Biochemical Characterization of PECAM-1 (CD31 Antigen) on Human Platelets

Summary

The platelet plasma membrane expresses several membrane glycoproteins with a high molecular weight. In this study we have investigated the properties of the CD31 antigen on platelets and endothelial cells using the monoclonal antibody (MoAb) RUU-PL 7E8. Comparative studies revealed that the CD31 antigen, PECAM-1 and endoCAM are the same protein. The CD31 antigen was immunoprecipitated with a molecular mass of 125 kDa nonreduced and 135 kDa reduced from Nonidet-P40 lysates of surface labeled human platelets. The relative position in two-dimensional nonreduced/reduced SDS-PAGE and IEF-PAGE, compared to other glycoproteins of similar molecular weight, was elucidated. The position of the CD31 antigen was clearly distinct from the position of the platelet membrane glycoproteins Ia, Ib, IIa, IIb, IIIa and the granule membrane protein GMP-140. Native resting platelets bound 7,760 ± 1,670 molecules/platelet, whereas thrombin-stimulated platelets bound 14,500 ± 3,790 molecules/platelet. Immunoelectron microscopy revealed the presence of the CD31 antigen on the membrane of both resting and thrombin-activated platelets. Immunofluorescence studies showed the presence of the CD31 antigen in the membrane of endothelial cells on sites of cell-cell contact, suggesting that the CD31 antigen might be involved in cell-cell interaction. In functional studies, MoAb RUU-PL 7E8 did not inhibit platelet aggregation, platelet adherence to the extracellular matrix of endothelial cells and purified collagen fibrils under flow conditions, nor was any influence found on endothelial cell detachment and growth.

• References

• 1 Phillips DR, Poh Agin PP. Platelet plasma membrane glycoproteins. Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis. J Biol Chem 1977; 252: 2121-6
• 2 O'Farrell PH. High resolution of two-dimensional electroforesis of proteins. J Biol Chem 1975; 250: 4007-21
• 3 Bienz D, Clemetson KJ. Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent. J Biol Chem 1989; 264: 507-14
• 4 McEver RP, Martin MN. A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 1984; 259: 9799-804
• 5 Pischel KD, Bluestein HG, Woods Jr VL. Platelet glycoproteins Ia, Ic, and Ha are physicochemically indistinguishable from the very late activation antigens adhesion-related proteins of lymphocytes and other cell types. J Clin Invest 1988; 81: 505-13
• 6 Hsu-Lin SC, Berman CL, Furie BC, August D, Furie B. A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets. J Biol Chem 1984; 259: 9121-6
• 7 Modderman PW. Cluster report: CD62. In: Leucocyte Typing IV, White Cell Differentiation Antigens.. Knapp WP, Dörken B, Gilks WR. et al. (eds) Oxford University Press, Oxford: 1989. pp 1038-9
• 8 Modderman PW. Cluster report: CD29. In: Leucocyte Typing IV, White Cell Differentiation Antigens.. Knapp W, Dörken B, Gilks WR. et al. (eds) Oxford University Press, Oxford: 1989. pp 1019-20
• 9 von dem Borne AEGKr, Modderman PW. Cluster report: CD31. In: Leucocyte Typing IV, White Cell Differentiation Antigens.. Knapp WP, Dörken B, Gilks WR. et al. (eds) Oxford University Press, Oxford: 1989. p 995
• 10 Newman PJ, Berndt MC, Gorski MC. et al. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science 1990; 247: 1219-22
• 11 Staunton DE, Marlin SD, Stratowa C, Dustin ML, Springer TA. Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell 1988; 52: 925-33
• 12 Köhler G, Milstein C. Derivation of specific antibody-producing tissue cultures and tumor lines by cell fusion. Eur J Immunol 1976; 6: 511-9
• 13 Metzelaar MJ, Sixma JJ, Nieuwenhuis HK. Activation dependent mAb recognizing a 140 kD platelet alpha granula membrane protein, expressed after activation. In: Leucocyte Typing IV White Cell Differentiation Antigens.. Knapp W, Dörken B, Gilks WR. et al. (eds) Oxford University Press, Oxford: 1989. pp 1039-40
• 14 Giltay JC, Brinkman HM, Modderman PW, von dem Borne AEGKr, 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
• 15 van Mourik JA, Leeksma OC, Reinders JH, de Groot PhG, Zandbergen-Spaargaren J. Vascular endothelial cells synthesize a plasma membrane protein indistinguishable from the platelet membrane glycoprotein IIa. J Biol Chem 1985; 260: 11300-6
• 16 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparing soluble chloroamide 1,3,4,6-tetra chloro-3a,6a-diphenylglycoluril. Biochem Biophys Res Commun 1978; 80: 849-57
• 17 Sixma JJ, Schiphorst ME. Identification of ectoproteins of human platelets. Combination of radioactive labelling and two-dimensional electroforesis. Biochim Biophys Acta 1980; 603: 70-83
• 18 Morrissey JH. Silverstain for proteins in Polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117: 307-10
• 19 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical cord veins. Identification of morphology and immunologic criteria. J Clin Invest 1973; 52: 2745-56
• 20 Tokuyasu KT. A study of positive staining of ultrathin frozen sections. J Ultrastruc Res 1978; 63: 287-307
• 21 Slot JW, Geuze HJ, Weerkamp AJ. Localisation of macromolecular components by application of the immunogold technique on cryosectioned bacteria. Meth Microbiol 1988; 20: 211-36
• 22 Sakariassen KS, Aarts PAMM, de Groot PhG, Houdijk WPM, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their cellular matrix and purified components. J Lab Clin Med 1983; 102: 522-35
• 23 Houdijk WPM, Sakariassen KS, Nievelstein PFEM, Sixma JJ. Role of factor VIII-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: 531-40
• 24 Sixma JJ, Nievelstein PFEM, Zwaginga J, de Groot PhG. Adhesion of blood platelets to the extra cellular matrix of cultured human endothelial cells. Ann N Y Acad Sci 1987; 516: 39-52
• 25 Albelda SM, Oliver PD, Romer LH, Buck CA. EndoCAM: a novel endothelial cell-cell adhesion molecule. J Cell Biol 1990; 110: 1227-37
• 26 Woods VL, Wolff LE, Keller DM. Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. J Biol Chem 1986; 261: 15242-51
• 27 Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 880-6
• 28 Coller BS. A new murine monoclonal antibody reports on activation-dependent change in the confirmation and/or micro-environment of the platelet glycoprotein IIb-IIIa complex. J Clin Invest 1985; 76: 101-8
• 29 Niiya K, Hodson E, Bader R. et al. Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood 1987; 70: 475-83
• 30 Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane glycoprotein Ilb-IIIa complex during platelet activation. J Biol Chem 1985; 260: 11107-14
• 31 Muller WA, Ratti CM, McDonnell SL, Cohn ZA. A human endothelial cell restricted externally exposed plasmalemmal protein that is enriched at the intracellular junctions of cultured human umbilical vein endothelial cells. J Exp Med 1989; 170: 399-414