A Monoclonal Antibody, VM64, Reacts with a 130 kDa Glycoprotein Common to Platelets and Endothelial Cells: Heterogeneity in Antibody Binding to Human Aortic Endothelial Cells

Summary

A new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia–IIa, Ib, IIb–IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 ± 3.5 × 10³ per platelet and on the surface of EC monolayer – 2.40 ± 0.32 × 10⁶ per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb–IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.

• References

• 1 Phillips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb–IIIa complex. Blood 1988; 71: 831-43
• 2 Ginsberg MH, Loftus JC, Plow EF. Cytoadhesins, integrins and platelets. Thromb Haemostas 1988; 59: 1-6
• 3 Hemler ME, Crouse C, Takada Y, Sonnenberg A. Multiple very late antigen (VLA) heterodimers in platelets. Evidence for distinct VLA2, VLA5 (fibronectin receptor), and VLA6 structures. J Biol Chem 1988; 263: 7660-5
• 4 Pischel KD, Bluestein HG, Woods VL. Platelet glycoproteins Ia, Ic, and IIa are physicochemically indistinguishable from very late activation antigens adhesion-related proteins of lymphocytes and other cell types. J Clin Invest 1988; 81: 505-13
• 5 Giltay JC, Brinkman H-JM, Modderman PW, von dem Borne Kr AEG, van Mourik JA. Human vascular endothelial cells express a membrane protein complex immunochemically indistinguishable from the platelet VLA-2 (glycoprotein Ia-IIa) complex. Blood 1989; 73: 1235-41
• 6 Sprandio JD, Shapiro SS, Thiagaraian P, McCord S. Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib. Blood 1988; 71: 234-7
• 7 Bonfanti R, Furie BC, Furie B, Wagner DD. PADGEM (GMP-140) is a component of Weibel-Palade bodies of human endothelial cells. Blood 1989; 73: 1109-12
• 8 McEver RP, Beckstead JH, Moore KL, Marshall-Carison L, Bainton DF. GMP-140, a platelet α-granule membrane protein is also synthesized by vascular endothelial cells and is localised in Weibel-Palade bodies. J Clin 1989; 84: 92-9
• 9 Favaloro EJ, Moraits N, Koutts J, Exner T, Bradstock KF. Endothelial cells and normal circulating haemopoietic cells share a number of surface antigens. Thromb Haemostas 1989; 61: 217-24
• 10 Newman PJ, Berndt MC, Gorski J, White II GC, Lyman S, Paddock C, Muller WA. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science 1990; 247: 1219-22
• 11 Jaffe EA. Culture and identification of large vessel endothelial cells. In: Biology of Endothelial Cells.. Jaffe EA. (ed) Martinus Nijhoff Publishers, The Hague: 1987. pp 3-19
• 12 Antonov AS, Lukashev ME, Romanov YA, Tkachuk VA, Repin VS, Smirnov VN. Morphological alterations in endothelial cells from human aorta and umbilical vein induced by forskolin and forbol 12-myristate-13-acetate: a synergistic action of adenylate cyclase and protein kinase C activators. Proc Natl Acad Sci USA 1986; 83: 9704-8
• 13 Antonov AS, Nikolaeva MA, Klueva TS, Romanov YA, Babaev VR, Bystrevskaya VB, Perov NA, Repin VS, Smirnov VN. Primary culture of endothelial cells from atherosclerotic human aorta. I. Indification, morphological and ultrastructural characteristics of two endothelial subpopulations. Atherosclerosis 1986; 59: 1-19
• 14 Meyer M, Herrman FN, Weissbach G, Wiedemann H, Lagutina NJ, Tchishova AI. Heterogenitôt der Protein- und Glycoprotein Anomalien beider hereditôren Thrombasthenie Glanzmann. Dtsch Gesundheitswesen 1983; 38: 1614-8
• 15 Patcheke H. Shape and functional properties of human platelets washed with citrate. Haemostasis 1981; 10: 14-27
• 16 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predetermined specificity. Nature 1975; 256: 495-7
• 17 Berndt MC, Gregory C, Kabral A, Zola H, Fournier D, Castaldi PA. Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane. Eur J Biochem 1985; 151: 637-49
• 18 Skinner MP, Fournier DJ, Andrews RK, Gorman JJ, Chesterman CN, Berndt MC. Characterization of human platelet GMP-140 as a heparin-binding protein. Biochem Biophys Res Commun 1990; 164: 1373-9
• 19 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloramide, 1, 3, 4, 6-tetrachloro-5, 6-diphenylglycoronyl. Biochem Biophys Res Commun 1987; 80: 849-57
• 20 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T₄ . Nature 1970; 227: 680-5
• 21 Towbin H, Stachelin T, Gordon J. Electrophoretic transfer of proteins from Polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 1979; 76: 4350-4
• 22 Phillips DR. Effect of trypsin on the exposed polypeptides and glycoproteins in the human platelet membrane. Biochemistry 1972; 11: 4582-8
• 23 Berndt MC, Gregory C, Chong BH, Zola H, Castaldi PA. Additional glycoprotein defects in Bernard-Soulier's syndrome: confirmation of genetic basis by parental analysis. Blood 1983; 62: 800-7
• 24 Hogg N, Horton MA. Myeloid antigens: a new and previously defined clusters. In: Leucocyte Typing III. White Cell Differentiation Antigens.. McMichael AJ. (ed) Oxford University Press, Oxford: 1988. pp 576-602
• 25 Knapp W, Rieber P, Dorken B, Schmidt RE, Stein H, von dem Borne Kr AEG. Towards a better definition of human leucocyte surface molecules. Immunol Today 1989; 10: 253-8
• 26 Albelda SM, Oliver PD, Komer LH, Buch CA. EndoCAM: a novel endothelial cell-cell adhesion molecule. J Cell Biol 1990; 110: 1227-37