TRIDEGIN, A NEW PEPTIDIC INHIBITOR OF FACTOR XIIIA, FROM THE BLOODSUCKING LEECH HAEMENTERIA-GHILIANII

1. Crude salivary gland extract of the giant Amazon leech, Haementeria ghilianii, contains an inhibitor of plasma factor XIIIa. 2. The inhib itory agent was purified to homogeneity by anion-exchange, cation-exch ange, gel-filtration and reverse-phase chromatography to yield a singl e band on SDS/PAGE with an apparent molecular mass of 7.3 kDa. It has been named tridegin. 3. Micro-sequencing of proteolytic fragments show ed tridegin to be a peptide of 66 amino acids. The sequence is unique with little similarity to other leech-derived proteins. 4. Inhibition of plasma factor XIIIa activity was confirmed by four independent meth ods: tridegin increased the solubility of fibrin clots ia-urea, inhibi ted ammonia produced from the incorporation of ethylamine into casein, inhibited the incorporation of 5'-(biotinamido)pentylamine into casei n and prevented gamma- dimer formation in clotting fibrinogen. 5. The IC50 of tridegin (approx. 9.2 nM) is very close to the concentration o f factor XIIIa used in the assay and in fact depends on its concentrat ion. This is the most potent inhibitor of factor XIIIa yet described. 6. Tridegin also inhibits platelet factor XIIIa (factor XIIIAa) with a similar potency to that of the plasma enzyme. 7. Tridegin also inhibi ts tissue transglutaminase but with lower potency and independently of the enzyme concentration. 8. Tridegin appears to be specific for tran sglutaminases, since it has no effect on the coagulation times of huma n plasma, on thrombin or factor Xa. Moreover it has no effect on other thiol-containing enzymes and has no ability to digest fibrinogen or c leave the isopeptide substrate, L-gamma-glutamyl-4-nitroanilide.