, Volume 97, Issue 5, pp 381-388

Immunocytochemical evidence for the translocation of α-granule membrane glycoprotein IIb/IIIa (integrin αIIbβ3) of human platelets to the surface membrane during the release reaction

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Summary

The localization of glycoprotein (GP) IIb/IIIa (integrin αIIbβ3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the pre-embedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0±2.7 gold particles/μm of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of α-granules, and the density of gold particles was 19.7±3.6 particles/μm. At 5 min, the α-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0±3.7 particles/μm; p<0.01). In immunostained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the α-granule membranes of resting platelets. At 30 s after thrombin stimulation the α-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that α-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS. Also the translocation of α-granule membrane GPIIb/IIIa gives rise to an actual increase in GPIIb/IIIa on the surface membrane during the release reaction induced by thrombin.