Trypsin Mediated Activation of Thrombin Activatable Fibrinolytic Inhibitor (Carboxypeptidase U): Potential Clinical Implications.

Thrombin activatable fibrinolytic inhibitor (TAFI) (EC 3.4.17.20) is now known to mediate the fibrinolytic deficit in various pathophysiologic conditions such as thrombotic stroke, acute coronary syndrome and pancreatic disorders. While such serine proteases as plasmin are known to activate TAFI into TAFIa, no information is available on the effect of trypsin (EC 3.4.21.4) on the activation process. Pancreatic diseases including inflammatory conditions, surgical interventions and malignancies are associated with hypercoagulable state and DIC. Fibrinolytic deficit is also commonly observed in these patients. In these conditions, pancreatic enzymes are released into circulation. While the pathogenic role of pancreatic leakage mediator is known the effect of such pancreatic enzymes, as trypsin on the conversion of TAFI to TAFIa has not been addressed. To test the hypothesis that the observed fibrinolytic deficit in pancreatic disorders may be related to the conversion of TAFI to TAFIa by trypsin, the effects of this serine protease were studied on immunopurified human TAFI utilizing proteomic profiling, SDS gel electrophoresis and functional TAFI evaluation employing a specific substrate for TAFIa. Immunopurified human TAFI was incubated with human trypsin at graded concentrations in standardized assays with appropriate controls. The activation of TAFI was monitored by using a TAFIa substrate (Benzoyl-(L)-lysyl-(e-benzyloxycarbonyl)-a-(L)-(2-quanidinoethylthio)-glycine) from Pentapharm Inc. (Basel Switzerland). Thrombin thrombomodulin complex (T-TM) was also used as a positive control for TAFI to TAFIa conversion. The proteomic profile of the cleavage products generated was profiled utilizing surface enhanced laser desorption ionization (SELDI) (Ciphergen, Freemont, CA). Additionally, SDS gel electrophoresis of the cleavage products with appropriate controls were also obtained. Trypsin and TAFI alone did not produce any hydrolysis of the TAFIa substrate. However, when trypsin was added to TAFI it produced a concentration dependent hydrolysis of the substrate, which was comparable to that observed upon the activation of TAFI by T-TM. The proteomic profile of trypsin activated TAFI revealed specific peaks at 45.2, 35.6, 24.4 and 19 kDa. In addition, there were several cleavage products at < 10 kDa molecular weight range, which was not seen with the activation of TAFI by T-TM. Similarly activation products as measured by SDS gel electrophoresis showed differential molecular profiles between trypsin and T-TM. These observations clearly suggest that trypsin is capable of activating TAFI into TAFIa independent of other processes such as the T-TM and plasmin mediated conversions. The observed fibrinolytic deficit in pancreatitus/pancreatic cancer may be due to the leakage of trypsin into circulation.