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)