Roles of fibrin(ogen) in conformational activation of hemostatic proteinase precursors

Prothrombin and plasminogen, two central hemostatic zymogens, are activated proteolytically by cleavage of an activation loop. The newly formed N-terminus inserts into a binding pocket and triggers formation of a functional active site. The activation products thrombin and plasmin respectively form and degrade fibrin, but physiological

regulation prevents uncontrolled clotting and promiscuous plasmin-mediated tissue degradation. The bacterial virulence factors, staphylocoagulase (SC) and streptokinase (SK), hijack this mechanism by inserting their own N-termini into the host zymogen pockets, and conformationally activating the catalytic site. The SK-plasminogen

complex proteolytically activates plasminogen to plasmin. Both the SC and SK complexes with the zymogens and the mature proteases cleave fibrin(ogen) but are impervious to host antithrombin and antiplasmin, and alternative methods are needed to zcontrol their unwanted activity. Our monoclonal antibodies (mAbs) against

the SC and SK N-termini block complex formation and activity, counteracting infection-related thrombosis and bacterial spreading in vivo. This illustrates mechanism-based mAb feasibility in an environment of increasing antibiotic resistance. SC and SK have additional, incompletely defined binding sites for fibrin(ogen) independent

of substrate recognition, that play a role in localization. Our proposal aims to identify unique SC and SK sequences, and conformational epitopes in their complexes with the zymogens, that promote binding of fibrin(ogen), both in substrate and anchoring modes. Our group has long-standing expertise with SC and SK-

mediated zymogen activation, and we recently made good progress identifying fibrin(ogen) fragment D binding to the C-terminal repeats of SC. However, interactions of the SK-plasmin(ogen) complexes with host fibrin(ogen) are still not well understood. Our short-term goals are to define fibrin(ogen) binding, enhancement of cofactor-

zymogen reactivity by fibrin(ogen), identify binding epitopes, and develop in vivo effective mAbs that will be added to our existing antibody arsenal. We combine our structure-function and mechanism expertise with that of experts in mAb development (Dr. Bill Church), and in application of mouse models of SC and SK action (Dr.

Peter Panizzi). Aim 1 will define dual interaction mechanisms of the SC-prothrombin complex with fibrin(ogen), with the goal of identifying suitable linear and conformational epitopes for blocking fibrin(ogen) binding. Aim 2 will delineate fibrin(ogen)-dependent plasminogen activation mechanisms of S. pyogenes SK variants that to

date are not well defined, with the same goal of identifying fibrin(ogen)-binding epitopes on the SK variants. Aim 3 will test our humanized mAbs targeting the N-termini of SC and SK in vivo, and select tight-binding anti- fibrin(ogen) binding site mAbs for in vivo studies. Long-term goals for future funding cycles are the development

of mAbs that that cross-react with a wide range of serotypes and allelic variants, and may qualify for pre-clinical and clinical testing. Cocktails of these mAbs would support the patient's hemostatic system by minimizing plasmin-mediated bacterial spreading and unwanted prothrombin activation without causing bacterial resistance.