβ115 and Lysβ117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the β subunit of Botrocetin is important for VWF binding, and Arg115 and Lys117 in the β subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argβ115Glu and Lysβ117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial Botrocetin that can inhibit the VWF–GPIb interaction.
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The von WiHebrand Factor Domain-Mediating Botrocetin4nduced Binding to Glycoprotein LB Lies Between Vat449 and Lys728
2013Co-Authors: Yoshihiro Fujimura, Zaverio M. Ruggeri, Linda Z. Holl, Theodore S. ZimmermanAbstract:The von willebrand factor domain-mediating Botrocetin-induced binding t
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Identification and recombinant analysis of Botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination.
Biochemistry, 2012Co-Authors: Yukiyo Yamamoto-suzuki, Yoshihiko Sakurai, Yoshihiro Fujimura, Masanori Matsumoto, Jiharu Hamako, Tetsuro Kokubo, Hitoshi Kitagawa, Sarkar M. A. Kawsar, Yuki Fujii, Yasuhiro OzekiAbstract:Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a Botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with Botrocetin subunits. Recombinant Botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural Botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of Botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these ...
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Binding site on human von Willebrand factor of bitiscetin, a snake venom-derived platelet aggregation inducer.
Biochemistry, 2002Co-Authors: Taei Matsui, Takayuki Nakayama, Tadashi Matsushita, Yoshihiro Fujimura, Jiharu Hamako, Koiti TitaniAbstract:Bitiscetin, a C-type lectin-like heterodimeric snake venom protein purified from Bitis arietans, binds to human von Willebrand factor (VWF) and induces the platelet membrane glycoprotein (GP) Ib-dependent platelet agglutination in vitro similar to Botrocetin. In contrast with Botrocetin which binds to the A1 domain of VWF, the A3 domain, a major collagen-binding site of VWF, was proposed to be a bitiscetin-binding site. In the competitive binding assay, neither bitiscetin nor Botrocetin had an inhibitory effect on the VWF binding to the immobilized type III collagen on a plastic plate. The anti-VWF monoclonal antibody NMC-4, which inhibits VWF-induced platelet aggregation by binding to α4 helix of the A1 domain, also inhibited bitiscetin binding to the VWF. Binding of VWF to the immobilized bitiscetin was competitively inhibited by a high concentration of Botrocetin. A panel of recombinant VWF, in which alanine-scanning mutagenesis was introduced to the charged amino acid residues in the A1 domain, showed...
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Glycoprotein Ib-von Willebrand Factor Interactions Activate Tyrosine Kinases in Human Platelets
Blood, 1997Co-Authors: Naoki Asazuma, Yoshihiro Fujimura, Shuji Miura, Kaneo Satoh, Yutaka Yatomi, Yukio Ozaki, Makoto Handa, Shoji KumeAbstract:von Willebrand factor (vWF ) in the presence of Botrocetin induces p72syk activation, assessed as its autophosphorylated level and in vitro kinase assays, the transient association of p72syk with p60c-src, and the translocation of p60c-src and p54/58lyn to cytoskeletal fractions. Jararaca glycoprotein Ib-binding protein (GPIb-BP), which specifically binds to GPIb, abolished these phenomena, suggesting that they are mediated by the vWF-GPIb interaction. These tyrosine kinase-related events were not inhibited by GRGDS peptide (plus EGTA), indicating that GPIIb/IIIa is not involved in the observed responses. Shc, an adaptor protein, was also tyrosine phosphorylated by the Botrocetin-vWF activation. When GPIb was immunoprecipitated with nonfunctional monoclonal antibodies (MoAbs) directed against GPIb, a kinase activity was found to associate with GPIb upon Botrocetin-vWF activation. On the other hand, anti-GPIb MoAbs that inhibit the vWF-GPIb interaction did not coprecipitate a kinase activity. Because the recovery of GPIb did not differ significantly, it is suggested that the excessive presence of inhibitory anti-GPIb MoAb dissociated a kinase activity from GPIb. Phosphoamino acid analysis showed that the kinase activity was that of a tyrosine kinase. The identity of the tyrosine kinase and the mode of interaction with the cytoplasmic region of GPIb await to be determined. Our findings suggest that the tyrosine kinase associated with GPIb serves at a most proximal step in the signal transduction pathway involved in the vWF-GPIb-induced platelet activation, which leads to other tyrosine kinase-related intracellular signals.
