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.