scholarly journals The binding domain of von Willebrand factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and collagen and resides between amino acid residues Leu 512 and Lys 673

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2310-2317 ◽  
Author(s):  
O Christophe ◽  
B Obert ◽  
D Meyer ◽  
JP Girma
Keyword(s):  
Von Willebrand Factor ◽  
Competitive Inhibition ◽  
Binding Domain ◽  
Functional Site ◽  
Platelet Glycoprotein ◽  
Functional Domain ◽  
Amino Acid Residues ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract A series of proteolytic fragments of human von Willebrand Factor (vWF) was purified to characterize the functional site that supports its interaction with sulfatides. SpIII, an N-terminal homodimer generated by V-8 protease (amino acids [AA] 1 to 1365), bound to sulfatides in a dose-dependent and saturable way. SpIII also totally inhibited the binding of vWF to sulfatides and SpIII binding was completely abolished by vWF. In contrast, SpII, the complementary C-terminal homodimer (AA 1366 to 2050), did not exhibit any binding affinity for sulfatides. Four purified fragments overlapping the sequence of SpIII were also tested for their ability to interact with sulfatides. An N-terminal monomeric 34-Kd fragment (P34, AA 1 to 272) generated by plasmin, a central monomer (SpI, AA 911 to 1365) produced by digestion with V-8 protease, and a tetrameric fragment III-T2 (comprising a pair of the two sequences AA 273 to 511 and AA 674 to 728) produced by secondary digestion of SpIII with trypsin did not interact with sulfatides. In contrast, a monomeric 39/34-Kd fragment produced by dispase (AA 480 to 718) bound specifically and with a high affinity to sulfatides and totally displaced vWF or SpIII binding. Conversely, binding of the 39/34-Kd species was totally abolished by vWF or SpIII. Thus, a functional site responsible for sulfatide binding was localized between AA 480 and 718 and comparison of the binding properties of the 39/34-Kd and III-T2 fragments indicated that the sequence 512 to 673 is necessary for the binding to sulfatides. Further mapping of this new functional domain of vWF, based on experiments of competitive inhibition of binding by either heparin or monoclonal antibodies directed toward vWF, showed that the site interacting with sulfatides is distinct from those involved in binding to platelet glycoprotein Ib, collagen, or heparin. This finding was confirmed by experiments using synthetic peptides which also indicated that the sequence comprising AA 569 to 584 is part of the sulfatide-binding domain or influences its activity.

scholarly journals The binding domain of von Willebrand factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and collagen and resides between amino acid residues Leu 512 and Lys 673

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2310-2317 ◽  
Author(s):  
O Christophe ◽  
B Obert ◽  
D Meyer ◽  
JP Girma
Keyword(s):  
Von Willebrand Factor ◽  
Competitive Inhibition ◽  
Binding Domain ◽  
Functional Site ◽  
Platelet Glycoprotein ◽  
Functional Domain ◽  
Amino Acid Residues ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

A series of proteolytic fragments of human von Willebrand Factor (vWF) was purified to characterize the functional site that supports its interaction with sulfatides. SpIII, an N-terminal homodimer generated by V-8 protease (amino acids [AA] 1 to 1365), bound to sulfatides in a dose-dependent and saturable way. SpIII also totally inhibited the binding of vWF to sulfatides and SpIII binding was completely abolished by vWF. In contrast, SpII, the complementary C-terminal homodimer (AA 1366 to 2050), did not exhibit any binding affinity for sulfatides. Four purified fragments overlapping the sequence of SpIII were also tested for their ability to interact with sulfatides. An N-terminal monomeric 34-Kd fragment (P34, AA 1 to 272) generated by plasmin, a central monomer (SpI, AA 911 to 1365) produced by digestion with V-8 protease, and a tetrameric fragment III-T2 (comprising a pair of the two sequences AA 273 to 511 and AA 674 to 728) produced by secondary digestion of SpIII with trypsin did not interact with sulfatides. In contrast, a monomeric 39/34-Kd fragment produced by dispase (AA 480 to 718) bound specifically and with a high affinity to sulfatides and totally displaced vWF or SpIII binding. Conversely, binding of the 39/34-Kd species was totally abolished by vWF or SpIII. Thus, a functional site responsible for sulfatide binding was localized between AA 480 and 718 and comparison of the binding properties of the 39/34-Kd and III-T2 fragments indicated that the sequence 512 to 673 is necessary for the binding to sulfatides. Further mapping of this new functional domain of vWF, based on experiments of competitive inhibition of binding by either heparin or monoclonal antibodies directed toward vWF, showed that the site interacting with sulfatides is distinct from those involved in binding to platelet glycoprotein Ib, collagen, or heparin. This finding was confirmed by experiments using synthetic peptides which also indicated that the sequence comprising AA 569 to 584 is part of the sulfatide-binding domain or influences its activity.

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.

Glycoprotein Ib Can Mediate Endothelial Cell Attachment to a von Willebrand Factor Substratum

1995 ◽  
Vol 73 (02) ◽  
pp. 309-317 ◽  
Author(s):  
Dorothy A Beacham ◽  
Miguel A Cruz ◽  
Robert I Handin
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Single Amino Acid ◽  
Cell Surface Expression ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryIntroduction of single amino acid substitutions into the C-terminal Arg-Gly-Asp-Ser (RGDS) site of von Willebrand Factor, referred to as RGD mutant vWF, selectively abrogated vWF binding to platelet glycoprotein IIb/IIIa (GpIIb/IIIa, αIIbβ3 and abolished human umbilical vein endothelial cell (HUVEC) spreading, but not attachment, to RGD mutant vWF (Beacham, D. A., Wise, R. J., Turci, S. M. and Handin, R. I. 1992. J. Biol. Chem. 167, 3409-3415). These results suggested that in addition to the vitronectin receptor (VNR, αvβ3), a second endothelial membrane glycoprotein can mediate HUVEC adhesion to vWF. HUVEC attachment to wild-type (WT) and RGD-mutant vWF was reduced by two proteins known to block the vWF-platelet glycoprotein Ib/IX (GpIb/IX) interaction, the monoclonal antibody AS-7 and the recombinant polypeptide, vWF-A1. The addition of cytochalasin B or DNase I to disrupt potential GPIbα-cytoskeletal interactions enhanced the immunoprecipitation of endothelial GPIbα, caused HUVEC to round up, and increased HUVEC adhesion to RGD mutant vWF. These results indicate that while the VNR is the primary adhesion receptor for vWF, endothelial GPIbα can mediate HUVEC attachment to vWF. GpIb-dependent attachment could contribute to HUVEC adhesion under conditions when cell surface expression of the VNR is downregulated, and VNR-dependent adhesion is reduced.

scholarly journals von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain

2019 ◽  
Vol 3 (7) ◽  
pp. 957-968 ◽  
Author(s):  
Changjie Zhang ◽  
Anju Kelkar ◽  
Sriram Neelamegham
Keyword(s):  
Von Willebrand Factor ◽  
Ex Vivo ◽  
Association Studies ◽  
Apolipoprotein A1 ◽  
Platelet Glycoprotein ◽  
Functional Domain ◽  
Self Association ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues (“Lock-VWF”) reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain (“ΔA1 proteins”) suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

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