Koiti Titani - One of the best experts on this subject based on the ideXlab platform.
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Binding site on human von Willebrand factor of bitiscetin, a snake venom-derived platelet aggregation inducer.
Biochemistry, 2002Co-Authors: Taei Matsui, Takayuki Nakayama, Tadashi Matsushita, Yoshihiro Fujimura, Jiharu Hamako, Koiti TitaniAbstract:Bitiscetin, a C-type lectin-like heterodimeric snake venom protein purified from Bitis arietans, binds to human von Willebrand factor (VWF) and induces the platelet membrane glycoprotein (GP) Ib-dependent platelet agglutination in vitro similar to Botrocetin. In contrast with Botrocetin which binds to the A1 domain of VWF, the A3 domain, a major collagen-binding site of VWF, was proposed to be a bitiscetin-binding site. In the competitive binding assay, neither bitiscetin nor Botrocetin had an inhibitory effect on the VWF binding to the immobilized type III collagen on a plastic plate. The anti-VWF monoclonal antibody NMC-4, which inhibits VWF-induced platelet aggregation by binding to α4 helix of the A1 domain, also inhibited bitiscetin binding to the VWF. Binding of VWF to the immobilized bitiscetin was competitively inhibited by a high concentration of Botrocetin. A panel of recombinant VWF, in which alanine-scanning mutagenesis was introduced to the charged amino acid residues in the A1 domain, showed...
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Interaction of von Willebrand factor with the extracellular matrix and glycocalicin under static conditions.
Journal of biochemistry, 1997Co-Authors: Taei Matsui, Tomoki Naoe, Yoshihiro Fujimura, Jiharu Hamako, Yasuhiro Ozeki, Masahiko Katayama, Shinji Kunishima, Tadashi Kamiya, Koiti TitaniAbstract:The binding of human von Willebrand factor (vWF) to a variety of extracellular matrix components immobilized on plates and the binding of vWF to platelet glycoprotein Ib (GPIb) after interacting with these matrix components were examined by means of an enzyme-linked immunosorbent assay. vWF preferably bound to type III collagen, whereas it did not significantly bind to type I, IV, V, or VI collagen, fibronectin, laminin, elastin, or proteoglycans. Soluble type III collagen did not bind to vWF coated on plates and showed a little effect on the vWF binding to the immobilized collagen, suggesting that solid-phase collagen is important for the interaction with vWF. When glycocalicin, the N-terminal carbohydrate-rich extracellular domain of GPIb alpha exhibiting the vWF-binding activity, was added to vWF bound to collagen type III, no significant binding of glycocalicin was observed, but it bound to vWF in the presence of Botrocetin, a vWF modulator protein isolated from Bothrops jararaca snake venom. These results indicate that vWF immobilized on collagen can interact with GPIb but that binding of vWF to the collagen matrix alone is insufficient for modulating vWF so that it interacts with GPIb under static conditions. Another unknown physiological modulator functionally mimicking Botrocetin or high-shear stress may be involved in the platelet adhesion to extracellular matrix in the early stage of hemostasis.
