Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.


Abstract

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.

Submission Details

ID: VmqwsMYL4

Submitter: Shu-Ching Ou

Submission Date: Oct. 25, 2018, 10:44 a.m.

Version: 1

Publication Details
Boffa MB;Bell R;Stevens WK;Nesheim ME,J Biol Chem (2000) Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor. PMID:10777585
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

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)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 Carboxypeptidase B2 Q96IY4 CBPB2_HUMAN