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

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.

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CHARACTERIZATION OF THE VON WILLEBRAND FACTOR-BINDING DOMAIN OF PLATELET MEMBRANE GLYCOPROTEIN Ib

10.1055/s-0038-1642925 ◽

1987 ◽

Cited By ~ 1

Author(s):

M Handa ◽

K Titani ◽

K Takio ◽

Z M Ruggeri

Keyword(s):

Von Willebrand Factor ◽

Binding Domain ◽

Platelet Membrane ◽

Membrane Glycoprotein ◽

Tryptic Fragment ◽

Amino Terminal ◽

Platelet Membrane Glycoprotein ◽

Limited Digestion ◽

Von Willebrand ◽

Willebrand Factor

We have previously obtained immunochemical evidence that the von Willebrand factor (vWF)-binding domain of the platelet membrane glycoprotein (GP) Ib is located near the amino terminus of the a subunit (Journal of Biological Chemistry 261: 12579-12585, 1986). We have now determined the complete amino acid sequence of the 45 kDa tryptic fragment of glycocalicin that contains this domain. Purified glycocalicin was subjected to limited digestion with trypsin and the proteolytic fragments were separated by size-exclusion high-pressure liquid chromatography. Two fragments of 45 kDa and 84 kDa, respectively, were obtained under nonreducing conditions. After reduction and S-carboxymethylation, the 84 kDa fragment was unchanged, while the 45 kDa fragment yielded two new fragments, one of 35 kDa and the other of 7 kDa. This finding proves the existence of a trypsin cleavage site within a disulfide loop. Two primary sets of overlapping fragments were obtained by cleavage of the carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of glycocalicin with Staphylococcus aureus V8 protease and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the sequence of the amino terminal 299 residues of the GP Ib a-chain. This includes the 45 kDa tryptic fragment containing the vWF-binding domain. This 299-residue sequence, corresponding approximately to two thirds of the α-chain polypeptide, is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215-299, with a cluster of ten negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at Ser/Thr residues) region corresponding to the previously described "macroglycopeptide" begins at residue 292. The determined sequence of glycocalicin contains a region with seven repeats, indicative of gene duplication, and is highly homologous to human leucine-rich α2-glycoprotein.

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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 ◽

10.1182/blood.v78.9.2310.bloodjournal7892310 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2310-2317 ◽

Cited By ~ 1

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.

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Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.84.16.5610 ◽

1987 ◽

Vol 84 (16) ◽

pp. 5610-5614 ◽

Cited By ~ 76

Author(s):

K. Titani ◽

K. Takio ◽

M. Handa ◽

Z. M. Ruggeri

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Von Willebrand Factor ◽

Binding Domain ◽

Platelet Membrane ◽

Membrane Glycoprotein ◽

Glycoprotein Ib ◽

Factor Binding ◽

Von Willebrand ◽

Willebrand Factor

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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 ◽

10.1182/blood.v78.9.2310.2310 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2310-2317 ◽

Cited By ~ 37

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.

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Glycoprotein Ib Has a Partial Role in Platelet-von Willebrand Factor Collagen Interaction

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647026 ◽

1988 ◽

Vol 60 (02) ◽

pp. 182-187 ◽

Cited By ~ 2

Author(s):

Morio Aihara ◽

Ken Tamura ◽

Ryuko Kawarada ◽

Keizou Okawa ◽

Yutaka Yoshida

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Ricinus Communis ◽

Protein S ◽

Platelet Membrane ◽

Membrane Glycoprotein ◽

Normal Plasma ◽

Ricinus Communis Agglutinin ◽

Von Willebrand ◽

Willebrand Factor

SummaryThe adhesion of human fixed washed platelets (FWP) to collagen was decreased after treatment with Serratia marcescens protease (SP), which removed 95% of the glycocalicin from platelet membrane glycoprotein (GP) lb. However, the diminished adhesion of SP treated FWP to collagen could still be increased in the presence of purified von Willebrand factor (vWF). This ability of vWF to increase FWP adhesion to collagen is defined as collagen cofactor (CCo). The adhesion of FWP to collagen was not affected by a monoclonal antibody (MAb) to GP Ilb/IIIa (10E5), that inhibits ADP and collagen induced platelet aggregation. On the other hand, it was decreased by 50% by a MAb to GP lb (6D1), that inhibits ristocetin induced platelet aggregation. Adhesion of FWP in buffer to collagen was completely inhibited by Ricinus communis agglutinin I or concanavalin A, while Lens culinalis agglutinin and wheat germ agglutinin showed 50% inhibition. The FWP adhesion to collagen in the presence of vWF (normal plasma) was unaffected by MAbs to GP Ilb/IIIa (10E5, P2, HPL1) but was decreased to 32-38% by MAbs to GP lb (6D1, AN51, HPL11). A MAb to vWF (CLB-RAg 35), that inhibits ristocetin induced binding of vWF to platelets, decreased the CCo of normal plasma by 70%. The MAb, CLB-RAg 201, that inhibits the binding of vWF to collagen, completely inhibited the CCo of normal plasma. In conclusion, our data suggest that (1) GP lb has a partial role in FWP adhesion to collagen; (2) the binding of vWF to collagen is required for the expression of CCo; (3) CCo is partly mediated through GP lb; but (4) other platelet membrane protein(s) besides GP lb or GP Ilb/IIIa must also be involved in FWP-vWF-collagen interactions.

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Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation

Blood ◽

10.1182/blood.v95.1.164 ◽

2000 ◽

Vol 95 (1) ◽

pp. 164-172 ◽

Cited By ~ 49

Author(s):

Mariagrazia De Luca ◽

David A. Facey ◽

Emmanuel J. Favaloro ◽

Mark S. Hertzberg ◽

James C. Whisstock ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Platelet Membrane ◽

Membrane Glycoprotein ◽

Platelet Membrane Glycoprotein ◽

And Function ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Abstract Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481–Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf–dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700–Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf–induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494–Leu-512; CR2, Phe-536–Ala-554; CR3, Arg-578–Glu-596; CR11 and CR15, Ala-564–Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700–Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)

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