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Purification and Characterization of Bitiscetin, a Novel von Willebrand Factor Modulator Protein from Bitis arietans Snake Venom
Biochemical and biophysical research communications, 1996Co-Authors: Jiharu Hamako, Yoshihiro Fujimura, Yasuhiro Ozeki, Masami Suzuki, Taei Matsui, Masayuki Ito, Kaori Makita, Koiti TitaniAbstract:Abstract We have screened 20 snake venoms and purified a novel snake venom protein, named bitiscetin, fromBitis arietansvenom that specifically binds to human von Willebrand factor (vWF) and induces platelet agglutination. Bitiscetin showed a heterodimeric structure composed of disulfide-linked α (16kDa) and β (13kDa) subunits on SDS-PAGE and showed a basic nature with pI value of 9.1, in contrast to Botrocetin (pI 4.6), a vWF modulator isolated from another snake (Bothrops jararaca) venom. Bitiscetin-induced platelet agglutination was dependent on vWF and platelet membrane glycoprotein (GP) Ib, but not on Ca2+and GPIIb/IIIa. vWF bound to bitiscetin but not to Botrocetin electroblotted to a PVDF membrane after SDS-PAGE and this binding was diminished after reduction of disulfide bonds of bitiscetin. Bitiscetin did not cross-react to anti-Botrocetin monoclonal antibodies. These results suggest that bitiscetin directly interacts with vWF and requires the protein conformation for its interaction as well as Botrocetin, but its interaction manner with vWF appears to be different from that of Botrocetin.
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Fibronectin and 130-kDa Molecule Complex Mimics Snake Venom Botrocetin-Like Structure Potentially Modulating Association between von Willebrand Factor and Vascular Vessel Wall
Journal of biochemistry, 1995Co-Authors: Masahiko Katayama, Yoshihiro Fujimura, Shuji Miura, Satomi Nagata, Sayuri Hirai, Taei Matusi, Ikunoshin Kato, Koiti TitaniAbstract:We established seven hybridomas secreting monoclonal antibodies (MoAbs) against the venom from Bothrops jararaca. Six of them were demonstrated to specifically recognize Botrocetin, a venom protein which binds with von Willebrand factor (vWf) and induces platelet agglutination. Two of them, BCT4-3 and BCT115-2 MoAbs, could significantly inhibit Botrocetin binding with plasma vWf. BCT4-3 could react slightly with a monolayer of human endothelial cells (ECs), and BCT4-3 binding to ECs was drastically enhanced by the coexistence of human plasma in a dose-dependent manner, indicating that a biological modulator structurally resembling Botrocetin is created initially on the EC surface complexed with some plasma proteins. Botrocetin-like components could be immuno-purified only by immobilized BCT4-3, but not by the other immobilized MoAbs, from umbilical vein extracts. Interestingly, the immunoisolated materials were identified to consist essentially of fibronectin (Fn) and a 130 kDa molecule, and this complex bound to vWf in the extracts. Depletion of Fn from plasma decreased BCT4-3 binding to ECs. The epitope of BCT4-3 expressed on the endothelial surface, comprising plasma Fn and the coisolated 130 kDa molecule, is proposed to be a physiological modulator structurally mimicking Botrocetin, and essentially supporting vWf-binding to injured endothelium and subsequently to circulating platelets.
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Isolation and characterization of jararaca GPIb-BP, a snake venom antagonist specific to platelet glycoprotein Ib.
