Limited proteolysis of human von Willebrand factor by Staphylococcus aureus V-8 protease: isolation and partial characterization of a platelet-binding domain

Biochemistry ◽  
1986 ◽  
Vol 25 (11) ◽  
pp. 3156-3163 ◽  
Author(s):  
Jean Pierre Girma ◽  
Michael W. Chopek ◽  
Koiti Titani ◽  
Earl W. Davie
Keyword(s):  
Staphylococcus Aureus ◽  
Von Willebrand Factor ◽  
Limited Proteolysis ◽  
Binding Domain ◽  
Partial Characterization ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Isolation and characterization of a collagen binding domain in human von Willebrand factor.

1986 ◽  
Vol 261 (32) ◽  
pp. 15310-15315 ◽  
Author(s):  
F I Pareti ◽  
Y Fujimura ◽  
J A Dent ◽  
L Z Holland ◽  
T S Zimmerman ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Domain ◽  
Collagen Binding ◽  
Isolation And Characterization ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Phage Display Functionally Defines Variants in the Von Willebrand Factor Platelet Binding Domain

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1033-1033
Author(s):  
Andrew Yee ◽  
Melda Arslantas Guzel ◽  
Manhong Dai ◽  
Fan Meng ◽  
David Ginsburg
Keyword(s):  
Phage Display ◽  
Von Willebrand Factor ◽  
Phage Display Library ◽  
Binding Activity ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Missense Variants ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Missense variants in the von Willebrand factor (VWF) platelet binding domain, A1, may pathologically hyperactivate or weaken interactions with its platelet receptor, GPIbα, and lead to von Willebrand disease (VWD) subtypes 2B or 2M, respectively. Variants identified in VWD patients and tested as recombinant VWF have supported genotype-phenotype associations and subtyping of VWD by genetic analyses. However, novel variants, most classified as variants of uncertain significance (VUS) are poorly defined. To functionally characterize a large subset of VWF A1 variants (P1254-L1460), we screened a phage display library for binding to a recombinant form of GPIbα used to clinically assess VWF platelet binding activity, GPIbM. Comprised of ~5x10 6 independent clones, the phage display library contained 1,427 unique, missense variants (~36% of all possible single amino acid substitutions) which could be scored for significant enrichment, depletion, or no change following selection for GPIbM binding. The enrichment of phage displayed VWD variants previously classified as VWD subtype 2B significantly segregated from reported 2M variants (mean fold change from preselected phage ~1.06 for 2B vs. ~0.68 for 2M, p < 0.005). To further validate these findings, five depleted, four unchanged, and seven enriched VWF A1 variants were introduced into the full length VWF sequence by site-directed mutagenesis and expressed by transient transfection of HEK293T cells. Conditioned media were collected and analyzed for VWF level (VWF:Ag) and activity (VWF:GPIbM). Of the sixteen variants examined, fourteen (87.5%) exhibited a VWF GPIbM:Ag ratio that was concordant with the phage display findings. Furthermore, the VWF GPIbM:Ag ratios were well correlated with the degree of enrichment by phage display (Pearson R = 0.69, p< 0.01). Taken together, these findings demonstrate phage display as a high content approach to measure and functionally define the platelet-binding activity of genetic variants within the VWF A1 domain. Disclosures Ginsburg: Takeda: Patents & Royalties.

The α1 helix-β13 strand spanning Leu214 to Val229 of platelet glycoprotein Ibα facilitates the interaction with von Willebrand factor: evidence from characterization of the epitope of monoclonal antibody AP1

