TRANSGLUTAMINASE INHIBITION BY 2-[(2-OXOPROPYL)THIO]IMIDAZOLIUM DERIVATIVES - MECHANISM OF FACTOR XIIIA INACTIVATION

The physiologic role of several transglutaminases could be more precis ely defined with the development of specific inhibitors for these enzy mes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For thes e purposes, the inactivation of fXIIIa and human erythrocyte transglut aminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which c omprise a novel class of transglutaminase inactivators, was studied. A s a specific example, 3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazoli um chloride (III) inactivated fXIIIa with an apparent second-order rat e 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 react ion rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [C-14]-isoto pic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio] imidazolium iod ide (II). Structural analyses demonstrated that acetonylation of the a ctive site cysteinyl residue of fXIIIa occurred along with the stoichi ometric release of the complementary fragment of the inactivator as th e 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 irrever sible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases an d thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio] imidazolium d erivatives and the related molecule 2-(1-acetonylthio) -5-methylthiazo lo-[2,3]-1,3,4-thiadiazolium perchlorate (I), when present at the time of clot formation at 1-10 mu M, enhanced the rates of tissue plasmino gen activator catalyzed clot lysis in vitro. These inactivators preven ted the fXIIIa-catalyzed covalent incorporation of alpha(2)-antiplasmi n into the at chain of fibrin and the formation of high molecular weig ht fibrin a chain polymers, providing the basis for the observed enhan cements in clot lysis rates.