Calcium and temperature regulation of the stability of the human platelet integrin GPIIb/IIIa in solution: an analytical ultracentrifugation study
✍ Scribed by G. A. Rivas; P. Usobiaga; J. González-Rodríguez
- Book ID
- 104778697
- Publisher
- Springer
- Year
- 1991
- Tongue
- English
- Weight
- 577 KB
- Volume
- 20
- Category
- Article
- ISSN
- 1432-1017
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✦ Synopsis
The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the e~Ib subunit (GPIIb, 136kDa) and the /~3 subunit (GPIIIa, 92 kDa), serves as the fibrinogen receptor at the surface of activated platelets. The degree of dissociation of the GPIlb/lIla heterodimer (s 020*, 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 Ca 2 +-and temperature-dependence correlated with Ca 2 +-binding to GPIIb/IIIa and its temperature dependence. At 21 °C half-maximal dissociation of GPIlb/IIIa occurs at 5.5 _+ 2.5 x 10 -8 M Ca 2+, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kdl 8 _ 3 x 10-8 M) (Rivas and Gonzfilez-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 °C, the stability of the heterodimer is apparently Ca 2 +-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 Ca 2+ 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 °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 perma-Abbreviations." GPIIb, GPIIIa, and GPIIb/Illa, glycoprotein IIb, IIIa, and the heterodimer formed by them, respectively; s °2o*, the sedimentation coefficient of the glycoprotein-detergent complexes determined at 20 °C, after extrapolation to zero-glycoprotein concentration Offprint requests to. J. Gonzfilez-Rodriguez nently stable monomers of GPIIb and GPIIIa and the reconstitution of the GPIIb/Illa heterodimer in Triton X100 solutions.