Antipeptide Monoclonal Antibodies to Defined Fibrinogen Aα Chain Regions: Anti-Aα 487-498, a Structural Probe for Fibrinogenolysis

Abstract

The fibrinogen αC domain (Aα 220-610) is one of the earliest targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the αC domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its Aα chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb F-104 was raised against a multiple antigenic peptide derivative modelled after the hydrophilic 12-residue sequence corresponding to Aα 487-498 within the αC domain. A sensitive solution phase competitive enzyme-linked immunosorbent assay (ELISA) was developed for MoAb F-104 that can be applied for the direct measurement of intact fibrinogen (purified or plasma; ED50%≈5 pmol Aα chain equivalents/mL), with negligible cross-reactive interference from peptide cleavage products released by plasmin from the COOH-terminal end of the Aα chain (<3%). Immunoblotting and ELISA studies to characterize the fate of the F-104 epitope during fibrinogenolysis in vitro indicated a rapid loss of fibrinogen-associated immunoreactivity that reflected the heterogeneity of plasmin cleavage sites within the αC domain; cleavage at the 493-494 arg-his bond destroyed the F-104 epitope, while cleavage at other sites released it in an altered, inaccessible, conformation within the structure of 35- to 40-kD and 17.5- to 18-kD Aα chain degradation products. Application of the F-104 ELISA to monitor the course of Aα chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that the loss of fibrinogen-associated F-104 immunoreactivity was a very early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a reagent for evaluating the creation of an effective lytic state early during therapy, information that could help determine the need for further clinical intervention. Thus, these studies illustrate a rational, targeted, approach towards the development of a novel antifibrinogen MoAb whose application as a structural probe for the region Aα 487-498 in vitro and in vivo can provide new insights into the various molecular forms of fibrinogen that circulate under physiologic conditions and in disease.