Multivalent Tranexamic Acid (TXA) and Benzamidine Derivatives for Serine Protease Inhibition

Blood coagulation and fibrinolysis pathways involve many serine proteases in a careful equilibrium. Disruption of this hemostatic balance can cause life-threatening thromboembolic and bleeding disorders that require therapeutic intervention. Heterobivalent molecules synthesized with both benzamidine (active site serine protease inhibitor) and tranexamic acid (TXA, kringle/lysine-site inhibitor) of increasing dPEG linker lengths (dPEG₄-dPEG₃₆) were synthesized and analyzed for plasmin, thrombin, and tissue plasminogen activator (tPA) inhibition using soluble enzymatic substrates. Linker lengths greater than the active and lysine binding site separation achieved improved inhibition with plasmin and tPA due to multivalent subsite binding effects. Despite TXA being a weak active site inhibitor, homomultivalent TXA (PAMAM⁸-TXA) demonstrated strong competitive plasmin inhibition (<i>K</i> _i = 2.5 ± 1.8 μM) due to the statistical rebinding effect. IC₅₀ values were also determined by assaying on physiologically relevant, fluorescently tagged, annular fibrin clots to capture the effect of kringle binding inhibition on fibrinolytic potential in the presence and absence of inhibitors.