Thrombosis and haemostasis, 1995Co-Authors: Yoshihiro Fujimura, Koiti Titani, Masami Suzuki, Shuji Miura, Eri Yoshida, Sachiyo Nishida, Yasuo Ikeda, Hiroko Shima, Yuta Taniuchi, Tomihisa KawasakiAbstract:A platelet glycoprotein Ib-binding protein (GPIb-BP) was isolated from the snake venom of Bothrops jararaca. Jararaca GPIb-BP showed a single band with M(r) of 30,000, and two distinct bands with M(r) of 17,000/13,000 under non-reducing and reducing conditions, respectively, on SDS-polyacrylamide gel electrophoresis. Jararaca GPIb-BP itself induced neither platelet aggregation nor serotonin release from platelets, but specifically bound to GPIb (40,629 +/- 2,521 molecules per normal platelet, with Kd 39.1 +/- 2.4 nM at saturation). The purified venom protein completely inhibited ristocetin- or Botrocetin-induced von Willebrand factor (vWF) binding, and blocked the bovine vWF binding to GPIb, with IC50 values ranging from 28 to 42 nM, without affecting the platelet aggregation induced by ADP or alpha-thrombin. 125I-jararaca GPIb-BP binding to GPIb was not altered by the presence of human alpha-thrombin. Jararaca GPIb-BP at a final concentration of 104 nM totally abolished vWF-dependent shear-induced platelet aggregation (SIPA) at a high shear stress, but had no effect on SIPA at a low shear stress. Reduced and S-carboxyamido-methylated jararaca GPIb-BP lost its inhibitory activity on SIPA. The NH2-terminal amino acid sequences of the subunits revealed a high degree of homology with those of several Ca(2+)-dependent lectins, especially to those of two functionally opposite venom proteins, Botrocetin (a vWF-modulator) and alboaggregin-B (a GPIb-modulator).
Dominique Meyer - One of the best experts on this subject based on the ideXlab platform.
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A new mutation, S1285F, within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and Botrocetin causing type 2M von Willebrand disease
British journal of haematology, 2003Co-Authors: Alain Stepanian, Jean-pierre Girma, A. S. Ribba, Jean-maurice Lavergne, Edith Fressinaud, Irène Juhan-vague, Claudine Mazurier, Dominique MeyerAbstract:Summary. We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or Botrocetin-induced binding of VWF to platelets in association with a decreased binding to Botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or Botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to Botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285-rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to Botrocetin and GPIb.
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Identification of a new type 2M von Willebrand disease mutation also at position 1324 of von Willebrand factor.
Thrombosis and haemostasis, 2002Co-Authors: Lysiane Hilbert, Dominique Meyer, A. S. Ribba, Edith Fressinaud, Claudine MazurierAbstract:Type 2M von Willebrand disease (VWD) refers to variants with decreased platelet-dependent function that is not associated with the loss of high molecular weight (HMW) von Willebrand factor (VWF) multimers. This category includes the so-called "phenotype B" responsible for inexistent ristocetin-induced but normal Botrocetin-induced binding of VWF to platelet glycoprotein lb. The missense mutation G1324S was identified in the first patient reported to display "phenotype B". We report here on the identification in four members of a French family of a missense mutation also affecting this glycine residue but changing it into an alanine residue. These individuals are heterozygous for this mutation and two of them display an additional quantitative VWF deficiency resulting from a stop codon at position 2470. After transient transfection in Cos-7 cells, the mutated recombinant protein harbouring the G1324A substitution was shown to exhibit normal multimers and inexistent ristocetin-induced but normal Botrocetin-induced binding to GPIb, confirming the classification of this new mutation as a type 2M VWD mutation.
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Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and Botrocetin by charged-to-alanine scanning mutagenesis.
The Journal of biological chemistry, 2000Co-Authors: Tadashi Matsushita, Dominique Meyer, J. Evan SadlerAbstract:At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or Botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp514, Asp520, Arg552, and Arg611(numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of 125I-Botrocetin was decreased by mutations at Arg629, Arg632, Arg636, and Lys667. Ristocetin-induced and Botrocetin-induced binding to GPIb both were decreased by mutations at Lys599, Arg629, and Arg632; among this group the K599A mutant was unique because 125I-Botrocetin binding was normal, suggesting that Lys599 interacts directly with GPIb. Ristocetin and Botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys534, Arg571, Lys572, Glu596, Glu613, Arg616, Glu626, and Lys642, whereas Botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg636 and Lys667. The binding of monoclonal antibody B724 involved Lys660 and Arg663, and this antibody inhibits125I-Botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the Botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of Botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys599 on helix 3.
