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H Suzuki, RL Kinlough-Rathbone, MA Packham, K Tanoue, H Yamazaki and JF Mustard
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
The association of fibrinogen with washed human platelets was examined by
immunocytochemistry during aggregation induced by adenosine diphosphate
(ADP) and during deaggregation. The platelets were suspended either in a
medium containing 2 mmol/L Ca2+ or in a medium containing no added Ca2+ (20
mumol/L Ca2+). Platelets were fixed at several times during aggregation and
deaggregation, embedded in Lowicryl K4M, sectioned, incubated with goat
antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and
prepared for electron microscopy. To determine whether the method detected
fibrinogen associated with the platelets, the platelets were pretreated
with chymotrypsin (10 U/mL) and aggregated by fibrinogen; gold particles
were apparent not only in the alpha granules but on the platelet surface
and between adherent platelets as well. In the medium with 2 mmol/L Ca2+,
ADP caused extensive aggregation of normal platelets in the presence of
fibrinogen (0.4 mg/mL), and gold particles were evident between the
adherent platelets and on the platelet surface; when the platelets
deaggregated, gold was no longer present on the surface. In a medium
without added Ca2+, ADP caused extensive aggregation in the presence of
fibrinogen, and large numbers of gold particles were on the platelet
surface and even more between adherent platelets. In this medium, the
platelets did not deaggregate, and by five minutes, the granules appeared
to be swollen or fused. In the absence of external fibrinogen, ADP caused
the formation of small aggregates, and fibrinogen was not detected between
adherent platelets. Thus, the association of fibrinogen with the platelet
surface enhances platelet aggregation but is not essential for the
ADP-induced formation of small aggregates. The association of fibrinogen
with platelets is greater under conditions in which platelets release their
granule contents and do not deaggregate because both endogenous and
exogenous fibrinogen take part in aggregation.
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