Botrocetin-induced vWF Binding Research Articles

Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.

We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation.The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.

Properties of Soluble His-Tagged rGPIbα1–483 Wild-Type and Increase-of-Function Mutants.

The extracellular domain of human platelet GPIbα contains the binding site for von Willebrand factor (vWF) within the amino-terminal 45 kDa region comprising approximately 300 amino acids. In the present studies, we have used CHO cells to synthesize a longer fragment of His1-Phe483, representing nearly the complete extracellular domain of GPIbα, with an additional 8 histidine residues added at the carboxyl end to provide recognition by his-tag reagents. Both wild-type and the increase-of-function mutants Gly233→Val233 and/or Met239→Val239 were employed. For studies of antibody binding, modulator-induced vWF binding, or antibody inhibition of this vWF binding, capture of the recombinant GPIbα could be accomplished by nickel chelate binding of the his-tag. Both botrocetin and ristocetin induced saturable binding of vWF by ELISA, as demonstrated by HRP-conjugated anti-vWF polyclonal antibody. In the case of the different increase-of-function mutants, significant vWF binding was seen even in the absence of added modulator. Inhibition of botrocetin-induced vWF binding was greater for wild-type than for the Met239→Val239 GPIbα mutant with some mabs (e.g., AS-2), whereas the degree of inhibition was essentially equivalent with other mabs (e.g., C-34). When a rabbit polyclonal anti-his antibody was used to immobilize the recombinant proteins instead of the nickel chelate system, binding of vWF and of conformation-dependent mabs was impaired. However, when this same antibody was biotinylated and then captured on a streptavidin plate, excellent capture and subsequent binding characteristics of the recombinant GPIbα were restored. With both this latter system and with the nickel chelate system, a highly sensitive functional vWF activity assay could be established, using botrocetin-induced binding of vWF from human plasma. Linearity of binding was observed between 0.1–0.6% normal plasma, with saturation occurring around 2%. Whereas higher concentrations of plasma actually appeared to displace bound GPIbα to the nickel chelate, the streptavidin-biotin-anti-his attachment method appeared more resistant to such effects. The ELISA-based assay systems developed in this study accordingly provide an and effective approach to study functional interactions of both wild-type and mutant forms of GPIbα with its ligand vWF and also with antibodies directed against different epitopes within the entire extracellular domain of this receptor molecule. The high sensitivity, reproducibility, and rapidity of the assay system also offer the possibility of a useful test for functional assay of plasma vWF.

Tyrosine Sulfation of Glycoprotein Ibα: ROLE OF ELECTROSTATIC INTERACTIONS IN VON WILLEBRAND FACTOR BINDING

Glycoprotein I(b)alpha (GP I(b)alpha), the ligand binding subunit of the platelet glycoprotein Ib-IX-V complex, is sulfated on three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279). This posttranslational modification is known to be critical for von Willebrand factor (vWF) binding; yet it remains unclear whether it provides a specific structure or merely contributes negative charges. To investigate this issue, we constructed cell lines expressing GP I(b)alpha polypeptides with the three tyrosine residues converted to either Glu or Phe and studied the ability of these mutants to bind vWF in the presence of modulators or shear stress. The mutants were expressed normally on the cell surface as GP Ib-IX complexes, with the conformation of the ligand-binding domain preserved, as judged by the binding of conformation-sensitive monoclonal antibodies. In contrast to their normal expression, both mutants were functionally abnormal. Cells expressing the Phe mutant failed to bind vWF in the presence of either ristocetin or botrocetin. These cells adhered to and rolled on immobilized vWF only when their surface receptor density was increased to twice the level that supported adhesion of cells expressing the wild-type receptor and even then only 20% as many rolled and rolled significantly faster than wild-type cells. Cells expressing the Glu mutant, on the other hand, were normal with respect to ristocetin-induced vWF binding and adhesion to immobilized vWF but were markedly defective in botrocetin-induced vWF binding. These results indicate that GP I(b)alpha tyrosine sulfation influences the interaction of this polypeptide with vWF primarily by contributing negative charges under physiological conditions and when the interaction is induced by ristocetin but contributes a specific structure to the botrocetin-induced interaction.

A Novel Monoclonal Antibody against the Extracellular Domain of GPIbβ Modulates vWF Mediated Platelet Adhesion

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.

