IMMUNOCYTOCHEMICAL LOCALIZATION OF FIBRINOGEN DURING THROMBIN-INDUCED AGGREGATION OF HUMAN PLATELETS

Abstract

The association of fibrinogen (Fbg) with washed platelets was studied during thrombin-induced aggregation in Tyrode-albumin solution with 2 mM Ca2+ or no added Ca2+. Platelets were fixed, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman Fbg, washed, reacted with gold-labelled rabbit anti-goat IgG and prepared for electron microscopy. To ensure that Fbg could be detected with this method, platelets were pretreated with chymotrypsin and aggregated with Fbg; gold particles were apparent on the surface and between adherent platelets and in the alpha granules. In a Ca2+-containing medium in the absence of external Fbg, washed platelets did not have Fbg on their surface although there was extensive gold labelling of the platelet alpha granules. Thrombin (0.05 U/ml) caused platelet aggregation, centralization and apparent fusion of alpha granules. By 60 sec large aggregates had formed, many platelets appeared degranulated but few gold particles were seen between adherent platelets. At 5 min, little Fbg remained in the aggregated platelets. In a few regions gold accumulated in fused granule material, and in occasional clusters between adherent platelets. In the presence of external Fbg (0.4 mg/ml) thrombin caused aggregation, centralization and fusion of granules, and discharge of granule contents. However, numerous gold particles were readily detectable between adherent platelets and on the platelet membrane. Fibrin formed and was abundantly labelled with immuno-gold. Similar findings were obtained in the medium without added Ca2+ (20 μM Ca2+). These results agree with observations obtained from measurements of 125I-Fbg binding and show that in the presence of external Fbg thrombin causes Fbg binding to platelets during aggregation. In the absence of added Fbg, thrombin aggregates platelets without extensive binding of released Fbg to the platelets.