Abstract
The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the αIIb subunit (GPIIb, 136 kDa) and the β3 subunit (GPIIIa, 92 kDa), serves as the fibrinogen receptor at the surface of activated platelets. The degree of dissociation of the GPIIb/IIIa heterodimer (s°20 *, 8.9 S) into its constituent glycoproteins (GPIIb, 5.8 S; and GPIIIa, 3.9 S) has been assessed by analytical ultracentrifugation in Triton X100 buffers, and its Ca2+- and temperature-dependence correlated with Ca2+-binding to GPIIb/IIIa and its temperature dependence. At 21°C half-maximal dissociation of GPIIb/IIIa occurs at 5.5 ± 2.5 × 10−8 M Ca2+, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kd1 8 ± 3 × 10−8 M) (Rivas and González-Rodríguez, 1991) and much lower than the Kd of the 3.4 medium affinity Ca-binding sites (Kd2 4 ± 1.5 × 10−5 M), which seems to demonstrate that the stability of the heterodimer in solution at room temperature is regulated by the degree of saturation of the high-affinity Ca-binding site. At 4°C, the stability of the heterodimer is apparently Ca2+-independent, while at room and physiological temperatures (15–37°C) the degree of dissociation of the heterodimer is regulated by the degree of dissociation of the high- and medium-affinity Ca-binding sites, respectively. On increasing the Ca2+ concentration up to 1 × 10−4 M after dissociation in Triton X100 solutions, the reconstitution of the GPIIb/IIIa heterodimer depends on the time and temperature at which the dissociated heterodimer was maintained, being almost complete within the first 5–10 min at 37°C and within the first 1–2 h at 21°C. After this time, a time- and temperature-dependent irreversible autoassociation of GPIIb (covalent) and GPIIIa (non-covalent) occurs, which hinders both the isolation of permanently stable monoamers of GPIIb and GPIIIa and the reconstitution of the GPIIb/IIIa heterodimer in Triton X100 solutions.
Similar content being viewed by others
References
Bennett JS, Vilaire MJ (1979) Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest 64:1393–1401
Born GVR, Cross MJ (1964) Effects of inorganic ions and of plasma proteins on the aggregation of blood platelets by ADP. J Physiol 170:397–414
Bowen TJ (1970) An introduction to ultracentrifugation. Wiley, London, pp 99–102
Brass LF, Shattil SJ (1984) Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets. J Clin Invest 73:626–632
Brass LF Shattil SJ, Kunicki TJ, Bennett JS (1985) Effect of calcium on the stability of the platelet membrane glycoprotein IIb-IIa complex. J Biol Chem 260:7875–7881
Eirin MT, Calvete JJ, González-Rodríguez J (1986) New isolation procedure and further biochemical characterization of glycoprotein IIb and IIIa from human platelet plasma membrane. Biochem J 240:147–153
Fabiato A (1981) Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle. J Gen Physiol 78:457–497
Fitzgerald LA, Phillips DR (1985) Calcium regulation of the platelet membrane glycoprotein IIb–IIIa complex. J Biol Chem 260:11366–11374
Forstner J, Manery JF (1971) Calcium binding to human erythrocyte membranes. Biochem J 124:563–571
Fujimura K, Phillips DR (1983) Calcium cation regulation of glycoprotein IIb-IIIa complex formation. J Biol Chem 258:10247–10252
Jennings LK, Phillips DR (1982) Purification of glycoproteins IIb and IIIa from human platelet plasma membrane and characterization of a calcium-dependent glycoprotein IIb-IIIa complex. J Biol Chem 257:10458–10466
Kieffer N, Phillips DR (1990) Platelet membrane glycoproteins: functions in cellular interactions. Ann Rev Cell Biol 6:329–357
Kunicki TJ, Pidard D, Rosa J-P, Nurden AT (1981) The formation of Ca2+-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis. Blood 58:268–278
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T 4. Nature 227:680–685
Markwell MAK, Haas SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210
Peerschke EI, Grant RA, Zucker MB (1980) Decreased association of 45calcium with platelets unable to aggregate due to thrombasthenia or prolonged calcium deprivation. Br J Haematol 46:247–256
Pidard D, Didry D, Kunicki TJ, Nurden AT (1986) Temperature-dependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. Blood 67:604–611
Plow EF, Ginsberg MH (1989) Cellular adhesion: GPIIb/IIIa as a prototypic adhesion receptor. Progr Hemost Thromb 9:117–156
Press WH, Flannery BP, Teukolsky SA, Watterling WH (1986) Numerical recipes. The art of scientific computing. Cambridge University Press, Cambridge
Rivas GA, González-Rodríguez J (1991) Calcium binding to human platelet integrin GPIIb/IIIa and to its constitutent glycoproteins. Effects of lipids and temperature. Biochem J 276:35–40
Rivas GA, Calvete JJ, González-Rodríguez J (1991 a) A large scale procedure for the isolation of integrin GPIlb/IIIa, the human platelet fibrinogen receptor. Prot Express Purif 2 (in press)
Rivas GA, Aznárez JA, Usobiaga P, Saiz J-L, Gonzalez-Rodriguez J (1991b) Molecular characterization of human platelet integrin GPIlb/IIIa and its constituent glycoproteins. Eur Biophys J 19:335–345
Rosa J-P, Kieffer N, Didry D, Pidard D, Kunicki TJ, Nurden AT (1984) The human platelet membrane glycoprotein complex GPIIb/IIIa expresses antigenic sites not expossed on the dissociated glycoproteins. Blood 64:1246–1253
Shattil SJ, Brass LF, Bennett JS, Pandhi P (1985) Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb–IIIa complex. Blood 66:92–98
Steiner B, Cousot D, Trzeciak A, Grillessen D, Hadvary P (1989) Ca2+-dependent binding of a synthetic Arg. Gy. Asp (RGD) peptide to a simple site on the purified glycoprotein GPIIb/IIIa complex. J Biol Chem 264:13102–13108
Zucker MA, Grant RA (1978) Nonreversible loss of platelet aggregability induced by calcium deprivation. Blood 52:505–514
Author information
Authors and Affiliations
Additional information
Abbreviations: GPIIb, GPIIIa, and GPIIb/IIIa, glycoprotein IIb, IIIa, and the heterodimer formed by them, respectively; s°20 *, the sedimentation coefficient of the glycoprotein-detergent complexes determined at 20°C, after extrapolation to zero-glycoprotein concentration
Offprint requests to: J. González-Rodríguez
Rights and permissions
About this article
Cite this article
Rivas, G.A., Usobiaga, P. & González-Rodríguez, J. Calcium and temperature regulation of the stability of the human platelet integrin GPIIb/IIIa in solution: an analytical ultracentrifugation study. Eur Biophys J 20, 287–292 (1991). https://doi.org/10.1007/BF00450564
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00450564