Hirudin and Thrombin

The saliva of the medicinal leech contains a battery of substances that interfere with the hemostatic mechanisms of the host. One of these compounds is hirudin, a potent anticoagulant, which maintains the fluidity of the ingested blood and is the most potent inhibitor of thrombin. Upon binding to thrombin, the cleavage of fibrinogen and subsequent clot formation are prevented. The potency and specificity of hirudin make it a useful antithrombin-III-independent alternative to heparin for the control of thrombosis. 

---

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. 

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. 

De Filippis, V., DeBoni, S., DeDea, E., Dalzoppo, D., Grandi, C., and Fontana, A. Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1-47 of hirudin as a probe of hirudin folding and thrombin recognition, Protein Sci., 13 1489-1502,2004. 

Fitzgerald, G. (1996). The human pharmacology of thrombin inhibition. Coronary Artery Dis. 7(12), 911-918. Markwardt, F. (1991). Hirudin and its derivatives as anticoagulant agents. Thromb. Haemost. 66, 141-152. 

Lepirudin is a recombinant derivative of hirudin (direct thrombin inhibitor secreted by salivary glands of leech). It inhibits thrombin directly and is mainly used in heparin induced thrombocytopenia. 

The direct thrombin inhibitors (DTIs) exert their anticoagulant effect by directly binding to the active site of thrombin, thereby inhibiting thrombin s downstream effects. This is in contrast to indirect thrombin inhibitors such as heparin and LMWH (see above), which act through antithrombin. Hirudin and bivalirudin are bivalent DTIs in that they bind at both the catalytic or active site of thrombin as well as at a substrate recognition site. Argatroban and melagatran are small molecules that bind only at the thrombin active site. 

The rate constants for the association of proteins with one another and with other macromolecules are profoundly influenced by the geometry of the interaction and by electrostatic factors. Only a small part of each protein may be involved in the formation of a protein-protein complex, which imposes a bad steric factor on the reaction. Accordingly, protein-protein association rate constants may be as low as 104 s 1 M x (Table 4.1). But there is very fast association at > 5 X 109 s-1 AT 1 at low ionic strength for proteins that have complementary charged surfaces, such as bamase with its polypeptide inhibitor barstarfwhose, properties are discussed in Chapter 19), thrombin with its polypeptide inhibitor hirudin, and ferricytochrome c with ferrocytochrome b5. 

Antman EM for the TIMI 9b Investigators, Hirudin in acute myocardial infarction thrombolysis and thrombin inhibition in myocardial infarction (TIMI) 9b trial, Circulation 1996 94 91 1-921. 

Antman E for the TIMI 9A Investigators. Hirudin in acute myocardial infarction safety report from the Thrombolysis and Thrombin Inhibition in Myocardial Infarction (TIMI) 9A Trial. Circulation 1994 90 1624-1630. 

Sarich TC, Wolzt M, Eriksson UG, et al. Effects of ximelagatran, an oral direct thrombin inhibitor, r-hirudin and enoxaparin on thrombin generation and platelet activation in healthy male subjects, J Am Coll Cardiol 2003 41 557-564,... 

Hirudin and its derivatives (bivalirudin, lepirudin) block the active center of thrombin. 

Nowak G. 2001. Clinical monitoring of hirudin and direct thrombin inhibitors. Semin. Thromb. Hemos-tas. 27 537-541. 

In view of the overwhelming evidence implicating thrombin as the primary mediator of arterial thrombosis (vide supra), numerous thrombin inhibitors have been evaluated in various experimental models of thrombosis [73,74] while hirudin and Hirulog have progressed to advanced stages of clinical development [75]. 

Using an arteriovenous shunt model of thrombosis in the rat, compounds 4 and 7 were evaluated for their ability to maintain patency of the extracorporeal circuit which serves as a thrombogenic surface [76]. Table 2 shows that on a gravimetric basis r-hirudin and inhibitor 4, were equipotent [r-hirudin EDis = 1.4 mg/kg 4 EDi5= 1.2 mg/kg i.v. bolus]. The thrombin inhibitor 7 was less effective in this assay having an EDi5= 6.3 mg/kg. 

In addition to its intrinsic reduced affinity for thrombin, the higher doses required for inhibitor 7 may be correlated to its pharmacokinetics and metabolic susceptibility. The t /2p of r-hirudin and inhibitor 7 were approximately the same in the rat (25-30 minutes) while for the low molecular weight inhibitor 7 it is less than 3 minutes. It is well known that the kidney serves as the primary site of elimination of many polypeptide based drugs [77]. Indeed hirudin is excreted intact in the urine of several species [78]. We found that differences in the rate of elimination between inhibitors 4 and 7 may be explained in part by differences in the metabolic susceptibility toward rat kidney membrane proteases. The principal result from these studies is that the truncated inhibitor 7 is much more proteolytically labile compared to 4 in vitro. In 7, the proteolytic products arise rmidly (< 5 min) firom the cleavage of the Phe -Glu bond followed by cleavage of Asp -Phe, both of which are sufficient to inactivate the peptide. In inhibitor 4, three principal proteolytic sites were identified, two of which are within the spacer residues 1) Leu -Gln 2) Asn -Asp and 3) Gln -Ser . However, intact compound could still be detected after 30 minutes. 

Vindigni A, De Filippis V, Zanotti G, et al. (1994). Probing the structure of hirudin from Hirudinaria manillensis by limited proteolysis Isolation, characterization and thrombin-inhibitory properties of N-terminal fragments. Eur. J. Biochem. 226 323-333. 

---