Abstract
An immunoglobulin M monoclonal antibody (IgM MAb; AP-6) recognizing the sequence 211-221
of glycoprotein (GP) IIIa enabled a study of the distribution of this epitope on unstimulated
or thrombin-activated platelets. Flow cytometry was used to evaluate the expression
of this epitope on platelets and immunogold staining on ultrathin sections to analyze
its distribution within the cell. There was little or no binding of AP-6 to unstimulated
platelets, but immunogold staining showed labeling associated with the membranes of
α-granules. The binding of AP-6 to thrombin-stimulated platelets was compared with
that of anti-RIBS MAbs and antifibrinogen polyclonal antibodies. An increased expression
of the AP6 epitope was observed on membranes of the surface-connected canalicular
system and at the periphery of the cell as early as 10 to 15 seconds after platelet
activation by thrombin. At the same time, binding of anti-RIBS MAbs confirmed that
at least part of the endogenous fibrinogen had left the α-granules and was bound to
platelet membranes. Staining with polyclonal antifibrinogen antibody also revealed
fibrinogen in vesicles resulting from granule fusion. Rapidly, the pool of internal
membranes with bound fibrinogen became exposed to the outside of the platelet, a process
involving the unfolding of membranes and pseudopod formation. Our results provide
evidence that activation of GP IIb-IIIa and binding of fibrinogen to platelet membranes
can occur before their expression at the platelet surface. They also suggest the presence
of an α-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.
Keywords:
Platelets - thrombin - GP IIb-IIIa complexes - activation-dependent epitopes - immunogold
staining
