Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets

Abstract

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 ...

Generation and Characterization of a Highly Stable Form of Activated Thrombin-activable Fibrinolysis Inhibitor

“…Although porcine pancreatic CPB cannot bind to plasminogen or fibrinogen, it is a very good attenuator of fibrinolysis, suggesting that no binding is required for the protection of the clot against lysis. It has also been shown that CPB is, like TAFIa, able to decrease the cofactor function of fibrinogen degradation products of t-PA-mediated plasminogen activation (24,25). So, it is more likely that there are other reasons yet to be identified, which may include a reduced rate of activation and inactivation, why TAFI-CPB-(293-401) is, despite its stability, not a good attenuator of clot lysis.…”

Polyphosphate modulates blood coagulation and fibrinolysis

“…Rapid formation of factor Va antagonizes the function of an important natural anticoagulant (TFPI), whereas accelerated thrombin generation likely enhances the function of a natural antifibrinolytic protein (TAFI). Although plasma contains low levels of free TFPI, both TAFI and TFPI are present in human platelets and are secreted following platelet activation (17,18). In addition, a large pool of active TFPI is sequestered on the endothelial surface in vivo (5), so the local concentration of active TFPI in blood clots can be considerably higher than in plasma.…”

Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis

“…It is produced mainly by the liver as a zymogen (proCPB) and is also detected in platelets (10). Activation of CPB occurs during thrombotic events (11), through the removal of a so-called activation peptide in the N terminus of proCPB (9).…”