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Mapping and functional studies of two alloantibodies developed in patients with type 3 von Willebrand disease.
Thrombosis and haemostasis, 2000Co-Authors: Hélène Tout, Dominique Meyer, Edith Fressinaud, Bernadette Obert, Anne Houllier, Chantal Rothschild, Jean-pierre GirmaAbstract:Inhibitors against von Willebrand factor (vWF) developed in two unrelated multitransfused patients (patients 1 and 2) with severe (type 3) von Willebrand disease (vWD) were analyzed. Both inhibitors were identified as antibodies of the IgG class by ELISA using immobilized purified vWF and either serum or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 antibody contained all subclasses of IgG (1, 2, 3 and 4) whereas antibody from patient 2 was a mixture of only IgG1 and 4. By ELISA using a series of immobilized purified proteolytic fragments of vWF, patient 1 antibody mainly bound to fragment SpIII and, to a lower extent, to fragments SpII and SpI; it poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 antibody only bound to fragments corresponding to the N-terminal portion of vWF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit vWF binding to its various ligands. Both antibodies blocked vWF binding to Factor VIII (FVIII), fibrillar type III collagen, bitiscetin and the subsequent induced binding to GPIb. Patient 1 antibody also blocked vWF binding to platelet GPIb when induced by ristocetin. However it failed to block vWF binding to GPIb when induced by Botrocetin as well as the binding of Botrocetin itself to vWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on vWF but the sites of vWF involved in its interaction with ristocetin. In contrast, we observed that patient 2 antibody blocked vWF binding to platelet GPIb induced by either agonist as well as vWF binding to Botrocetin. Finally, the effect of the antibodies was tested on vWF binding to GPIIb/IIIa. As expected from the mapping experiments, only IgG from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed alloantibodies with similar properties to those of polyclonal antibodies but with distinct effects on the functions of vWF.
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Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor
Blood, 1998Co-Authors: H Depraetere, Dominique Meyer, Jean-pierre Girma, Nadine Ajzenberg, Hans Deckmyn, Catherine Lacombe, Dominique BaruchAbstract:Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of alphaIIb beta3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to alphaIIb beta3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds-1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds-1, DSP was increased from 5.9% +/- 3.5% in the absence of vWF to 32.7% +/- 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to alphaIIb beta3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds-1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of Botrocetin, and MoAb 9, an inhibitor of vWF binding to alphaIIbbeta3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab')2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcgamma receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a Botrocetin-binding site on vWF, MoAb 724 mimics the effect of Botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.
Michael C. Berndt - One of the best experts on this subject based on the ideXlab platform.
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A role for glycosphingolipid-enriched microdomains in platelet glycoprotein Ib-mediated platelet activation
Journal of thrombosis and haemostasis : JTH, 2007Co-Authors: W Jin, Michael C. Berndt, Katsue Suzuki-inoue, Kaneo Satoh, Makoto Handa, Osamu Inoue, Noriko Tamura, Shinya Goto, Yukio OzakiAbstract:Summary. Background: Glycoprotein (GP) Ib, a platelet von Willebrand factor (VWF) receptor, plays a crucial role in thrombosis and hemostasis. As recent reports have suggested that GPIb partially locates in a particular region, designated as glycosphingolipid-enriched microdomains (GEMs), we hypothesized that GEMs play a central role in GPIb-mediated platelet activation. Methods: Platelets were stimulated by VWF/Botrocetin to activate platelets through GPIb. GEMs and non-GEMs were isolated by sucrose density gradient ultracentrifugation and the location of signaling molecules characterized. The role of GEMs-mediated signaling in platelet behavior was tested by platelet aggregation and by platelet interaction with immobilized VWF under flow conditions when GEMs were disrupted by methyl-β-cyclodextrin (MβCD). Results: GPIb was partially translocated to GEMs upon VWF/Botrocetin stimulation. Immunoprecipitation of GPIb in GEMs and non-GEMs revealed that the tyrosine kinases, Src and Lyn, were associated with GPIb only in GEMs after GPIb-stimulation, and not in non-GEMs. Activation of PLCγ2 was more intense in GEMs than non-GEMs. Disruption of GEMs by MβCD strongly inhibited tyrosine phosphorylation of Syk and PLCγ2. Functional studies revealed that stable adhesion of platelets to a VWF-coated surface under flow was impaired by GEM disruption by MβCD. Conclusion: The combined results suggest that GEMs play an important role in GPIb-mediated platelet activation.
