Visualization of activation-dependent epitopes on glycoprotein IIb-IIIa complexes of platelets stimulated by thrombin: immunogold staining of ultrathin sections.
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 alpha-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 alpha-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 alpha-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.
Humbert
M
M
URA 1464 CNRS, Institut Fédératif Coeur-Vaisseaux-Thrombose, Hôpital Cardiologique, Pessac, France.
Kunicki
T J
TJ
Bihour
C
C
Winckler
J
J
Nurden
A T
AT
Nurden
P
P
D016824:Antigens, Human Platelet / Q000276:immunology; D001792:Blood Platelets / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D015539:Platelet Activation / Q000187:drug effects / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology*; D013917:Thrombin / Q000494:pharmacology
D016824:Antigens, Human Platelet; D000939:Epitopes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D013917:Thrombin