Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme that after activation downregulates fibrinolysis. Platelets are known to contain antifibrinolytic factors that are secreted during platelet activation. Therefore, the presence of TAFI in platelets was analyzed. TAFI was identified in platelets in a concentration of about 50 ng/1 ؋ 10 9 platelets and was secreted on platelet activation. Thrombin-mediated activation of platelet-derived TAFI resembled that of plasma-derived TAFI with respect to stimulation by thrombomodulin and spontaneous loss of activity at 37°C. The different glycosylation of platelet-derived TAFI compared with plasma-derived TAFI suggests that platelet-derived TAFI is synthesized in the megakaryocyte. This suggestion was substantiated by the detection of mRNA in the megakaryocytic cell lines DAMI and CHRF, representing the intermediate and late stages of megakaryocyte development. These results establish the presence of TAFI in platelets and suggest a role for platelet-derived TAFI in the protection of the clot against fibrinolysis. IntroductionThrombin activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase U, EC 3.4.17.20) provides a balance between coagulation and fibrinolysis. 1 Because the concentration of TAFI in plasma (70-250 nM) is 10-to 30-fold below its Michaelis constant (K M ) for activation by thrombin, the amount of TAFIa formed during activation is dependent on the TAFI concentration. 2,3 TAFI levels vary considerably between individuals, and individual differences in clot lysis times could be attributed to variations in TAFI levels. 3 Increased TAFI levels are associated with thromboembolic disease, and polymorphic variations in the TAFI gene have been linked to plasma TAFI levels. [4][5][6][7][8] These observations indicate an important role for the concentration of TAFI in plasma on the regulation of fibrinolysis and the occurrence of thromboembolic disease.Because platelets are known to increase local concentrations of coagulation and fibrinolytic factors by releasing the contents of their ␣-granules on activation, this study was initiated to evaluate if TAFI is present in platelets and could contribute to the variations in plasma TAFI levels. Study design Immunofluorescence microscopyCytospins of gel-filtered platelets, fixed with 2% para-formaldehyde, were permeabilized with 1% saponine and subsequently incubated with monoclonal antibody (Moab) 9H10 against TAFI and a fluorescein isothiocyanate (FITC)-labeled secondary goat antimouse antibody. 3 Detection of TAFI in platelets by ELISAGel-filtered platelets were prepared as described. 9 Secretion of TAFI from gel-filtered platelets was determined by enzyme-linked immunosorbent assay (ELISA). 3 Determination of TAFIa activity in plateletsGel-filtered platelets were incubated for 60 minutes with thrombin (50 nM), thrombomodulin (10 nM), CaCl 2 (5 mM), and hippuryl-Arg (5 mM) after which the amount of hippuric acid was determined in the supernatant as ...