Immunocytochemical localization of fibrinogen during thrombin-induced
aggregation of washed human platelets
H Suzuki, RL Kinlough-Rathbone, MA Packham, K Tanoue, H Yamazaki and JF Mustard
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Because thrombin aggregates afibrinogenemic platelets and platelets from
patients with the gray platelet syndrome and because antibodies to
fibrinogen inhibit thrombin-induced aggregation only at low concentrations
of thrombin, the role of fibrinogen in the formation of thrombin-induced
aggregates was investigated further with human platelets washed and
resuspended in Tyrode-albumin solution containing apyrase, either with or
without added Ca2+ (2 mmol/L). Samples for immunocytochemical assessment of
fibrinogen distribution were taken at several times (up to five minutes)
after aggregation induced by 0.5 U/mL of thrombin. Glutaraldehyde-fixed
samples were embedded in Lowicryl K4M, sectioned, incubated with goat
antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and
prepared for electron microscopy. By 10 seconds, small aggregates formed,
and granules were centralized; alpha granules were heavily labeled with
immunogold, but the platelet surface was not. As large aggregates formed,
granule swelling or fusion occurred, and in some areas granule material
seemed to be in contact with the exterior. In these experiments with no
added fibrinogen, there were some clusters of gold particles on the
platelet surfaces remote from sites of granule discharge, but there were
large areas where platelets were in close contact with little or no
fibrinogen detectable between them. No fibrin was visible up to five
minutes after the addition of thrombin, which indicated that fibrinogen
from the granules does not readily become available for fibrin formation in
the ambient fluid. Similar results were obtained in media with and without
added Ca2+. Thus at least some aggregation in response to thrombin can
occur without the participation of released fibrinogen, and much of the
granule fibrinogen appears to remain localized at sites where granules fuse
with the plasma membrane or the open canalicular system. Incubation of
unstirred samples with thrombin for ten minutes resulted in the formation
of small aggregates, extensive gold label in regions connected to the
exterior of the platelets, but very little gold labeling of the platelet
membrane and no visible fibrin formation. When the platelets were
aggregated in the presence of external fibrinogen, the morphological
changes within the platelets were the same, but fibrinogen rapidly became
associated with the entire platelet surface, and visible fibrin formed
within 30 seconds in the medium containing 2 mmol/L Ca2+.(ABSTRACT
TRUNCATED AT 400 WORDS)
Volume 71,
Issue 5,
pp. 1310-1320,
05/01/1988
Copyright © 1988 by The American Society of Hematology
