Localizaton of fibrin converted by thrombin from released platelet fibrinogen

Abstract

Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane “ghosts” for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII- detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.

Measurement of Fibrinogen Concentrations in Suspensions of Washed Rabbit and Human Platelets by Radioimmunoassays

“…Plasma fibrinogen concentrations and the total fibrinogen content of platelets were determined by using radioimmunoassays that had been developed to measure the low concentrations of fibrinogen found in the suspending media of washed rabbit and human platelets. The plasma fibrinogen concentration of 2.6 mg/mL found for human plasma is well within the normal range of 2-4 mg/mL, and the platelet content of 63.7 pg/109 platelets is at the lower end of the range reported by other investigators (36,37). Measurement of p-thromboglobulin concentrations gave a value for total platelet content of 33.8 pg/109 platelets, in agree ment with previously reported values (31,38,39).…”

“…The thrombin treatment must have caused release of fibrinogen from the a-granules, and since thrombin-degranulated platelets aggregated to the same extent as control platelets upon ADP stimulation (15), some of the released fibrinogen appears to have bound to the platelet membrane in a configuration that allowed it to support aggrega tion upon addition of ADP. This hypothesis is consistent with the reports of several investigators that a-granule proteins, including fibrinogen, fibronectin, thrombospondin and von Willebrand factor, are expressed on the surface of human platelets after stimulation with thrombin (18,35,36,(40)(41)(42)(43)(44)(45). It is reasonable to conclude that when platelets are stimulated with ADP rather than thrombin, small amounts of fibrinogen are released and become membrane-bound and available to support aggregation.…”

Comparative Study of Size, Total Protein, Fibrinogen and 5-HT Content of Human and Canine Platelet Density Subpopulations

“…The amount of fibrinogen in human platelets measured by electroimmunoassay was in the range reported by other investigators (28,29). Fibrinogen, mainly localized in alpha-granules (29)(30)(31)(32) constitutes 7 .4 and 9.0% of the total protein content of platelets in humans and dogs, respectively.…”