Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool

Abstract

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes

“…This suggests that fibrinogen-mediated binding of Efb to platelets is integrin α IIb β 3 -dependent, whereas Efb binding in the presence of thrombin without exogenous fibrinogen proceeds via a different mechanism and is integrin α IIb β 3 -independent. Indirectly, this suggests that the levels of endogenous fibrinogen released by washed platelets upon thrombin stimulation are significantly lower than 3 mg/ml, the exogenous fibrinogen concentration used in our experiments and the average concentration in plasma ( 33 ); this is consistent with previous studies ( 34 , 35 ) involving the fibrinogen concentration released by washed platelets in vitro . In summary, our data suggest a dual mechanism of binding of Efb by platelets: an extracellular fibrinogen/integrin α IIb β 3 -dependent mechanism, and a fibrinogen-independent mechanism mediated by at least one unidentified platelet surface receptor.…”

“…6 B ) (which occurs in the absence of high extracellular concentrations of fibrinogen). This conclusion was reached in experiments on washed platelets; previous studies highlighted that the concentration of fibrinogen (<100 μg/ml) released by washed platelets in vitro ( 34 , 35 ) is at least an order of magnitude lower than the average plasma concentration of fibrinogen in healthy blood donors of ∼3 mg/ml ( 33 ). Interestingly, several platelet functional responses depend on high concentrations of fibrinogen that cannot be attained by simple release by platelets.…”

Direct Analysis of Plasma Fibrinogen-Derived Fibrinopeptides by High Performance Liquid Chromatography: Investigation of Aα-Chain N-Terminal Heterogeneity

“…hepatocytes, while we have demonstrated an increased AP of fibrinogen contained in platelet o-granules (22). Thken together with the results of the present study, these results provide good grounds for suggesting that the degree of phosphorylation and to a lesser extent N-terminal degradation of FPA may depend in part upon the age of the overall pool of fibrinogen in the circulation.…”

A New Genetic Fibrinogen Variant (Fibrinogen Erfurt I) Structurally Characterized by an Abnormal Bβ-Chain and Present both in Plasma and Platelets

“…Fibrinogens Oslo I and Oslo III, characterized by a B(3-and a y-chain abnormality, and fibrinogen Manchester with an amino acid substitution in the Aa-chain were observed both in plasma and blood platelets (21,22). The fibrinogen Erfurt I is also present in both pools.…”