Duplication of the Antistasin-Like Structure Resulted in a New Anticoagulant Protein in the Medicinal Leech

Blood-sucking organisms produce various anticoagulant proteins that prevent blood clotting in their prey. Even in well-studied species like <i>Hirudo medicinalis</i>, many such proteins remain unidentified. We previously described a novel cysteine-rich anticoagulant (CRA), a distant homolog of antistasin. Later, we discovered another, much larger homolog in the medicinal leech. Its amino acid sequence is also highly cysteine-rich. Analysis of cysteine patterns showed four antistasin-like domain motifs, with one of them strongly disrupted. Since both antistasin and CRA contain two such domains, the new protein represents a duplicated antistasin-like structure. We cloned its cDNA, expressed the recombinant protein in <i>Escherichia coli</i>, purified it by metal-chelate chromatography, refolded it, and tested its anticoagulant properties. Using standard clinical assays-activated partial thromboplastin time, prothrombin time, and thrombin time-we found that the protein inhibited coagulation in all tests, though to varying degrees. These findings suggest that different antistasin-like anticoagulants in the leech enable it to block both intrinsic and extrinsic coagulation pathways, while hirudin inhibits the final step of clot formation. The combination of different anticoagulant proteins allows the leech to effectively prevent the prey's blood from clotting during feeding.