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von Willebrand factor mediates platelet spreading through glycoprotein Ib and αIIbβ3 in the presence of Botrocetin and ristocetin, respectively
Journal of thrombosis and haemostasis : JTH, 2006Co-Authors: Owen J. T. Mccarty, Michael C. Berndt, Simon D. J. Calaminus, Laura M. Machesky, Stephen P. WatsonAbstract:Summary. Background: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin αIIbβ3. Objective: To evaluate the ability of GPIb and αIIbβ3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and Botrocetin. Results: In the presence of Botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of αIIbβ3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon αIIbβ3 and PI3-kinase. Furthermore, αIIbβ3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of Botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, Botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an αIIbβ3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an αIIbβ3-dependent manner. Conclusions: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF–GPIb interactions in the context of platelet regulation.
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the roles of adp and txa2 in Botrocetin vwf induced aggregation of washed platelets
Journal of Thrombosis and Haemostasis, 2004Co-Authors: J Liu, Michael C. Berndt, Tamara I. Pestina, Shirley A. Steward, Carl W. Jackson, T K GartnerAbstract:Summary. Background: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a cascade of events leading to αIIbβ3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination-induced GPIbα-mediated platelet activation have not been fully described. Methods: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Gαq, or αIIbβ3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, αIIbβ3 and Ca2+ in Botrocetin/VWF-induced signaling. Results: Our data demonstrate that Botrocetin/VWF/GPIbα-mediated agglutination results in calcium-independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbα-elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2-induced αIIbβ3 activation and ADP signaling. TXA2 or ADP can activate αIIbβ3, but both are required for α-granule secretion and aggregation. Botrocetin/VWF-induced dense granule secretion is Gαq-dependent. α-Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an αIIbβ3-dependent manner. Conclusions: In contrast to adhesion or shear stress-induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates αIIbβ3 requires TXA2. Agglutination-elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination-elicited GPIb signaling causes αIIbβ3 activation by a mechanism that is distinct from those used by adhesion, or shear stress-induced GPIb signaling.
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Snake venom toxins affecting platelet function.
Methods in molecular biology (Clifton N.J.), 2004Co-Authors: Robert K. Andrews, Elizabeth E. Gardiner, Michael C. BerndtAbstract:Botrocetin from the South American pit viper Bothrops jararaca was described as an activator of von Willebrand factor-dependent platelet aggregation by Read, Shermer, and Brinkhous in 1978 (1). Subsequently, Botrocetin has been widely used as an important in vitro modulator in the analysis of von Willebrand factor and platelet aggregation. Botrocetin has since been identified as a heterodimer of the C-type lectin family of snake venom proteins (~25 kDa nonreduced, ~14 kDa reduced), the primary sequence and crystal structure have been determined, and specific binding sites within the A1 domain of von Willebrand factor have been identified (2–8). Interestingly, members of the metalloproteinase-disintegrin family of snake venom proteins—jararhagin, jaracetin, and one-chain Botrocetin—are functionally related to two-chain Botrocetin, and also interact