Confirmation that GP Ib-IX complexes have a reduced surface distribution on platelets activated by thrombin and...

In 1990 we reported that GP Ib-IX complexes accumulated within the surface-connected canalicular system (SCCS) of thrombin-stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Ib alpha in electron microscopy with immunogold staining performed on ultrathin sections of resin-embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti-GP Ib-IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either alpha-thrombin or TRAP-14-mer peptide. In all cases a decreased density of GP Ib-IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP-14-mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib-IX complexes which follow thrombin-induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP-14-mer peptide.