von Willebrand Factor without the A2 Domain Is Resistant to Proteolysis

Summary

von Willebrand factor (vWF) is a complex multimeric plasma glycoprotein, that plays a critical role in the mediation of platelet adhesion to the damaged vascular wall, and functions as a carrier protein for factor VIII. vWF has a domain structure consisting of repeated A, B, C, and D domains. The A1 domain is involved in binding to the platelet receptor glycoprotein (GP) lb, and the A3 domain has a binding site for collagen. A function of the A2 domain has not been described, although point mutations identified in von Willebrand disease (vWD) type 2A patients are localized in this domain. To study the role of the A2 domain a deletion mutant was constructed which lacked the A2 domain, ΔA2- vWF. Previous studies have shown that this approach is a powerful tool to study the function of a domain in a protein since it does not affect the activity of other domains. After expression in baby hamster kidney (BHK) cells, ΔA2-vWF was compared to wild-type (WT) vWF, and to ΔAl-vWF (Lankhof et al., Blood 86: 1035,1995). Ristocetin induced platelet binding was slightly increased but botrocetin induced platelet binding was normal as was binding to heparin and collagen type III. Adhesion studies to surface coated purified ΔA2-vWF or to ΔA2-vWF preincubated on collagen under flow conditions showed no abnormalities. Incubation with normal human plasma showed that ΔA2-vWF like WT-vWF was not sensitive to proteolysis. After addition of urea, WT-vWF becomes sensitive to the protease, indicating that unfolding of the molecule is necessary for exposure of the cleavage site. ΔA2- vWF tested under the same conditions was resistant, indicating that the protease sensitive site is located in the A2 domain.

