von Willebrand factor (vWF) mediates platelet adhesion at sites of vascular damage. It acts as a bridge between receptors on platelets and collagens present in the connective tissue. Two collagen binding sites have been identified on the A1 and A3 domain of the vWF subunit. To study the functional importance of these binding sites, we have made two deletion mutants that lack the A1 domain (residues 478-716; ΔA1-vWF; Sixma et al. Eur. J. Biochem. 196,369,1991 [1]) or the A3 domain (residues 910-1113; ΔA3-vWF). After transfection in baby hamster kidney cells overexpressing furin, the mutants were processed and secreted efficiently. Ristocetin or botrocetin induced platelet binding was normal for ΔA3-vWF as was binding to heparin and factor VIII. As reported by Sixma et al. (1) ΔAl-vWF still binds to collagen type III, indicating that the A3 domain is sufficient for the interaction. In the current study, we investigated the binding of ΔA3-vWF to collagen type III. When preincubated on collagen type III it did not support platelet adhesion under flow conditions, whereas it was able to support platelet adhesion when coated directly to a glass surface. The binding of 125I-ΔA3-vWF to collagen was specific but maximal binding was about 40 times less compared to 125I-vWF. When added at 25 times excess, ΔA3-vWF did not compete with 125I-vWF for binding to collagen type III, whereas ΔAl-vWF did. The binding of 125I-ΔA3-vWF could be blocked by excess unlabeled vWF but not by ΔA1-vWF. In conclusion, we demonstrate that the A3 domain in vWF contains the major collagen binding site. The major binding site present on the A3 domain and the minor site present on A1 bind to different sites on collagen.
References
1
Sixma JJ,
Schiphorst ME,
Verweij CL,
Pannekoek H.
Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin. Eur J Biochem 1991; 196: 369-375
3
Rand JH,
Wu X-X,
Potter BJ,
Uson RR,
Gordon RE.
Co-localization of von Willebrand factor and type VI collagen in human vascular subendothelium. Am J Pathol 1993; 142: 843-850
4
Denis C,
Baruch D,
Kielty CM,
Ajzenberg N,
Christophe O,
Meyer D.
Localization of von Willebrand factor binding domains to endothelial extracellular matrix and to type VI collagen. Arterioscler Thromb 1993; 13: 398-406
5
Ross JM,
Mclntire LV,
Moake JL,
Rand JH.
Platelet adhesion and aggregation on human type VI collagen surfaces under physiological flow conditions. Blood 1995; 85: 1826-1835
6
Kessler CM,
Floyd CM,
Rick ME,
Krizek DM,
Lee SL,
Gralnick HR.
Collagen-Factor VUI/von Willebrand factor protein interaction. Blood 1984; 63: 1291-1298
8
Houdijk WPM,
Schiphorst ME,
Sixma JJ.
Identification of functional domains bn von Willebrand factor by binding of tryptic fragments to collagen and to platelets in the presence of ristocetin. Blood 1986; 67: 1498-1503
9
Roth GJ,
Titani K,
Hoyer LW,
Hickey MJ.
Localization of binding sites within human von Willebrand factor for monomeric type III collagen. Biochemistry 1986; 25: 8357-8361
10
Pared FI,
Niija K,
McPherson JM,
Ruggeri ZM.
Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen type I and III. JBC 1987; 262: 13835-13841
11
Pared FI,
Fujimura Y,
Dent JA,
Holland LZ,
Zimmerman TS,
Ruggeri ZM.
Isolation and characterization of a collagen binding domain in human von Willebrand factor. JBC 1986; 261: 15310-15315
12
Mohri H,
Yoshioka A,
Zimmerman TS,
Ruggeri ZM.
Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites. JBC 1989; 264: 17361-17367
13
Kalafatis M,
Takahashi Y,
Girma J-P,
Meyer D.
Localization of a collagen-interactive domain of human von Willebrand factor between amino acid residues Gly 911 and Glu 1365. Blood 1987; 70: 1577-1583
14
Azuma H,
Dent JAe,
Sugimoto M,
Ruggeri ZM,
Ware J.
Independent assembly and secretion of a dimeric adhesive domain of von Willebrand factor containing the glycoprotein Ib-binding site. JBC 1991; 266: 12342-12347
16
Pietu G,
Meulien P,
Cherel G,
Diaz J,
Baruch D,
Courtney M,
Meyer D.
Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein lb, collagen and heparin binding domains. Biochem Biophys Res Comm 1989; 164: 1339-1347
17
Cruz MA,
Yuan H,
Lee JR,
Wise RJ,
Handin RI.
