Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation.
The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.
Freund
K F
KF
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
Doshi
K P
KP
Gaul
S L
SL
Claremon
D A
DA
Remy
D C
DC
Baldwin
J J
JJ
Pitzenberger
S M
SM
Stern
A M
AM
D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004285:Dogs; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology*; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D013438:Sulfhydryl Compounds / Q000737:chemistry; D011503:Transglutaminases / Q000037:antagonists & inhibitors*
D007093:Imidazoles; D013438:Sulfhydryl Compounds; C088946:1,3,4,5-tetramethyl-2-((2-oxopropyl)thio)imidazolium; C088947:1,3-dimethyl-2-((2-oxopropyl)thio)imidazolium; D011503:Transglutaminases; D005978:Glutathione; D003545:Cysteine; D007460:Iodoacetamide