• References

• 1 Sixma JJ, VanZanten GH, Banga JD, Nieuwenhuis HK. De Groot PG. Platelet adhesion. Semin Hemat 1995; 32: 1-6
• 2 Ruggeri ZM, Ware J. von Willebrand factor. FASEB J 1993; 7: 308-316
• 3 Shelton-Inloes BB, Titani K, Sadler JE. cDNA sequence for human von Willebrand factor reveals five types of repeated domains and five possible protein sequence polymorphisms. Biochemistry 1986; 25: 3164-3171
• 4 Bonthron DT, Handin RI, Kaufman RJ, Wasley LC, Orr EC, Mitsock LM, Ewenstein B, Loscalzo J, Ginsburg D, Orkin SH. Structure of pre-pro-von Willebrand factor and its expression in heterologous cells. Nature 1986; 324: 270-273
• 5 Verweij CL, Diergaarde P, Hart M, Pannekoek H. Full-length von Willebrand factor (vWF) cDNA encodes a highly repetitive protein, considerable larger than the mature vWF subunit. EMBO J 1986; 5: 1839-1847
• 6 Bork P, Rohde K. More von Willebrand factor type A domains? Sequence similarities with malaria thrombospondin-related anonymous protein, dihydropyridine-sensitive calcium channel and intertrypsin inhibitor. Biochem J 1991; 279: 908-910
• 7 Bork P. The modular architecture of vertebrate collagens. FEBS Lett 1992; 307: 49-54
• 8 Colombatti A, Bonaldo P, Doliana R. Type A modules: interacting domains found in several non-fibrillar collagens and in other extracellular matrix proteins. Matrix 1993; 13: 297-306
• 9 Perkins SJ, Smith KF, Williams SC, Haris PI, Chapman D, Sim RB. The secondary structure of the von Willebrand factor type A domain in factor B of human complement by Fourier transform infrared spectroscopy. Its occurrence in collagen types VI, VII, XII and XIV, the integrins and other proteins by averaged structure predictions. J Mol Biol 1994; 238: 104-119
• 10 Kamata T, Wright R, Takada Y. Critical threonine and aspartic acid residues within the I domains of 2 integrins for interactions with intercellular adhesion molecule 1 (ICAM-1) and C3bi. J Biol Chem 1995; 270: 12531-12535
• 11 Edwards CP, Champe M, Gonzalez T, Wessinger ME, Spencer SA, Presta LG, Berman PW. Bodary SC: Identification of amino acids in the CD1 la I-domain important for binding of the leukocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1). J Biol Chem 1995; 270: 12635-12640
• 12 Girma J-P, Ribba A-S, Meyer D. Structure-function relationship of the Al domain of von Willebrand factor. Thromb Haemost 1995; 74: 156-160
• 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 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
• 15 Dong Z. Sadler JE: Interactions of human von Willebrand factor type A domains with collagen, heparin and extracellular matrix. Blood 1994; 84: 388a (abstr).
• 16 Lankhof H, Van Hoeij M, Schiphorst ME, Bracke M, Wu Y-P, Ijsseldijk MJW, Vink T, DeGroot PHG, Sixma JJ. A3 domain is essential for interaction of von Willebrand factor with collagen type III. Thromb Haemost 1996; 75: 950-958
• 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. J Biol Chem 1995; 270: 10822-10827
• 18 Dent JA, Berkowitz SD, Ware J, Kasper CK, Ruggeri ZM. Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci USA 1990; 87: 6306-6310
• 19 Dent JA, Galbusera M, Ruggeri ZM. Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit. J Clin Invest 1991; 88: 774-782
• 20 Lyons SE, Bruck ME, Bowie Ej W, Ginsburg D. Impaired intracellular transport produced by a subset of type IIA von Willebrand disease mutations. J Biol Chem 1992; 267: 4424-4430
• 21 Sixma JJ, Sakariassen KS, Beeser-Visser NH. Adhesion of platelets to human artery subendothelium. Effect of factor VUI-von Willebrand factor of various multimeric composition. Blood 1984; 63: 128-139
• 22 Sakariassen K, Fressinaud E, Girma JP, Baumgartner HR, Meyer D. Mediation of platelet adhesion to fibrillar collagen in flowing blood by a proteoltytic fragment of human von Willebrand factor. Blood 1986; 67: 1515-1518
• 23 Wu YP, van Breugel HHFI, Lankhof H, Wise RJ, Handin RI, De Groot PG, Sixma JJ. Platelet adhesion to multimeric and dimeric von Willebrand factor and to collagen type III preincubated with von Willebrand factor. Arterioscler Thromb Vase Biol 1996; 16: 611-620
• 24 Sixma JJ, Ijsseldijk MJW, Hindriks G, Van Zanten H-G, Gorecki M, Panet A, Garfinkel LI, De Groot PG. Adhesion of blood platelets is inhibited VCL, a recombinant fragment (leucine⁵⁰⁴to lysine⁷²⁸) of von Willebrand factor. Arterioscler Thromb Vase Biol 1996; 16: 64-71
• 25 Lankhof H, Wu Y-P, Vink T, Schiphorst ME, Zerwes H-G, De GrootPG, Sixma JJ. Role of the glycoprotein Ib-binding Al repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor. Blood 1995; 86: 1035-1042
• 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
• 27 Yon J, Fried M. Precise gene fusion by PCR. Nucl Acids Res 1989; 17: 4895
• 28 Koppelman SJ, Van Hoeij M, Vink T, Lankhof H, Schiphorst ME, Damas C, Vlot AJ, Wise R, Bouma BN, Sixma JJ. Requirements of von willebrand factor to protect factor VIII from inactivation by activated protein C. Blood 1996; 87: 2292-2300
• 29 Furlan M, Robles R, Lammle B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 1996; 87: 4223-42234
• 30 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
• 31 Sakariassen KS, Aarts PAMM, DeGroot 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
• 32 VanZanten GH, Saelman EUM, Schut-Hese KM, Wu Y-P, Slootweg PJ, Nieuwenhuis HK, DeGroot PG, Sixma JJ. Platelet adhesion to collagen type IV under flow conditions. Blood 1996; 88: 3862-3871
• 33 Houdijk WPM, Girma J-P, van MourikJA, Sixma JJ, Meyer D. Comparison of tryptic fragments of von Willebrand factor involved in binding to thrombin-activated platelets with fragments involved in ristocetin-induced binding and binding to collagen. Thromb Haemost 1986; 56: 391-396
• 34 Kessler CM, Floyd CM, Rick ME, Krizek DM, Lee SL, Gralnick HR. Collagen-Fac- tor VUI/von Willebrand factor protein interaction. Blood 1984; 63: 1291-1298
• 35 Ginsburg D, Sadler JE. Von Willebrand disease: A database of point mutations, insertions, and deletions. Thromb Haemost 1993; 69: 177-184
• 36 Cruz MA, Handin RI, Wise RJ. The interaction of the von Willebrand factor-Al domain with platelet glycoprotein MX. J Biol Chem 1993; 268: 21238-21245
• 37 Schwartzbauer JE, Mulligan RC, Hynes RO. Efficient and stable expression of recombinant fibronectin polypeptides. Proc Natl Acad Sci USA 1987; 84: 754-758
• 38 Doolittle RF. The multiplicity of domains in proteins. Annu Rev Biochem 1995; 64: 287-314
• 39 Lee J-O, Rieu P, Amaout MA, Liddington R. Crystal structure of the A domain from the subunit of integrin CR3 (CD1 lb/CD18). Cell 1995; 80: 631-638
• 40 Mohri H, Fujimura Y, Shima M, Yoshioka A, Houghten RA, Ruggeri ZM, Zimmerman TS. Structure of the von Willebrand factor domain interacting with glycoprotein Ib. J Biol Chem 1988; 263: 17901-17904
• 41 Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood 1996; 87: 4235-4244
• 42 Stoddart Jr JH, Andersen J, Lynch DC. Clearance of normal and type 2A von Willebrand factor in the rat. Blood 1996; 88: 1692-1699