Cloning and Expression of Canine Glycoprotein Ibα

Summary

The interaction of the glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (vWF) is critical in initiation of haemostasis and thrombosis through platelet adhesion to damaged endothelium. The binding site for vWF resides within the GPIbα subunit of the complex. To further define the physiological function of platelet GPIbα we cloned and expressed the canine GPIbα cDNA. A canine platelet cDNA library was constructed and screened with a randomly primed ³²P-labeled 1041-base-pair restriction fragment of the human GPIbα cDNA. Analysis of 23 clones demonstrated that the canine GPIbα cDNA is 2530 nucleotides in length and includes a short 5’ untranslated segment of 42 nucleotides followed by a signal peptide of 16 amino acids, a mature peptide of 645 amino acids and a 3’ noncoding region of 455 nucleotides. A single intron of 142 nucleotides, 6 nucleotides upstream from the ATG translation initiation codon was identified in the canine gene in a similar location to that present in the human gene.

Chinese hamster ovary cells that stably express human GPIbα and GPIX were transfected with the canine GPIbα cDNA. Canine GPIbα was expressed on the surface of these cells and bound vWF in the presence of botrocetin. The binding of vWF was inhibited by an anti-vWF human monoclonal antibody known to inhibit vWF binding to GPIbα. The results of this investigation will allow the development of reagents to study the physiological function of GPIbα in an animal model.

