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Promotion of binding of von Willebrand factor to platelet glycoprotein Ib by dimers of ristocetin.

ABSTRACT: In the absence of high shear forces, the in vitro binding of human von Willebrand factor (vWF) to its platelet receptor glycoprotein Ib (GPIb) can be promoted by two well-characterized mediators, botrocetin and ristocetin. Using purified vWF and GPIb, we have investigated the mechanisms by which ristocetin mediates this binding. Specific binding of vWF monomers to GPIb occurred with a 1:1 stoichiometry, but high-affinity binding required the participation of two ristocetin dimers. Binding was strongly dependent on pH and inhibited by low poly-L-lysine concentrations, indicating ristocetin-dependent charge neutralization during the interaction. With increasing ristocetin concentrations, vWF binding depended progressively less on the involvement of its A1 loop, which is compatible with a model in which the two ristocetin dimers bridge the vWF-GPIb complex on secondary sites. In agreement with this model, the ristocetin-dimer-promoted stabilization of vWF on GPIb was abolished by low concentrations of poly(Pro-Gly-Pro), which is known to complex ristocetin dimers. Mechanistic analysis of the inhibition of vWF binding by the recombinant vWF fragment Leu504-Ser728 (VCL), which covers the entire A1 loop, revealed an affinity of VCL for GPIb comparable with that of the botrocetin-vWF complex for GPIb, and identified a specific but 20-fold lower affinity of VCL in the presence of ristocetin. The proline-rich peptides flanking the vWF A1 loop, Cys474-Val489 and Leu694-Asp709, inhibited vWF binding semispecifically by competitively interfering with the formation of the GPIb-vWF complex rather than by complexation of free ristocetin dimers. In conclusion, ristocetin-promoted binding of vWF to its GPIb receptor results from charge neutralization and interactions involving proline residues in the vicinity of the natural interaction sites present on both GPIb and the A1 domain of vWF.

SUBMITTER: Hoylaerts MF

PROVIDER: S-EPMC1136540 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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Project description:Essentials New VWF activity assays are increasingly used but information on their comparability is limited. This is an ISTH SSC-organized study (expert labs, 5 countries) to compare all available assays. VWF activity by six assays correlated well with each other. The new assays show improved characteristics - minor differences are noted. SUMMARY:Background Several new assays have become available to measure von Willebrand factor (VWF) activity. The new assays appear to have improved performance characteristics compared with the old reference standard, ristocetin cofactor activity (VWF:RCo), but information is limited about how they compare with VWF:RCo and each other. Methods The von Willebrand factor Subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) designed a collaborative study involving expert laboratories from several countries to compare available tests with each other and with VWF:RCo. Eight laboratories from five countries were provided with blinded samples from normal healthy individuals and well-characterized clinical cases. Laboratories measured VWF activity using all tests available to them; data from six laboratories, not affected by thawing during transportation, are included in this study. Results All tests correlated well with VWF:RCo activity (r-values ranged from 0.963 to 0.989). Slightly steeper regression lines for VWF:Ab and VWF:GPIbM were clinically insignificant. The new assays showed improved performance characteristics. Of the commercially available assays, the VWF:GPIbR using the AcuStar system was the most sensitive and could reliably detect VWF activity below 1 IU dL-1 . The lower limit of the measuring interval for the VWF:GPIbM and the VWF:GPIbR assays was in the 3-4 and 3-6 IU dL-1 range, respectively. Inter-laboratory variation was also improved for most new assays. Conclusion All VWF activity assays correlated well with each other and the VWF:RCo assay. The slight differences in characteristics found in the COMPASS-VWF study will assist the VWF community in interpreting and comparing activity results.

Project description:Glycoprotein (GP: HIS 1 -PRO 265 ) Ibɑ is a receptor protein expressed on the surface of the platelet. Its N-terminus domain binds with the A1 domain (ASP 1269 -PRO 1472 ) of its ligand protein von Willebrand factor (VWF) and plays a unique role in platelet adhesion under blood flow conditions. Single amino acid substitutions at residue 233 from glycine (G) to alanine (A), aspartic acid (D), or valine (V) are known to cause biochemically distinct functional alterations known as equal, loss, and gain of function, respectively. However, the underlying physical characteristics of VWF binding with GPIbɑ in wild-type and the three mutants exerting different biological functions are unclear. Here, we aimed to test the hypothesis: biological characteristics of macromolecules are influenced by small changes in physical parameters. The position coordinates and velocity vectors of all atoms and water molecules constructing the wild-type and the three mutants of GPIbɑ (G233A, G233D, and G233V) bound with VWF were calculated every 2 × 10 -15 seconds using the CHARMM (Chemistry at Harvard Macromolecular Mechanics) force field for 9 × 10 -10 seconds. Six salt bridges were detected for longer than 50% of the calculation period for the wild-type model generating noncovalent binding energy of -1096 ± 137.6 kcal/mol. In contrast, only four pairs of salt bridges were observed in G233D mutant with noncovalent binding energy of -865 ± 139 kcal/mol. For G233A and G233V, there were six and five pairs of salt bridges generating -929.8 ± 88.5 and -989.9 ± 94.0 kcal/mol of noncovalent binding energy, respectively. Our molecular dynamic simulation showing a lower probability of salt bridge formation with less noncovalent binding energy in VWF binding with the biologically loss of function G233D mutant of GPIbɑ as compared with wild-type, equal function, and gain of function mutant suggests that biological functions of macromolecules such as GPIbɑ are influenced by their small changes in physical characteristics.

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.

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Promotion of binding of von Willebrand factor to platelet glycoprotein Ib by dimers of ristocetin.

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