Anti-thrombins

Details Anti-thrombins Anti-thrombins

1991-07-10

1995-12-05

Warden, Jill

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

530324, C07K 1400, A61K 3816

Patent

active

054729424

DESCRIPTION:

BRIEF SUMMARY
The present invention is concerned with novel anti-thrombins and, in particular, novel anti-thrombins derived from leech tissue and leech secretions.
Hirudin is a well known and well characterised polypeptide, which is known to be specific for thrombin, and which is obtained as an extract from leeches of the species Hirudo medicinalis. The polypeptide has a relatively low molecular weight (ca.7000) and is comprised of 65 amino acids. The sequence of first isoform of hirudin has been determined by Dodt, Muller, Seemuller and Chang ("The complete amino acid sequence of hirudin, a thrombin-specific inhibitor"; FEBS 165 (1984): pp180-184) to be as follows (SEQ ID NO:1): ##STR2## where Tyr at position 63 is a sulphated derivation.
Two variants of hirudin have also been characterised and the amino acid sequence determined. A first variant, as described by Dodt, Machleidt, Seemuller, Maschler and Fritz ("Isolation and characterisation of hirudin isoinhibitors and sequence analysis of hirudin PA"), Biol. Chem. Hoppe-Seyler, 367 (1986) pp803-811. This variant, designated SEQ ID NO:2, differs from the one described previously, in the following respects:


______________________________________ Ile- at position 1 instead of -val- thr- at position 2 instead of -val- lys- at position 24 instead of -gln- asn- at position 33 instead of -asp- lys- at position 35 instead of -glu- gly- at position 36 Instead of -lys- asn- at position 47 instead of -lys- glu- at position 49 instead of -gln- asn- at position 53 instead of -asp- ______________________________________
A second variant, as described by Harvey, Degryse, Stefani, Schamber et al ("Cloning and expression of a cDNA coding for the anti-coagulant hirudin from the bloodsucking leech, Hirudo medicinalis"), Proc. Nat. Acad. Sci. U.S.A. (1986) pp1084-1088. This is identical to the first-mentioned variant from positions 1 to 32 and then has the following differences from the first-mentioned hirudin: (SEQ ID NO:3):


______________________________________ gln- at position 33 instead of -asp- lys- at position 35 instead of -glu- asp- at position 36 instead of -lys- gln- at position 53 instead of -asp- pro- at position 58 Instead of -glu- asp- at position 62 instead of -glu- asp- at position 64 instead of -leu- glu- at position 65 instead of -gln- ______________________________________
As indicated above, hirudin has been derived from leeches of the species Hirudo medicinalis. Hirudinaria manillensis is similar to Hirudo medicinalis in that they are both able to feed on amphibian and mammalian blood. However, Hirudinaria is evolutionaryly more advanced than Hirudo medicinalis. It cannot be predicted with any certainty whether or not an active substance present in the secretions of a first species of leech is likely to be found in a different species, and (if substances of similar activities are found) whether they are likely to have substantially identical amino acid sequences, or markedly different amino acid sequences.
We have now isolated a novel anti-thrombin from Hirudinaria manillensis, the anti-thrombin having the following amino acid sequence SEQ ID NO:4: ##STR3##
Comparison with the hirudin sequence indicates approximately 62% homology (that is, about 38% differences), which is a surprisingly substantial difference. There are, in particular, significant differences at the important C-terminus, and the following clear differences:


______________________________________ at position 13 (-tyr- instead of -leu-); at position 17 (-val.- instead of -glu-); at position 24 (-glu- instead of -gln-); at position 26 (-asp- instead of -asn-); at position 27 (-asn- instead of -lys-); at position 29 (-asn- instead of -ile-); at position 30 (-pro- or -cys- instead of -leu-); at position 31 (-gln- instead of -gly-); at position 32 (-leu- Instead of -ser-); at position 33 (-ser- instead of -asp-); at position 34 (-ser- instead of -gly-); at position 35 (-ser- instead of -glu-); at position 36 (-gly- instead of -lys-); at

REFERENCES:
patent: 4390630 (1983-06-01), Sawyer et al.
patent: 4820516 (1989-04-01), Sawyer et al.
patent: 5114922 (1992-05-01), Maschler et al.
patent: 5139944 (1992-08-01), Sawyer et al.

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