Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii

Abstract

1. Crude salivary gland extract of the giant Amazon leech, Haementeria ghilianii, contains an inhibitor of plasma factor XIIIa. 2. The inhibitory agent was purified to homogeneity by anion-exchange, cation-exchange, gel-filtration and reverse-phase chromatography to yield a single band on SDS/PAGE with an apparent molecular mass of 7.3 kDa. It has been named tridegin. 3. Micro-sequencing of proteolytic fragments showed 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 methods: tridegin increased the solubility of fibrin clots in urea, inhibited ammonia produced from the incorporation of ethylamine into casein, inhibited the incorporation of 5'-(biotinamido)pentylamine into casein and prevented gamma-dimer formation in clotting fibrinogen. 5. The IC50 of tridegin (approx. 9.2 nM) is very close to the concentration of factor XIIIa used in the assay and in fact depends on its concentration. 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 inhibits tissue transglutaminase but with lower potency and independently of the enzyme concentration. 8. Tridegin appears to be specific for transglutaminases, since it has no effect on the coagulation times of human plasma, on thrombin or factor Xa. Moreover it has no effect on other thiol-containing enzymes and has no ability to digest fibrinogen or cleave the isopeptide substrate, L-gamma-glutamyl-4-nitroanilide.

Natural selection and the molecular basis of electrophoretic variation at the coagulation F13B locus

“…However, only one species, the Amazonian leech Haementeria ghilianii, is known to produce an anticoagulant that inhibits factor XIIIa. 39,40 This species is of limited geographic distribution and is therefore unlikely to have contributed to the worldwide geographic patterns observed at the F13B locus. However, the possibility remains that modification of FXIII activity may compensate for the attenuation of some other coagulation factors (eg, thrombin, factor Xa), which are more typically targeted by the anticoagulants of hematophagic organisms.…”

Sol Sherry Lecture in Thrombosis

“…45 A protein isolated from the giant amazon leech was described to possess similar properties, although its mode of interaction with factor XIIIa is not yet known. [81][82][83] Positive results were obtained with a monoclonal antibody directed against the thrombin cleavage site of factor XIII, which blocked the activation of the zymogen. 84 Another interesting approach utilizes a monoclonal antibody against ␣ 2 PI.…”

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one‐step purification of leech proteins

“…Because affinity chromatography is ideally suited to the purification of high value biopharmaceutical proteins , and many natural thrombin-specific inhibitors such as Hirudin, granulin-like peptide, bufrudin, Hillarin, Haemadin, Theromin (Salzet, 2002), Tridegin (Finney et al, 1997), and Calin (Harsfalvi et al, 1995) have been isolated from leech, we chose the medicinal leech Hirudo nipponia as the protein source to evaluate the capacity of our library for enrichment, purification, and depletion of leech proteins. Here we report the results.…”