Hirudin

Hirudin is a leech-derived anticoagulant that functions by directly inhibiting thrombin. A range of blood-sucking animals contain substances in their saliva that specifically inhibit some element of the blood coagulation system (Table 12.4). 

With the advent of modern medical principles, the medical usage of leeches waned somewhat. In more recent years, however, they did stage a limited comeback. They were occasionally used to drain blood from inflamed tissue, and in procedures associated with plastic surgery. 

The presence of an anticoagulant in the saliva of the leech, Hirudo medicinalis, was first described in 1884. However, it was not until 1957 that the major anticoagulant activity present was purified and named hirudin. Hirudin is a short (65 amino acid) polypeptide, of molecular mass 7000 Da. The tyrosine residue at position 63 is unusual in that it contains a sulfate group. The molecule appears to have two domains. The globular N-terminal domain is stabilized by three disulfide linkages, whereas the C-terminal domain is more elongated and exhibits a high content of acidic amino acids. 

Hirudin exhibits its anticoagulant effect by tightly binding thrombin, thus inactivating it. In addition to its critical role in the production of a fibrin clot, thrombin displays several other (non-enzymatic) biological activities important in sustaining haemostasis. These include  

Polypeptide Molecular mass (Da) Producer Haemostatic effect disrupted 

A bite from any such parasite is characterized by prolonged host bleeding. This property led to the documented use of leeches as an aid to blood-letting as far back as several hundred years BC. The method was particularly fashionable in Europe at the beginning of the ninteenth century. Many doctors at that time still believed that most illnesses were related in some way to blood composition and blood letting was a common, if uneffective, therapy. The Napoleonic Army surgeons, for example, used leeches to withdraw blood from soldiers suffering from conditions as diverse as infections and mental disease. 

Hirudin 7000 Hirudo medicinalis Binds to and inhibits thrombin 

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Grtitter, M.G., et al. Crystal structure of the thrombin-hirudin complex a novel mode of serine protease inhibition. EMBO J. 9 2361-2365, 1990. 

Hirudin is a polypeptide derived from the saliva of the leech Hirudo medicinalis that binds to the blood serine proteinase, thrombins, and thus blocks clot formation. 

The prototype of this class is hirudin, which was originally isolated from the salivary glands of the medicinal leech, Hirudo medicinalis. Hirudin itself is not commercially available, but recombinant technology has permitted production of hirudin derivatives, namely lepirudin and desirudin.29,38,41 Lepirudin has a short half-life of approximately 40 minutes after IV administration and 120 minutes when given SC. Elimination of lepirudin is primarily renal therefore, doses must be adjusted based on the patient s renal function. The dose should be monitored and adjusted to achieve an aPTT ratio of 1.5 to 2.5 times the baseline measurement. Lepirudin is currently approved for use in patients with HIT and related thrombosis. Up to 40% of patients treated with lepirudin will develop antibodies to the drug.29,38,41... 

Plasminogen Activators. PAs may prove helpful in increasing fibrinolysis however, plasminogen activators may be most effective in conjunction with hirudin or synthetic hirudin analogues. 

The most potent thrombin inhibitor is hirudin, originally isolated from the salivary glands of the medicinal leech Hirudo medicinalis. Its inhibition constant is in the femtomolar (10-15 M) range (57). It is a 65-amino-acid tyrosine-sulfated single-chain polypeptide. Recombinant hirudin differs from native hirudin by the absence of the sulfate group on tyrosine 63 (Tyr-63) and is referred to as desulfato hirudin. The loss of this sulfate group reduces the thrombin inhibitory potency by 10-fold. 

The coupling of hirudin to polyethylene glycol (PEG) increases its half-life. PEG-hirudin is also less susceptible to proteolytic degradation (60). 

Bivalent inhibitors of thrombin have been synthesized to bind the anion-binding exosite and active (catalytic) site of thrombin simultaneously. By coupling the carboxy terminal fragment of hirudin to a tripeptide (D-Phe-Pro-Arg) by including a spacer molecule, both the anion exosite and the catalytic site are blocked. An example of such a molecule is Hirulog, which has 20 amino acids and has a Kj of 2 nM (61). Its ability to block the active site has been questioned, since thrombin has been shown to cleave the Arg-Pro bond of Hirulog slowly in vivo (58). In addition to hirudin and hirudin-like compounds, three other classes of site-directed thrombin inhibitors deserve mention. 

Direct thrombin inhibitors such as hirudin, Hirulog, the peptide aldehyde efegatran, and peptidomimetic compound argatroban have undergone clinical trials. Their application in the prevention and treatment of deep vein thrombosis contin-... 

In vitro platelet activation is dependent on the anticoagulant that is used for blood collection. In one study it was demonstrated that PF4 levels in platelet-poor plasma isolated after incubation without any stimuli for 1 hour at 37°C were as follows conventional heparin, 1180 ng/ml hirudin, 469 ng/ml citrate, 440 ng/ml and EDTA, 217 ng/ml (110). EDTA appears to suppress platelet degranulation. PF4 levels obtained with a low-molecular-weight heparin preparation called Frag-min were, however, comparable to those obtained with hirudin (110). 

Finally, with the use of direct thrombin inhibitors such as hirudin for anticoagulant therapy, laboratory tests such as APTT may not be sensitive enough to follow therapy. 

Markwardt F. Hirudin and derivatives as anticoagulant agents, Thromb Haemost 1991 66, 141-52. 

Rydel T. J., Ravichandran K. G., Tulinsky A., et al. The structure of a complex of recombinant hirudin and human alpha-thrombin. Science 1990 249,277-80. 

Eriksson B. I., Ekmann S. T Kalebo P., et al. Prevention of deep-vein thrombosis after total hip replacement Direct thrombin inhibition with recombinant hirudin, CCG 39393. Lancet 1996 347,635-9. 

Nowak G., Bucha E. A new method for the therapeutical monitoring of hirudin. Thromb Haemost 1993 69, 1306. 

Hirudin Oleosin promoter/ nos terminator Fused to native oleosin B. napus 1% of seed protein 21... 

Hirudin Polypeptide Leech saliva, genetic engineering 7 000... 

Dicoumarol and related molecules are generally used over prolonged periods, whereas heparin is used over shorter periods. Hirudin has recently been approved for general medical use, while ancrod remains under clinical investigation. 

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