We have used site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in order to identify the thrombin cleavage site in activated TAFI (TAFIa) and to determine the relative contribution of proteolytic cleavage and thermal instability in regulation of TAFIa activity in clots. Arg-330 of TAFIa had been proposed to be the thrombin cleavage site based on studies with trypsin, but mutation of this residue to Gln did not prevent thrombin-mediated cleavage nor did mutation to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gln abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gln substitutions decreased the thermal stability of TAFIa. The antifibrinolytic potential of the TAFI mutants in vitro correlates with the thermal stability of their respective TAFIa species, indicating that this property plays a key role in regulating the activity if TAFIa. Incubation of TAFIa under conditions that result in complete thermal inactivation of the enzyme accelerates subsequent thrombin- and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFIa activity. Collectively, these studies point to a role for the thermal instability, but not for proteolytic cleavage, of TAFIa in regulation of its activity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa.
Submitter: Shu-Ching Ou
Submission Date: Oct. 25, 2018, 10:44 a.m.
|Number of data points||85|
|Assays/Quantities/Protocols||Experimental Assay: Kinetic Parameters: kcat ; Experimental Assay: Thermodynamics of Inactivation: Standard Enthalpy Change ; Experimental Assay: Thermodynamics of Inactivation: Standard Entropy Change ; Experimental Assay: Kinetic Parameters: Km ; Experimental Assay: Half-Life (25°C) ; Experimental Assay: Half-Life (30°C) ; Experimental Assay: Half-Life (33°C) ; Experimental Assay: Half-Life (37°C) ; Derived Quantity: Kinetic Parameters: kcat/Km|
|Libraries||TAFI_Hippuryl-Lys variants ; TAFI_FA-Ala-Lys variants|
|Structure ID||Release Date||Resolution||Structure Title|
|4P10||2014-08-13||2.0||pro-carboxypeptidase U In Complex With 5-(3-aminopropyl)-1-propyl-6,7-dihydro-4H-benzimidazole-5-carboxylic acid|
|3LMS||2010-02-16||2.5||Structure of human activated thrombin-activatable fibrinolysis inhibitor, TAFIa, in complex with tick-derived funnelin inhibitor, TCI.|
|3D68||2008-07-01||2.8||Crystal structure of a T325I/T329I/H333Y/H335Q mutant of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI-IIYQ)|
|5HVF||2016-06-22||2.85||Crystal Structure of Thrombin-activatable Fibrinolysis Inhibitor in Complex with an Inhibitory Nanobody (VHH-i83)|
|5HVH||2016-06-22||3.0||Crystal Structure of Thrombin-activatable Fibrinolysis Inhibitor in Complex with two Inhibitory Nanobodies|
|5HVG||2016-06-22||3.05||Crystal Structure of Thrombin-activatable Fibrinolysis Inhibitor in Complex with an Inhibitory Nanobody (VHH-a204)|
|3D66||2008-07-01||3.1||Crystal structure of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)|
|3D67||2008-07-01||3.4||Crystal structure of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) in complex with 2-guanidino-ethyl-mercaptosuccinic acid (GEMSA)|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|