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).

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 19th 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 ineffective, therapy. The Napoleonic Army surgeons, for example, used leeches to withdraw blood from soldiers suffering from conditions as diverse as infections and mental disease.

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:

• it is a potent inducer of platelet activation and aggregation;

• it functions as a chemoattractant for monocytes and neutrophils;

• it stimulates endothelial transport.

Table 12.4 Some substances isolated from bloodsucking parasites which inhibit their host's haemostatic mechanisms. All are polypeptides of relatively low molecular mass

Polypeptide

Molecular mass (Da)

Producer

Haemostatic effect disrupted

Hirudin

7 000

Hirudo medicinalis

Binds to and inhibits thrombin

Rhodniin

11 100

Rhodnius prolixus

Binds to and inhibits thrombin

Antistatin

15 000

Haementeria officinalis

Inhibits factor Xa

Tick anticoagulant

6 800

Ornithodoros moubata

Inhibits factor Xa

peptide (TAP)

Calin

55 000

Hirudo medicinalis

Inhibits platelet adhesion

Decorsin

4 400

Macrobdella decora

Inhibits platelet adhesion

One molecule of hirudin binds a single molecule of thrombin with very high affinity (Kd ~ 10 ~12 mol T1). Binding and inactivation occur as a two-step mechanism. The C-terminal region of hirudin first binds along a groove on the surface of thrombin, resulting in a small conformational change of the enzyme. This then facilitates binding of the N-terminal region to the active site area (Figure 12.8). Binding of hirudin inhibits all the major functions of thrombin. Fragments of hirudin can also bind thrombin, but will generally only inhibit some of thrombin's range of activities. For example, binding of an N-terminal hirudin fragment to thrombin inhibits only the thrombin catalytic activity.

Hirudin displays several potential therapeutic advantages as an anticoagulant. These include:

• it acts directly upon thrombin;

• it does not require a cofactor;

• it is less likely than many other anticoagulants to induce unintentional haemorrhage;

Although the therapeutic potential of hirudin was appreciated for many years, insufficient material could be purified from the native source to support clinical trials, never mind its widespread medical application. The hirudin gene was cloned in the 1980s, and it has subsequently been expressed in a number of recombinant systems, including E. coli, Bacillis subtilis and Saccharomy-ces cerevisiae. A recombinant hirudin (tradename Refludan) was first approved for general medical use in 1997. The recombinant production system was constructed by insertion (via a plasmid) of a synthetic hirudin gene into a strain of S. cerevisiae. The yeast cells secrete the product, which is then purified by various fractionation techniques (Figure 12.9). The recombinant molecule displays a slightly altered amino acid sequence compared with the native product. Its first two amino acids, leucine and threonine, replace two valines of native hirudin. It is also devoid of the sulfate

Hirudin

N terminal region

Hirudin

N terminal region

Thrombin

Inactive Hirudin-Thrombin complex

Thrombin

Figure 12.8 Binding of hirudin to thrombin, thus inactivating this activated coagulation factor

Figure 12.9 Overview of the production of Refludan (recombinant hirudin). The exact details of many steps remain confidential for obvious commercial reasons. A number of QC checks are carried out on the final product to confirm the product's structure. These include amino acid composition, HPLC analysis and peptide mapping

Figure 12.9 Overview of the production of Refludan (recombinant hirudin). The exact details of many steps remain confidential for obvious commercial reasons. A number of QC checks are carried out on the final product to confirm the product's structure. These include amino acid composition, HPLC analysis and peptide mapping group normally present on tyrosine63. Clinical trials, however, have proven this slightly altered product to be both safe and effective. The final product is presented in freeze-dried form with the sugar mannitol representing the major added excipient. The product, which displays a useful shelf-life of 2 years when stored at room temperature, is reconstituted with saline or WFI immediately prior to its i.v. administration. A second recombinant product (tradename revasc, also produced in S. cerevisiae) has also been approved.

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