Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets.
Previous studies have shown a decreased binding of monoclonal antibodies (MoAbs) to glycoprotein (GP) Ib-IX complexes on thrombin-stimulated platelets, but the reason for this is poorly understood. We have used (1) immunofluorescence procedures and flow cytometry, and (2) immunogold staining and electron microscopy to investigate this phenomenon. Washed platelets were incubated with alpha-thrombin, adenosine diphosphate, or ionophore A23187 for increasing lengths of time. For alpha-thrombin, but not the other agonists, flow cytometry confirmed a dose- and time-dependent decrease in the binding of MoAbs specific for GP Ib alpha (AP-1, Bx-1), GP IX (FMC 25), or to the complex itself (SZ 1). Immunoglold staining performed using standard transmission or scanning electron microscopy high-lighted surface areas devoid of bound antibody. However, a quantitatively normal immunofluorescence was restored if paraformaldehyde-fixed, thrombin-stimulated platelets were permeabilized with Triton X-100 (Sigma Chemical Co, St Louis, MO) before MoAb addition, while immunogold staining was now seen to be concentrated within the interior of the platelet. Glutaraldehyde-fixed samples were then embedded in the resin Lowicryl K4M (Taab Laboratories Equipment Ltd, Aldermaston, England) and immunogold staining performed on thin sections using a polyclonal antibody to glycocalicin. An increased presence of GP Ib-IX complexes within surface-connected membrane systems of the thrombin-stimulated platelets was confirmed. Interestingly, GP Ib-IX movement was opposite to the thrombin-induced externalization of internal pools of GP IIb-IIIa complexes and of the alpha-granule membrane GP, GMP-140.
Hourdillé
P
P
Section Pathologie Cellulaire de l'Hémostase, Hôpital Cardiologique, Pessac, France.
Heilmann
E
E
Combrié
R
R
Winckler
J
J
Clemetson
K J
KJ
Nurden
A T
AT
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005404:Fixatives; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D017830:Octoxynol; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011092:Polyethylene Glycols; D011108:Polymers; D013917:Thrombin / Q000494:pharmacology*
D005404:Fixatives; D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D011108:Polymers; D005557:Formaldehyde; D000001:Calcimycin; D011092:Polyethylene Glycols; D000244:Adenosine Diphosphate; D017830:Octoxynol; D013917:Thrombin; C003043:paraform