[Rigbi Meir]

RIGBI, MEIR

Born 1917, Beirut; Ph.D. 1958, Hebrew Univ.; Lect. 1965; Sen. Lect. 1969; Assoc. Prof. 1975; Emeritus 1987.

Research Interests:
Proteinase inhibition. Identification of inhibitor reactive sites.
Isolation and characterization of bioactive components of leech saliva, in particular, inhibitors of coagulation enzymes and of platelet aggregation.
Research Projects:

Bioactive components of leech saliva:

  1. A specific inhibitor of coagulation Factor Xa.
  2. A specific low-molecular weight inhibitor of platelet activating factor (PAF).
  3. Apyrase (ATP diphosphohydrolase), an inhibitor of platelet aggregation.
Key words: Factor Xa inhibitor; leech saliva; apyrase (ATP diphosphohydrolase), platelet aggregation inhibition; PAF antagonist, thrombin, superoxide anion

Abstracts of Current State of Research:

Two specific inhibitors of coagulation Factor Xa

A specific Factor Xa inhibitor was isolated from dilute leech saliva (DLS). With a synthetic DNA primer based on the N-terminal amino acid sequence of the salivary inhibitor (SI), the DNA sequence of SI was obtained by PCR amplification of leech mRNA. Using a leech cDNA library, screened with a radiolabeled probe containing the DNA sequence of SI, a cDNA clone of a novel FXa inhibitor (NI) was obtained and expressed in E.coli. SI and NI possess, respectively, 85 and 33 amino acids, with 14 and 22 cysteines, presumably in disulfide linkage. They are 42 percent homologous and do not cross-react immunologically. Both are slow, tight-binding inhibitors. Ki app. is in the 10 nM range with free F.Xa, and in the 0.1 nM range with the reconstituted prothrombinase complex. Addition of DLS to totally inhibited FXa by heparin-antithrombin releases FXa activity, suggesting that SI modulates FXa inhibition by heparin-antithrombin. Both SI and NI are highly specific for FXa. Out of six proteinases tested, only trypsin was inhibited, with Ki app. in the nanomolar range. Other properties of SI and NI are, respectively: half-life in mice 7.5 and 80 minutes; activated partial thromboplastin time doubling in human plasma, at 0.14 and 1.0 mg/ml. NI reduced arterial thrombus formation in baboons.

The research was begun in the US in 1987 in collaboration with Dr. Craig M. Jackson. It was continued in Israel, with the support of Biotechnology General (Israel) Ltd. (BTG) and in cooperation with its research staff. BTG is in the process of developing NI as an antithrombotic drug.

Apyrase

Two apyrases (isozymes) were isolated from the saliva of the medicinal leech, of molecular weights over 400kDa and 49 kDa respectively. The 49 kDa enzyme was studied. Its pH activity range is 6.5 to 8.0, with an optimum around pH 7.5. Its kinetic parameters towards ATP and ADP were determined, and so was its 13-amino acid N-terminal sequence. The enzyme inhibits platelet aggregation induced by ADP as well as by arachidonic acid, platelet activating factor (PAF) and epinephrine. Potato apyrase is more specific than the leech apyrase towards ADP-induced aggregation. Leech apyrase may become an antithrombotic for use in special cases. The manuscript is in preparation.

Platelet Activating Factor (PAF) antagonist and an ornithine-rich peptide

Lyophilized dilute leech saliva was fractionated by gel-permeation chromatography on Bio-Gel P-2 (cut-off > 2kDa). One low-molecular weight fraction (Fr. II) inhibited platelet aggregation induced by PAF and thrombin but not by other aggregating agents. It also inhibited superoxide anion formation in activated neutrophils. Fr. II is amphipathic. Another low- molecular-weight fraction consisted of a peptide rich in ornithine.

Liquid-liquid partitioning of Fr.II on Extrelut yielded the purified PAF antagonist (PAFA) which inhibited PAF-induced aggregation only. On incubation with a mixture of phospholipases A2, B, C, and D, it lost 59 to 72 percent of its activity. PAFA is thus identified as a phosphoglyceride. As a platelet aggregation inhibitor, it is more potent than BN-52051 and CV-3988, but less potent than WEB-2086. PAFA may become an important compound for the treatment of thromboembolic disorders and inflammation.

Recent Publications (from 1995 onwards):

Rigbi, M. et al. (1995) F.Xa inhibitor from the saliva of the leech Hirudo medicinalis. Abstracts. 15th Congress of the International Society on Thrombosis and Haemostasis, Jerusalem, June 11-16, Abstract No. 1558.

Orevi, M., Eldor, A. and Rigbi, M. (1995) Characterization of the PAF antagonist from the saliva of the leech Hirudo medicinalis. Abstracts. 15th Congress of the International Society on Thrombosis and Haemostasis, Jerusalem, June 11-16, Abstract No. 582.

Orevi, M., Eldor, A., Gödeke, M.E., and Rigbi, M. (1996) Purification and partial characterization of the PAF antagonist from the saliva of the leech Hirudo medicinalis. Platelet Activating Factor and Related Lipid Mediators 2. (Nigam et al. eds.). Plenum Press, New York, pp. 73-77.

Rigbi, M., Orevi, M. and Eldor, A. (1996) Platelet aggregation and coagulation inhibitors in leech saliva and their roles in leech therapy (A review). Seminars in Thrombosis and Hemostasis, 22: 273-278.

Eldor, A., Orevi, M. and Rigbi, M. (1996) The role of the leech in medical therapeutics (A review). Blood Reviews, 10: 201-209.

Rigbi, M. et al. (1996) Two factor Xa inhibitors from the medicinal leech. Abstracts. 11th International Congress on Proteolysis and Protein Turnover, Turku. Finland, September 8-11, p. 200.

Orevi, M., Eldor, A., Giguzin I. and Rigbi M. (2000) Jaw anatomy of the blood-sucking leeches, Hirudinea Limnatis nilotica and Hirudo medicinalis, and its relationship to their feeding habits. J. Zool. Lond., 250: 121-127.


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