Platelet Factor 4 Inhibits Thrombomodulin-dependent Activation of Thrombin-activatable Fibrinolysis Inhibitor (TAFI) by Thrombin

Laurent O Mosnier

doi:10.1074/jbc.m110.147959

Abstract

Thrombomodulin (TM) is a cofactor for thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) and thereby helps coordinate coagulation, anticoagulation, fibrinolysis, and inflammation. Platelet factor 4 (PF4), a platelet α-granule protein and a soluble cofactor for TM-dependent protein C activation, stimulates protein C activation in vitro and in vivo. In contrast to stimulation of protein C activation, PF4 is shown here to inhibit activation of TAFI by thrombin-TM. Consequences of inhibition of TAFI activation by PF4 included loss of TM-dependent prolongation of clot lysis times in hemophilia A plasma and loss of TM-stimulated conversion of bradykinin (BK) to des-Arg(9)-BK by TAFIa in normal plasma. Thus, PF4 modulates the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, thereby preventing the generation of the antifibrinolytic and anti-inflammatory activities of TAFIa. To block the inhibitory effects of PF4 on TAFI activation, heparin derivatives were tested for their ability to retain high affinity binding to PF4 despite having greatly diminished anticoagulant activity. N-acetylated heparin (NAc-Hep) lacked detectable anticoagulant activity in activated partial thromboplastin time clotting assays but retained high affinity binding to PF4 and effectively reversed PF4 binding to immobilized TM. NAc-Hep permitted BK conversion to des-Arg(9)-BK by TAFIa in the presence of PF4. In a clot lysis assay on TM-expressing cells using hemophilia A plasma, NAc-Hep prevented PF4-mediated inhibition of TAFI activation and the antifibrinolytic functions of TAFIa. Accordingly, NAc-Hep or similar heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis.

Highlights

Platelet Factor 4 (PF4) or derivatives thereof are promising candidates to stimulate endogenous generation of APC as PF4 can act as a soluble cofactor for protein C activation in the presence of TM [13,14,15]
PF4 stimulates TM-dependent activation of protein C, the following question arises: what effect will PF4 have on TM-dependent thrombin-activatable fibrinolysis inhibitor (TAFI) activation? The results of this study indicate that PF4 stimulates the TM-dependent activation of protein C, whereas PF4 inhibits the activation of TAFI by the thrombin-TM complex
When washed platelets were used as the source of PF4, TM-dependent TAFI activation was inhibited at increasing platelet concentrations (Fig. 1E), similar to that observed in the presence of purified PF4

Summary

Introduction

Platelet Factor 4 (PF4) or derivatives thereof are promising candidates to stimulate endogenous generation of APC as PF4 can act as a soluble cofactor for protein C activation in the presence of TM [13,14,15]. In the absence of sufficient thrombin generation in hemophilia A plasma, TAFI activation, and TAFIa-mediated clot protection are dependent on the presence of TM [20].

Results

Conclusion