with the von Willebrand factor A1 domain (4,9,10). Jaracetin and one-chain Botrocetin are variably processed forms of jararhagin and are found in the same viper species as the C-type lectin family, two-chain Botrocetin. In this regard, recent evidence suggests that C-type lectin proteins and metalloproteinase-disintegrins may be derived from a common gene encoding a much larger precursor protein (11). An increasing number of C-type lectin proteins and metalloproteinases from cobra or viper venoms have been reported that selectively target either von Willebrand factor or its platelet receptor, glycoprotein (GP) Ibα of the GPIb-IX-V complex (some examples are shown in Table 1). These include the cobra venom metalloproteinase-disintegrin, mocarhagin from the Mozambiquan spitting cobra Naja mocambique mocambique (Naja mossambica mossambica) that cleaves GP Ibα within an anionic sequence containing three sulfated tyrosines (19,24,25). Mocarhagin also cleaves the neutrophil receptor PSGL-1 (P-selectin glycoprotein ligand-1) within an analogous sequence, and therefore has antiinflammatory activity in addition to being antithrombotic (18). In contrast, kaouthiagin from Naja kaouthia cleaves von Willebrand factor (21). A variety of C-type lectin proteins including alboaggregin-B (Table 1) bind to GPIbα and inhibit von
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The roles of ADP and TXA2 in Botrocetin/VWF‐induced aggregation of washed platelets
Journal of thrombosis and haemostasis : JTH, 2004Co-Authors: J Liu, Michael C. Berndt, Tamara I. Pestina, Shirley A. Steward, Carl W. Jackson, T K GartnerAbstract:Summary. Background: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a cascade of events leading to αIIbβ3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination-induced GPIbα-mediated platelet activation have not been fully described. Methods: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Gαq, or αIIbβ3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, αIIbβ3 and Ca2+ in Botrocetin/VWF-induced signaling. Results: Our data demonstrate that Botrocetin/VWF/GPIbα-mediated agglutination results in calcium-independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbα-elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2-induced αIIbβ3 activation and ADP signaling. TXA2 or ADP can activate αIIbβ3, but both are required for α-granule secretion and aggregation. Botrocetin/VWF-induced dense granule secretion is Gαq-dependent. α-Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an αIIbβ3-dependent manner. Conclusions: In contrast to adhesion or shear stress-induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates αIIbβ3 requires TXA2. Agglutination-elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination-elicited GPIb signaling causes αIIbβ3 activation by a mechanism that is distinct from those used by adhesion, or shear stress-induced GPIb signaling.
Jean-pierre Girma - One of the best experts on this subject based on the ideXlab platform.
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A new mutation, S1285F, within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and Botrocetin causing type 2M von Willebrand disease
British journal of haematology, 2003Co-Authors: Alain Stepanian, Jean-pierre Girma, A. S. Ribba, Jean-maurice Lavergne, Edith Fressinaud, Irène Juhan-vague, Claudine Mazurier, Dominique MeyerAbstract:Summary. We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or Botrocetin-induced binding of VWF to platelets in association with a decreased binding to Botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or Botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to Botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285-rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to Botrocetin and GPIb.
