Immunocytochemical studies on co-localization of -granule membrane IIb 3 integrin and intragranular fibrinogen of human platelets and their cell-surface expression during the thrombin-induced release reaction

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Abstract
We employed a double‐staining method to immunocytochemically study the role of α‐granule membrane αIIbβ3 integrin in the expression of intragranular fibrinogen (Fbg) and albumin on the surface of thrombin‐activated human platelets. In unstimulated platelets, αIIbβ3 was distributed along the α‐granule membrane as well as on the cell‐surface membrane, including the open canalicular system (OCS), while Fbg and albumin were exclusively and evenly detected in the granules. At 30 s after activation by 0.1 U ml−1 thrombin under non‐stirred conditions, a small amount of Fbg in the granules was redistributed in association with α‐granule membrane αIIbβ3, suggesting that co‐localization between αIIbβ3 and Fbg occurs at this period during platelet activation. At 1–5 min, the shape of the platelets changed from discoid to spheroid with pseudopodia and intact α‐granules were no longer observed in these cells. Fibrinogen associated with αIIbβ3 became increasingly expressed on the membranes of both the swollen OCS and the cell surface. Nevertheless, abundant Fbg still remained in the lumen of the swollen OCS, the membrane of which had already been depleted of αIIbβ3. By western blot analysis using anti‐human Fbg antibody, we detected no Fbg in the surrounding medium of the thrombin‐stimulated platelets for 5 min. Unlike Fbg, abundant albumin in the α‐granules was released into the surrounding medium without association with the membrane of either the α‐granules or the cell surface. These results suggest that a small portion of the intragranular Fbg may bind to an Fbg binding site expressed on the activated αIIbβ3 on the α‐granule membrane, resulting in the formation of an αIIbβ3–Fbg complex, which is then moved through the OCS membrane and expressed on the cell‐surface membrane. Thus, α‐granule membrane αIIbβ3 may act as a carrier of intragranular Fbg.

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