Platelet adhesion at high shear rates: The roles of von Willebrand factor/GPIb and the β1 integrin α2β1

We have previously described a monomeric rvWf fragment, Leu504-Lys728 that contains one disulfide bond linking Cys509-Cys695. This fragment, VCL, has previously been shown to inhibit vWf-ristocetin, asialo-vWf, and botrocetin-induced vWf binding and aggregation of platelets. VCL inhibited 50% of vWf binding to heparin, but it did not inhibit vWf binding to type I collagen. At a high shear force (2600 −1 sec), VCL inhibited platelet adhesion to the subendothelial surface of human umbilical arteries. The maximum inhibition of platelet adhesion was 83 ± 4% at a VCL concentration of 7.6μmol/L. Various monoclonal anti-Very Late Activation antigens (VLA) antibodies were added to the VCL and tested for their ability to enhance the inhibition of platelet adhesion at high shear forces. Of all of the VLA antibodies tested, only the anti-VLA-2 antibody (176D7) inhibited platelet aggregation in the absence of VCL and enhanced the inhibition of platelet adhesion in the presence of VCL. The VLA-2 antibody and VCL together inhibited 96 ± 4% of platelet adhesion at high shear forces.

Protein tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and von Willebrand factor

The interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) induced by ristocetin or botrocetin resulted in associated platelet aggregation, protein tyrosine phosphorylation (PTP) of a 64 kDa protein, as detected by a monoclonal antibody against phosphotyrosine (PY-20), and intracellular Ca 2+ elevation that is largely dependent upon Ca 2+ influx in human platelets. It is of interest that 75–80, 97 and 125 kDa proteins which are strongly tyrosine-phosphorylated in platelet activation induced by thrombin and other agonists were not detected. Neither vWF nor a coaggregating agent (ristocetin or botrocetin) alone induced aggregation, [Ca 2+] i elevation or the 64 kDa PTP. NMC-4, an antibody which inhibits both ristocetin- or botrocetin-induced vWF binding to GPIb, abolished the appearance of the 64 kDa PTP as well as other responses, suggesting that it is specifically induced by the GPIb-vWF interaction. Aspirin, or ONO-3708, a competitive inhibitor of thromboxane A 2, did not modify the 64 kDa PTP, while [Ca 2+] i elevation was moderately suppressed. Depletion of extracellular Ca 2+ or RGD peptides suppressed neither the 64 kDa PTP nor aggregation. H-7, a protein kinase C inhibitor, did not inhibit the 64 kDa PTP, while staurosporine, a potent protein kinase inhibitor, inhibited the 64 kDa PTP and Ca 2+ influx, but not aggregation, in a dose-dependent manner. It is suggested that the 64 kDa PTP is associated with platelet aggregation induced by the interaction between GPIb and vWF.

Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib

Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

Studies on Anti-von Willebrand Factor (vWF) Monoclonal Antibody NMC-4, Which Inhibits Both Ristocetin- and Botrocetin-Induced vWF Binding to Platelet Glycoprotein Ib

Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

Antiplatelet Glycoprotein lb Monoclonal Antibody (OP-Fl) Totally Abolishes Ristocetin-Induced von Willebrand Factor Binding, but Has Minimal Effect on the Botrocetin-Induced Binding

We describe here a new antiplatelet glycoprotein (GP) Ib monoclonal antibody (MoAb) designated OP-F1 (IgG1 kappa). Both OP-F1 and a well-characterized anti-GPIb MoAb, AP-1, totally abolished ristocetin-induced von Willebrand factor (vWF) binding to platelets and desialylated vWF binding to platelets at an IgG concentration of 2-8 micrograms/ml. AP-1 also blocked snake venom botrocetin-induced vWF binding at a similar IgG concentration, whereas OP-F1 had a minimal effect on botrocetin-induced binding. At a higher IgG concentration (150 micrograms/ml), OP-F1 inhibited botrocetin-induced binding by 50%. AP-1 (IgG) did not interfere with binding of [125I]OP-F1 (IgG) to platelets. Thus, the epitope involved in the binding of OP-F1 or AP-1 appears to be quite different. These results suggest that the vWF binding site(s) on the GPIb molecule generated by these inducers is in close proximity but not completely identical.

