A monoclonal antibody to the fibrinogen g chain alters clot structure and properties by producing thinner fibrin fibers arranged in bundles

Abstract number: OC373

Scheiner^* T., Jirouskova† M., Nagaswami‡ C., Coller† B. S., Weisel‡ J. W.

^*Institute of Chemical Technology, Czech Republic; †Rockefeller University, USA; ‡University of Pennsylvania School of Medicine, USA

We previously reported that hamster monoclonal antibody 7E9, which reacts with the C-terminus of the gchain of mouse fibrinogen, inhibits factor XIIIa (FXIIIa)-mediated cross-linking, platelet adhesion to fibrinogen, and platelet-mediated clot retraction, and facilitates thrombolysis. To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab')2, and 7E9 Fab on clot structure using electron microscopy and measurements of clot physical properties. By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab')2, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentration of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter, P < 0.001) arranged in unusual bundles surrounding large pores. These clots were visibly insubstantial and gossamer-like, with markedly increased permeation (~25-fold increase in buffer passage at constant pressure), a ~50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and a ~38% increase in tan d (P < 0.05; a reflection of the clots ability to undergo irreversible deformation). Thrombin-induced clot's formed in the presence of 7E9 IgG demonstrated increased absorbance and enhanced fibrinolysis, reflecting the unusual clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not recapitulated by control hamster IgG, monoclonal antibodies to fibrinogen a-chains, or 7E9 F(ab')2 or 7E9 Fab fragments, indicating a requirement for both the binding properties and mass of 7E9 IgG. These data indicate that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa-mediated cross-linking, clot retraction, fibrin structure, and fibrinolysis, raising the possibility that it may offer multiple benefits as an antithrombotic agent.