A turbidimetric method for measuring fibrin formation and fibrinolysis at solid-liquid interfaces

Abstract

We have developed a rapid and sensitive method by which to quantitate proteolysis of fibrin(ogen) at interfaces. Microscopic polystyrene-divinylbenzene beads coated with a mixed monomolecular film of lecithin and fibrinogen aggregate in aqueous media following exposure to thrombin or enzymes of thrombin-like activity. This aggregation is a consequence of interbead association of fibrin. As an indirect measure of the rate of fibrin formation, the rate of aggregation of beads can be used advantageously to assay enzymes and enzyme regulators pertinent to coagulation. Since the apparent absorbance of monodisperse beads is greater than that of bead aggregates, determination of the rate of change of apparent absorbance of a stirred dispersion of beads following addition of enzyme or enzyme-regulator mixture is a convenient and simple means by which to quantitate the rate of bead aggregation. Using a simple spectrophotometer or aggregometer, the method can be used to quantitate as little as 0.0005 NIH unit of thrombin. Aggregates of fibrin-coated beads can be disaggregated by several proteinases, most notably plasmin. Thus, just as bead aggregation can be used to quantitate effectors of fibrin formation, dissociation of aggregates of fibrin-coated beads can be used to quantitate effectors of fibrinolysis. Using disaggregation as a measure of fibrinolysis, the method is sensitive to as little as 0.005 unit of plasmin. Fibrin(ogen)-coated beads should prove a useful tool for studying proteolysis of fibrin(ogen) in general, and adsorbed fibrin(ogen) in particular.