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 3971-3978 ◽  
Author(s):  
Yuandong Peng ◽  
Michael C. Berndt ◽  
Miguel A. Cruz ◽  
José A. López
Keyword(s):  
Von Willebrand Factor ◽  
Chinese Hamster ◽  
Platelet Glycoprotein ◽  
Rolling Velocity ◽  
Platelet Binding ◽  
The Many ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The glycoprotein Ib-IX-V (GP Ib-IX-V) complex mediates platelet binding to von Willebrand factor (VWF) through its largest polypeptide, GP Ibα. Of the many GP Ibα monoclonal antibodies described, AP1 is of particular interest because it blocks static VWF binding induced by 2 modulators, ristocetin and botrocetin, and platelet adhesion to VWF surfaces under flow. We mapped the AP1 binding site to a region encompassing Arg218 to Tyr228, comprising the α1 helix and β13 strand defined by the GP Ibα crystal structure. AP1 binding absolutely required Arg218, Asp222, and Glu225. We evaluated the ability of cells expressing mutants of this region to bind VWF under static conditions in the presence of modulators, and to attach to and roll on a VWF matrix under flow. These data indicate that 2 regions within the sequence Arg218 to Tyr228 have important roles in VWF binding: the α1 helix has a regulatory role and the β turn and β13 strand bind VWF directly. Despite this, the only effect of a synthetic peptide corresponding to Leu214 to Val229 was to slightly increase the rolling velocity of GP Ibα-expressing Chinese hamster ovary (CHO) cells on VWF. This region thus appears to be more important for maintaining the regional conformation of GP Ibα, thereby facilitating the interaction with VWF. (Blood. 2004;104:3971-3978)

Ristocetin and Botrocetin Involve Two Distinct Domains of von Willebrand Factor for Binding to Platelet Membrane Glycoprotein lb

1990 ◽  
Vol 64 (02) ◽  
pp. 326-332 ◽  
Author(s):  
J P Girma ◽  
Y Takahashi ◽  
A Yoshioka ◽  
J Diaz ◽  
D Meyer
Keyword(s):  
Von Willebrand Factor ◽  
Synthetic Peptides ◽  
Competitive Inhibition ◽  
Final Concentration ◽  
Binding Domain ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe have evidence that ristocetin and botrocetin mediate binding of von Willebrand Factor (vWF) to platelet glycoprotein lb (GPIb) through two distinct domains on the vWF molecule. This was established by using monoclonal antibodies (MAbs) to vWF and synthetic peptides derived from the sequence of vWF. MAb 322 and MAb NMC/vW 4 both recognize native vWF as well as fragments containing the GPIb-binding domain of vWF, obtained with the following enzymes: trypsin (116 kDa), V-8 pro tease (Spill, 320 kDa) and V-8 protease plus subtilisin (33-28 kDa). Nevertheless, the lack of reciprocal displacement between the two MAbs in experiments of competitive inhibition for binding to vWF demonstrate that their respective epitopes are separate. Both MAbs inhibit 125I-vWF binding to platelet membrane GPIb and vWF-dependent platelet agglutination induced by ristocetin. However, only MAb NMC/vW4 inhibits these functions in the presence of botrocetin and when ristocetin-induced platelet agglutination is inhibited by MAb 322, botrocetin is still able to restore the agglutination. The involvement of two distinct domains of vWF for binding to GPIb in the presence of ristocetin or botrocetin was confirmed in experiments of binding of 125I-vWF to platelets using as competitor synthetic peptides corresponding to the GPIb binding domain of vWF (Cys 474 to Pro 488 and Ser 692 to Pro 708). At a final concentration of 2.5 mM both peptides inhibit more than 90% of the binding of vWF to ristocetin-treated platelets but are unable to modify this binding in the presence of botrocetin. In conclusion our data suggest that botrocetin and ristocetin involve distinct sites on vWF for binding to GPIb.

Characterization of Partial Gene Deletions in Type III von Willebrand Disease with Alloantibody Inhibitors

1994 ◽  
Vol 72 (02) ◽  
pp. 180-185 ◽  
Author(s):  
David J Mancuso ◽  
Elodee A Tuley ◽  
Ricardo Castillo ◽  
Norma de Bosch ◽  
Pler M Mannucci ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Pcr Analysis ◽  
Gene Deletions ◽  
Factor Gene ◽  
Type Iii ◽  
Large Deletions ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor gene deletions were characterized in four patients with severe type III von Willebrand disease and alloantibodies to von Willebrand factor. A PCR-based strategy was used to characterize the boundaries of the deletions. Identical 30 kb von Willebrand factor gene deletions which include exons 33 through 38 were identified in two siblings of one family by this method. A small 5 base pair insertion (CCTGG) was sequenced at the deletion breakpoint. PCR analysis was used to detect the deletion in three generations of the family, including two family members who are heterozygous for the deletion. In a second family, two type III vWD patients, who are distant cousins, share an -56 kb deletion of exons 22 through 43. The identification and characterization of large vWF gene deletions in these type III vWD patients provides further support for the association between large deletions in both von Willebrand factor alleles and the development of inhibitory alloantibodies.

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