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The arginine-552-cysteine (R1315C) mutation within the A1 loop of von Willebrand factor induces an abnormal folding with a loss of function resulting in type 2A-like phenotype of von Willebrand disease: study of 10 patients and mutated recombinant vo
Blood, 2001Co-Authors: Anne-sophie Ribba, Jean-pierre Girma, Jean-maurice Lavergne, Edith Fressinaud, Irène Juhan-vague, Lysiane Hilbert, Catherine Boyer-neumann, Catherine Ternisien, Jenny Goudemand, Claudine MazurierAbstract:The study identified 10 patients from 6 families with prolonged bleeding time, decreased von Willebrand factor (vWF) ristocetin cofactor activity (RCoF) to vWF:Ag (antigen) ratio, and reduced ristocetin-induced platelet agglutination as well as ristocetin- or Botrocetin-induced binding of plasma vWF to platelet glycoprotein Ib (GpIb). In addition, all patients showed a decrease of intermediate-molecular-weight (intermediate-MW) and high-molecular-weight (HMW) multimers of vWF. In the heterozygous state, a cysteine-to-threonine (C → T) transversion was detected at nucleotide 4193 of the VWF gene of all patients and lead to the arginine (R)522C substitution in the A1 loop of vWF mature subunit (R1315C in the preprovWF). By in vitro mutagenesis of full-length complementary DNA (cDNA) of vWF and transient expression in COS-7 cells, the mutated C552 recombinant vWF (C552rvWF) was found to exhibit decreased expression, abnormal folding, and lack of intermediate-MW and HMW multimers. In addition, direct binding of Botrocetin to C552rvWF, as well as ristocetin- and Botrocetin-induced binding of C552rvWF to GpIb, was markedly decreased. Although being localized in an area of the A1 loop of vWF where most of the type 2B mutations that induce a gain-of-function have been identified, the R552C mutation induces a 2A-like phenotype with a decrease of intermediate-MW and HMW multimers as well as a loss-of-function of vWF in the presence of either ristocetin or Botrocetin.
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Mapping and functional studies of two alloantibodies developed in patients with type 3 von Willebrand disease.
Thrombosis and haemostasis, 2000Co-Authors: Hélène Tout, Dominique Meyer, Edith Fressinaud, Bernadette Obert, Anne Houllier, Chantal Rothschild, Jean-pierre GirmaAbstract:Inhibitors against von Willebrand factor (vWF) developed in two unrelated multitransfused patients (patients 1 and 2) with severe (type 3) von Willebrand disease (vWD) were analyzed. Both inhibitors were identified as antibodies of the IgG class by ELISA using immobilized purified vWF and either serum or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 antibody contained all subclasses of IgG (1, 2, 3 and 4) whereas antibody from patient 2 was a mixture of only IgG1 and 4. By ELISA using a series of immobilized purified proteolytic fragments of vWF, patient 1 antibody mainly bound to fragment SpIII and, to a lower extent, to fragments SpII and SpI; it poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 antibody only bound to fragments corresponding to the N-terminal portion of vWF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit vWF binding to its various ligands. Both antibodies blocked vWF binding to Factor VIII (FVIII), fibrillar type III collagen, bitiscetin and the subsequent induced binding to GPIb. Patient 1 antibody also blocked vWF binding to platelet GPIb when induced by ristocetin. However it failed to block vWF binding to GPIb when induced by Botrocetin as well as the binding of Botrocetin itself to vWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on vWF but the sites of vWF involved in its interaction with ristocetin. In contrast, we observed that patient 2 antibody blocked vWF binding to platelet GPIb induced by either agonist as well as vWF binding to Botrocetin. Finally, the effect of the antibodies was tested on vWF binding to GPIIb/IIIa. As expected from the mapping experiments, only IgG from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed alloantibodies with similar properties to those of polyclonal antibodies but with distinct effects on the functions of vWF.
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Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor
Blood, 1998Co-Authors: H Depraetere, Dominique Meyer, Jean-pierre Girma, Nadine Ajzenberg, Hans Deckmyn, Catherine Lacombe, Dominique BaruchAbstract:Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of alphaIIb beta3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to alphaIIb beta3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds-1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds-1, DSP was increased from 5.9% +/- 3.5% in the absence of vWF to 32.7% +/- 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to alphaIIb beta3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds-1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of Botrocetin, and MoAb 9, an inhibitor of vWF binding to alphaIIbbeta3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab')2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcgamma receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a Botrocetin-binding site on vWF, MoAb 724 mimics the effect of Botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.
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Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor
Blood, 1998Co-Authors: H Depraetere, Dominique Meyer, Jean-pierre Girma, Nadine Ajzenberg, Hans Deckmyn, Catherine Lacombe, Dominique BaruchAbstract:Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of Botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a Botrocetin-binding site on vWF, MoAb 724 mimics the effect of Botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.