• References

• 1 Roth GJ. Developing relationships: arterial platelet adhesion, glycoprotein Ib, and leucine-rich glycoproteins. Blood 1991; 77: 5-19.
• 2 Alevriadou BR, Moake JL, Turner NA, Ruggeri ZM, Folie BJ, Phillips MD, Schreiber AB, Hrinda ME, McIntire LV. Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand Factor binding to platelets. Blood 1993; 81: 1263-76.
• 3 López JA, Chung DW, Fujikawa K, Hagen FS, Davie EW, Roth GJ. The α and β chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence. Proc Natl Acad Sci 1988; 85: 2135-9.
• 4 López JA. The platelet glycoprotein Ib-IX complex. Blood Coag and Fibrin 1994; 5: 97-119.
• 5 Modderman PW, Admiraal LG, Sonnenberg A, von dem Borne AE. Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane. J Biol Chem 1992; 267: 364-9.
• 6 George JN, Nurden AT, Phillips DR. Molecular defects in interaction of platelets with the vessel wall. N Engl J Med 1984; 311: 1084-98.
• 7 Sakariassen KS, Nievelstein PFEM, Coller BS, Sixma JJ. The role of platelet membrane glycoprotein Ib and IIb-IIIa in platelet adherence to human artery subendothelium. Br J Haem 1986; 63: 681-91.
• 8 Coller BS, Gralnick HR. Studies on the mechanism of ristocetin-induced platelet agglutination: effects of structural modification of ristocetin and vancomycin. J Clin Invest 1977; 60: 302-12.
• 9 Read MS, Shermer RW, Brinkhous KM. Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. Proc Natl Acad Sci 1978; 75: 4514-8.
• 10 van Zanten GH, de Graaf S, Slootweg PJ, Heijnen HFG, Connolly TM, de Groot PG, Sixma JJ. Increased platelet deposition on atherosclerotic coronary arteries. J Clin Invest 1994; 93: 615-32.
• 11 Ruggeri ZM. The Platelet Glycoprotein Ib-IX complex. Progress in Haemostasis and Thrombosis 1991; 10: 35-67.
• 12 Read MS, Potter JY, Brinkhous KM. Venom coagglutinin for detection of von Willebrand factor activity in animal plasmas. J Lab Clin Med 1983; 101: 74-82.
• 13 Ware J, Russell S, Ruggeri ZM. Cloning of the murine platelet glycoprotein Ibα gene highlighting species-specific platelet adhesion. Blood Cells, Molecules, and Diseases 1997; 23: 292-301.
• 14 Kenny D, Brooks HL, Warltier DC. Enhanced alpha adrenoceptor-mediated vasoconstriction by dietary omega-3 fatty acids in chronically instrumented dogs. Am J Physiol 1990; 258: H1660-7.
• 15 Kenny D, Wynsen JC, Brooks HL, Warltier DC. Dipyridamole-induced decrement of functional recovery of postischemic reperfused myocardium in conscious dogs with well-developed coronary collateral circulation. Am Heart J 1991; 121: 1339-47.
• 16 Folts JD, Crowell EB, Rowe GG. Platelet aggregation in partially obstructed vessels and its elimination with asprin. Circulation 1976; 54: 365-70.
• 17 Bush LR, Shebuski RJ. In vivo models of arterial thrombosis and thrombolysis. FASEB J 1990; 4: 3087-98.
• 18 Fitzgerald DJ, Hanson M, FitzGerald GA. Systemic lysis protects against the effects of platelet activation during coronary thrombolysis. J Clin Invest 1991; 88: 1589-95.
• 19 Davis LG, Dibner MD, Battey JF. Plasmid “mini-prep” method, in Basic methods in molecular biology. New York: Elsevier Science; 1986: 102-4.
• 20 Sanger F. Determination of nucleotide sequence in DNA. Science 1981; 214: 1205-10.
• 21 Kenny D, Newman PJ, Morateck PA, Montgomery RR. A dinucleotide deletion in glycoprotein Ibα results in defective membrane anchoring and circulating soluble glycoprotein Ibα in a novel form of Bernard-Soulier syndrome. Blood 1997; 90: 2626-33.
• 22 Scott JP, Montgomery RR. The rapid differentiation of type IIb von Willebrand’s disease from platelet-type (pseudo-) von Willebrand’s disease by the “neutral” monoclonal antibody binding assay. Am J Clin Path 1991; 96: 723-8.
• 23 Kawai Y, Montgomery RR. Endothelial cell processing of von Willebrand proteins. Ann NY Acad Sci 1987; 509: 60-6.
• 24 Andrews RK, Booth WJ, Gorman JJ, Castaldi PA, Berndt MC. Purification of botrocetin from Bothrops jaracara venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex. Biochemistry 1989; 28: 8317-26.
• 25 López JA, Weisman S, Sanan DA, Sih T, Chambers M, Li CQ. Glycoprotein (GP) Ibα is the critical subunit linking GP Ibα and GP IX in the GP Ib-complex. J Biol Chem 1994; 269: 23716-21.
• 26 Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielson M. Lipofection: a highly efficent, lipid mediated DNA-transfection procedure. Proc Natl Acad Sci 1987; 84: 7413-7.