Interaction of the von Willebrand factor (vWF) with collagen. Localization of the primary collagen-binding site by analysis of recombinant vWF A domain polypeptides. JBC 1995; 270: 10822-10827
18
Takagi J,
Sekiya F,
Kasahara K,
Inada Y,
Saito Y.
Inhibition of platelet-collagen interaction by propolypeptide of von Willebrand factor. JBC 1989; 264: 6017-6020
19
Voorberg J,
Fontijn R,
van Mourik JA,
Pannekoek H.
Domains involved in multimer assembly of von Willebrand factor (vWF): Multimerization is independent of dimerization. EMBO J 1990; 9: 797-803
20
van de Ven WJM,
Voorberg J,
Fontijn R,
Pannekoek H,
van den Ouwen-land AMW,
van Duijnhoven HLP,
Roebroek AJM,
Siezen RJ.
Furin is a subtilisin like proprotein processing enzyme in higher eukaryotes. Mol Biol Rep 1990; 14: 265-275
21
Wise RJ,
Barr PJ,
Wong PA,
Kiefer MC,
Brake AJ,
Kaufman RJ.
Expression of a human proprotein processing enzyme: Correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site. Proc Natl AcadSci USA 1990; 87: 9378-9382
23
Pecenka V,
Dvorak M,
Travnicek M.
Simple and efficient method for cloning of large DNA fragments with identical ends into plasmid. Nucl Acids Res 1988; 16: 4179
25
Verweij CL,
Hart M,
Pannekoek H.
Expression of variant von Willebrand factor (vWF) cDNA in heterologous cells: requirement of the pro-polypeptide in vWF multimer formation. EMBO J 1987; 6: 2885-2890
26
Hilbert L,
Gaucher C,
De Romeuf C,
Horellou MH,
Vink T,
Mazurier C.
Leu 697 Val mutation in mature von Willebrand factor is responsible for type IIB von Willebrand disease. Blood 1994; 83: 1542-1550
29
Lankhof H,
Wu Y-P,
Vink T,
Schiphorst ME,
Zerwes H-G,
De Groot PG,
Sixma JJ.
Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor. Blood 1995; 86: 1035-1042
30
van Duijnhoven HLP,
Creemers JWM,
Kranenborg MGC,
Timmer EDJ,
Groeneveld A,
Van den Ouweland AMW,
Roebroek AJM,
van de Ven WJM.
Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin. Hybridoma 1993; 11: 71-86
31
Sodetz JM,
Pizzo SV,
McKee PA.
Relationship of sialic acid to function and in vivo survival of human factor VUI/von Willebrand factor protein. JBC 1977; 252: 5538-5546
34
Keller PM,
Schultz LD,
Condra C,
Karczewski J,
Connolly TM.
An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression. JBC 1992; 267: 6899-6904
36
Houdijk WPM,
Sakariassen KS,
Nievelstein PFEM,
Sixma JJ.
Role of Factor VUI-von Willebrand factor and Fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar human collagen types I and III. J Clin Invest 1985; 75: 1-10
37
Saelman EUM,
Nieuwenhuis HK,
Hese KM,
De Groot PG,
Heijnen HFG,
Sage EH,
Williams S,
McKeown L,
Gralnick HR,
Sixma JJ.
Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa. Blood 1994; 83: 1244-1250
38
Wu Y-P,
van Breugel HHFI,
Lankhof H,
Wise RJ,
Handin RI,
De Groot PG,
Sixma JJ.
Platelet adhesion and von Willebrand factor. Platelet adhesion to multimeric and dimeric von Willebrand factor and to collagen type III preincubated with von Willebrand factor. Arterioscl Thromb Vase Biol 1995 submitted
39
Sakariassen KS,
Bolhuis PA,
Sixma JJ.
Human blood platelet adhesion to artery subendothelium is mediated by factor VUI-von Willebrand factor bound to the subendothelium. Nature 1979; 279: 636-638
40
Sakariassen KS,
Aarts PAMM,
De Groot PG,
Houdijk WPM,
Sixma JJ.
A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 1983; 102: 522-535
41
Koppelman SJ,
Van Hoeij M,
Vink T,
Lankhof H,
Schiphorst ME,
Damas C,
Vlot AJ,
Wise RJ,
Bouma BN,
Sixma JJ.
Requirements of von Willebrand factor to protect factor VIII from inactivation by activated protein C. Blood 1995 in press
