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Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes
within the membrane systems of activated human platelets
P Hourdille, E Heilmann, R Combrie, J Winckler, KJ Clemetson and AT Nurden
Section Pathologie Cellulaire de l'Hemostase, Hopital Cardiologique,
Pessac, France.
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.
Volume 76,
Issue 8,
pp. 1503-1513,
10/15/1990
Copyright © 1990 by The American Society of Hematology
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