Modulation of Substrate Specificity of the Thrombin-Thrombomodulin Complex for Protein C Versus TAFI Activation by Platelet Factor 4 and Non-Anticoagulant Heparin-Derivatives.

Abstract 3181

Poster Board III-108

The endothelial cell receptor thrombomodulin (TM) is an essential cofactor for thrombin-mediated activation of protein C and the generation of activated protein C (APC), an enzyme with anticoagulant and multiple cytoprotective activities. TM also stimulates thrombin-mediated activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI), resulting in anti-fibrinolytic and anti-inflammatory effects. Based on these properties of TM, several potential strategies for therapeutic intervention have been proposed. In hemophilia patients with inhibitory antibodies, stimulation of TM-dependent TAFI activation might diminish bleeding complications by protection of clots against premature lysis. Alternatively, stimulation of endogenous APC generation in patients with severe sepsis might prove to augment the beneficial effects of APC, as pharmacologic applications of APC in these patients increased survival. Platelet Factor 4 (PF4), a platelet alpha-granule protein, acts as a soluble cofactor for protein C activation by the thrombin-TM complex and stimulated endogenous generation of APC in vivo. Here we show that in contrast to stimulation of protein C activation, PF4 dose-dependently inhibited activation of TAFI by thrombin-TM in a purified system, in normal pooled plasma and on TM-expressing cells. Functional consequences of inhibition of TAFI activation by PF4 were determined in hemophilia A plasma by analysis of clot resistance to tPA-induced fibrinolysis. In hemophilia A plasma, activation of TAFI was dependent on TM due to the ineffective amplification of thrombin generation via the tenase complex. PF4 diminished TM-dependent prolongation of clot lysis times but did not affect clot lysis in the absence of TM. Inhibition of TM-dependent prolongation of clot lysis time by PF4 was dependent on both the TM and tPA concentration used, consistent with the proposed threshold mechanism for inhibition of fibrinolysis by TAFIa. To determine the effect of PF4 on TM-dependent TAFI activation in normal plasma, the inactivation of bradykinin (BK) in plasma by TAFIa during tissue factor induced coagulation was analyzed. Addition of TM to plasma accelerated BK cleavage, i.e., generation of des-Arg⁹-BK, consistent with TM-mediated stimulation of TAFI activation and subsequent BK cleavage by TAFIa. PF4 neutralized the TM-enhanced BK inactivation and reversed BK and des-Arg⁹-BK levels to those seen in the absence of TM. Thus, PF4 alters the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, such that PF4 prevents generation of TAFIa's anti-fibrinolytic and anti-inflammatory activities. To find an antidote for PF4's inhibitory effects on TAFI activation, heparin-derivatives were screened for high affinity PF4 binding but greatly reduced anticoagulant activity. N-acetylated heparin (NAc-Hep), but not N-desulfated without N-acetylation or N-acetylated and O-desulfated heparins, retained high affinity binding to PF4 and effectively blocked PF4 binding to immobilized TM. Consistent with high affinity binding of NAc-Hep to PF4, sulfylamine group oxygens were relatively minor determinants of the interactions between heparin and PF4 based on the available structural model. NAc-Hep did not show detectable anticoagulant activity in APTT clotting assays but restored BK conversion to des-Arg⁹-BK by TAFIa in the presence of PF4, similar to that seen in the absence of PF4. In addition, NAc-Hep reversed PF4-mediated inhibition of TAFI activation and TAFIa's antifibrinolytic functions in clot lysis assays on TM-expressing cells using hemophilia A plasma. Thus, NAc-Hep reversed PF4-mediated inhibition of TAFI activation. In summary, PF4 modulates the substrate-specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation. NAc-Hep, a non-anticoagulant heparin derivative with high affinity for PF4, effectively reversed PF4-mediated inhibition of TM-dependent TAFI activation and restored TAFIa's antifibrinolytic and anti-inflammatory functions. Accordingly, NAc-Hep or similar non-anticoagulant heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis.