The Basis for Fibrinogen Cedar Rapids (γ R275C) Fibrin Network Structure

Abstract

Fibrinogen ‘Cedar Rapids’ is a heterozygous dysfibrinogenemia characterized by delayed and abnormal fibrin polymerization. The specific molecular defect (γ R275C) is relatively common, but in only one case, fibrinogen Tokyo II, has the ultrastructural basis for defective clot formation been determined. This report reflects similar structural studies on Cedar Rapids fibrinogen and fibrin. Crosslinked fibrinogen molecules and fibrils, were prepared at 1 mg/ml in the presence of factor XIIIa (100 u/ml). When γ chains had become ˜10 to 20% crosslinked to γ dimers, samples were diluted with Hepes buffered saline, pH 7, to a fibrinogen concentration of 5 to 10 μg/ml. Three μl was then injected into 3 μl buffer on a carbon-coated EM grid, the specimen allowed to attach for one minute, fluid-exchanged several times with 150 mM NH4 acetate solution, frozen in liquid nitrogen, freeze-dried, and imaged at the Brookhaven STEM facility using a 40 kv probe focused at 0.25 nm. Fibrin for scanning EM (SEM) was formed directly on carbon-formvar coated gold grids. Clots that had formed overnight were fixed with 2.5% glutaraldehyde in 0.1 M Hepes, pH 7 buffer containing 0.2% tannic acid, washed with buffer, dehydrated, CO2 critical point dried, coated with 7.5 nm platinum, and imaged in a JOEL Field Emission SEM operated at 5 kV.