Therostasin, a novel clotting factor Xa inhibitor from the rhynchobdellid leech, Theromyzon tessulatum

Therostasin is a potent naturally occurring tight-binding inhibitor of mamm alian Factor Xa (K-i, 34 pM), isolated from the rhynchobdellid leech Therom yzon tessulatum, Therostasin is a cysteine-rich protein (8991 Dal consistin g of 82 amino acid residues with 16 cysteine residues. Its amino acid seque nce has been determined by a combination of techniques, including Edman deg radation, enzymatic cleavage, and matrix-assisted laser desorption/ionizati on time-of-flight mass spectrometry (MALDI-TOF MS) on the native and s-beta -pyridylethylated compound. Sequence analysis reveals that it shares no si gnificant homology with other Factor Xa inhibitors except for the putative reactive site. Moreover, it contains a signature pattern for proteins of th e endothelin family, potent vasoconstrictors isolated in mammal and snake v enom. Therostasin cDNA (825 bp) codes for a polypeptide of 82 amino acid re sidues preceded by 19 residues, representing a signal peptide sequence. As for the other known inhibitors of Factor Xa, therostasin is expressed and s tored in the cells of the leech salivary glands.