• 27 Wenger RH, Kieffer N, Wicki AN, Clemetson KJ. Structure of the human blood platelet membrane glycoprotein Ibα gene. Biochem Biophys Res Comm 1988; 156: 389-95.
• 28 Meyer S, Kresbach G, Häring P, Schumpp-Vonach B, Clemetson KJ, Hadváry P, Steiner B. Expression and characterization of functionally active fragments of the platelet glycoprotein Ib-IX complex in mammalian cells. J Biol Chem 1993; 268: 20555-62.
• 29 López JA, Leung B, Reynolds CC, Li CQ, Fox JEB. Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits. J Biol Chem 1992; 267: 12851-9.
• 30 Kobe B, Deisenhofer J. The leucine-rich repeat: a versatile binding motif. Trends in Biochem Sci 1994; 19: 415-21.
• 31 Hickey MJ, Hagen FS, Yagi M, Roth GJ. Human platelet glycoprotein V: Characterization of the polypeptide and the related Ib-V-IX receptor. Proc Natl Acad Sci 1993; 90: 8327-31.
• 32 Kenny D, Jónsson OG, Morateck PA, Montgomery RR. Naturally occurring mutations in glycoprotein Ibα that result in defective ligand binding and synthesis of a truncated protein. Blood 1998; 92: 175-83.
• 33 López JA, Chung DW, Fujikawa K, Hagen FS, Papayannopoulou T, Roth GJ. Cloning of the α chain of human platelet glycoprotein Ib: A transmembrane protein with homology to leucine-rich α₂ glycoprotein. Proc Natl Acad Sci 1987; 84: 5615-9.
• 34 Cruz MA, Petersen E, Turci SM, Handin RI. Functional analysis of a recombinant glycoprotein Ibα polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein IB-IX complex and collagen. J Biol Chem 1992; 267: 1303-9.
• 35 Murata M, Ware J, Ruggeri ZM. Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ibα demonstrating negatively charged residues involved in von Willebrand factor binding. J Biol Chem 1991; 266: 15474-80.
• 36 Katagiri Y, Hayashi Y, Ymamoto K, Tanoue K, Kosaki G, Yamazaki H. Localization of von Willebrand factor and thrombin-interactive domains on human platelet glycoprotein Ib. Thromb Haemost 1990; 63: 122-6.
• 37 Vincente V, Houghten RA, Ruggeri ZM. Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand factor binding. J Biol Chem 1990; 265: 274-80.
• 38 Scott JP, Montgomery RR, Retzinger GS. Dimeric ristocetin flocculates protein, binds to platelets, and mediates von Willebrand factor-dependent agglutination of platelets. J Biol Chem 1991; 266: 8149-55.
• 39 Marchese P, Murata M, Mazzucato M, Pradella P, De Marco L, Ware J, Ruggeri ZM. Identification of three tyrosine residues of glycoprotein Ibα with distinct roles in von Willebrand factor and α-thrombin binding. J Biol Chem 1995; 270: 9571-8.
• 40 Dong J, Li CQ, López JA. Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding. Biochemistry 1994; 33: 13946-53.
• 41 Hortin G, Folz R, Gordon JI, Strauss AW. Characterization of sites of tyrosine sulfation in proteins and criteria for predicting their occurrence. Biochem Biophys Res Com 1986; 141: 326-33.
• 42 López JA, Ludwig EH, McCarthy BJ. Polymorphism of human glycoprotein Ibα results from a variable number of tandem repeats of a 13-amino acid sequence in the mucin-like macroglycopeptide region. J Biol Chem 1992; 267: 10055-61.
• 43 Ishida F, Saji H, Maruya E, Furihata K. Human platelet-specific antigen, Sib^a is associated with the molecular weight polymorphism of glycoprotein Ibα. Blood 1991; 78: 1722-7.
• 44 Ishida F, Furihata K, Ishida K, Yan J, Kitano K, Kiyosawa K, Furuta S. The largest variant of platelet glycoprotein Ibα has four tandem repeats of 13 amino acids in the macroglycopeptide region and a genetic linkage with methionine 145. Blood 1995; 86: 1357-60.
• 45 Rixon MW, Chan WY, Davie WW. D. W. C. Characterization of a complementary deoxyribonucleic acid coding for the α chain of human fibrinogen. Biochemistry 1983; 22: 3237-44.
• 46 Murakawa M, Okamura T, Kamura T, Shibuya T, Harada M, Niho Y. Diversity of primary structures of the carboxy-terminal regions of mammalian fibrinogen Aα-chains. Characterization of the partial nucleotide and deduced amino acid sequences in five mammalian species; rhesus monkey, pig, dog, mouse and syrian hamster. Thromb Haemost 1993; 69: 351-60.
• 47 Ware J, Russell SR, Marchese P, Ruggeri ZM. Expression of human platelet glycoprotein Iba in transgenic mice. J Biol Chem 1993; 268: 8376-82.
• 48 The Epic Investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high risk coronary angioplasty. N Engl J Med 1994; 330: 956-61.