CALCIUM AND TEMPERATURE REGULATION OF THE STABILITY OF THE HUMAN PLATELET INTEGRIN GPIIB/IIIA IN SOLUTION - AN ANALYTICAL ULTRACENTRIFUGATION STUDY

The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the alpha-IIb subunit (GPIIb, 136 kDa) and the beta-3 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-degrees-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-degrees-C half-maximal dissociation of GPIIb/IIIa occurs at 5.5 +/- 2.5 x 10(-8) M Ca2+, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kd1 8 +/- 3 x 10(-8) M) (Rivas and Gonzalez-Rodriguez, 1991) and much lower than the Kd of the 3.4 medium affinity Ca-binding sites (Kd2 4 +/- 1.5 x 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-degrees-C, the stability of the heterodimer is apparently Ca2+-independent, while at room and physiological temperatures (15-37-degrees-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 x 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-degrees-C and within the first 1-2 h at 21-degrees-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 monomers of GPIIb and GPIIIa and the reconstitution of the GPIIb/IIIa heterodimer in Triton X100 solutions.