Enhanced botrocetin‐induced type iib von willebrand factor binding to platelet glycoprotein ib initiates hyperagglutination of normal platelets

Botrocetin, a protein isolated from the venom of the snake Bothrops jararaca, induces platelet aggregation/agglutination by von Willebrand factor (vWF) binding to the membrane glycoprotein (GP) Ib, an action resembling that of ristocetin. However, some differences in the interaction between vWF and platelet GPIb induced by these two substances have been reported. We have recently shown that the GPIb binding domain on the vWF molecule, in both instances, resides in the tryptic 52/48 kDa fragment extending from amino acid residue 449 to 728 of the constituent subunit. In the present report, we demonstrate that botrocetin does not induce agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome congenitally lacking GPIb and GPIX as well as GPV, a finding similar to that shown with ristocetin. A monoclonal antibody against GPIb (AP-1) inhibits either ristocetin- or botrocetin-dependent vWF binding to formalin-fixed platelets from normal individuals. Therefore, botrocetin-induced vWF binding to formalin-fixed platelets may reflect the interaction between vWF and platelet GPIb. To strengthen this concept, we have now found that heightened botrocetin-induced type IIB vWF binding to platelet GPIb causes hyperagglutination of normal platelets.

Cleavage of Human von Willebrand Factor by Porcine Pancreatic Elastase

von Willebrand factor (vWF) was purified from pooled normal plasma, radiolabeled with iodine then cleaved by porcine pancreatic elastase. Cleavage was monitored by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-agarose electrophoresis, crossed immuno-electrophoresis, ristocetin and botrocetin cofactor activities, and ristocetin and botrocetin induced binding to fixed washed platelets. Cleavage of vWF by porcine elastase was dependent on both the concentration of porcine elastase and period of incubation. Incubation of vWF with concentrations of porcine elastase greater than 20 U/mL for 30 minutes resulted in loss of the 240 Kd vWF subunit and a corresponding loss of the high molecular weight multimers and formation of fragments with molecular weights on reduced SDS-PAGE of 150, 125, 115 Kd and minor bands at 44, 28, and 24 Kd and a band at 20 Kd, which increased in intensity as either the concentration of porcine elastase or the period of incubation increased. More than 50% of the ristocetin cofactor activity was lost during a five-minute incubation of vWF with 0.56 U/mL porcine elastase when no detectable structural changes in the vWF molecule had occurred. Botrocetin cofactor activity was more resistant. Similarly botrocetin induced vWF binding to platelets was retained by a fraction produced by digestion of vWF with porcine elastase, which contained predominantly a 20 Kd fragment of vWF. This fragment retained insignificant amounts of ristocetin related functions and therefore represents a useful piece of the vWF molecule for further exploration of the site involved in botrocetin induced binding to platelets.

Cleavage of human von Willebrand factor by porcine pancreatic elastase

von Willebrand factor (vWF) was purified from pooled normal plasma, radiolabeled with iodine then cleaved by porcine pancreatic elastase. Cleavage was monitored by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-agarose electrophoresis, crossed immuno- electrophoresis, ristocetin and botrocetin cofactor activities, and ristocetin and botrocetin induced binding to fixed washed platelets. Cleavage of vWF by porcine elastase was dependent on both the concentration of porcine elastase and period of incubation. Incubation of vWF with concentrations of porcine elastase greater than 20 U/mL for 30 minutes resulted in loss of the 240 Kd vWF subunit and a corresponding loss of the high molecular weight multimers and formation of fragments with molecular weights on reduced SDS-PAGE of 150, 125, 115 Kd and minor bands at 44, 28, and 24 Kd and a band at 20 Kd, which increased in intensity as either the concentration of porcine elastase or the period of incubation increased. More than 50% of the ristocetin cofactor activity was lost during a five-minute incubation of vWF with 0.56 U/mL porcine elastase when no detectable structural changes in the vWF molecule had occurred. Botrocetin cofactor activity was more resistant. Similarly botrocetin induced vWF binding to platelets was retained by a fraction produced by digestion of vWF with porcine elastase, which contained predominantly a 20 Kd fragment of vWF. This fragment retained insignificant amounts of ristocetin related functions and therefore represents a useful piece of the vWF molecule for further exploration of the site involved in botrocetin induced binding to platelets.