[22] Limulus Proclotting Enzyme

This chapter describes the purification and properties of Limulus proclotting enzyme. Exposure of the Limulus hemocytes to bacterial endotoxins, which are lipopolysaccharides (LPS), results in the activation of the intracellular clotting system leading to gel formation. This phenomenon is known to be one of the self-defense systems of the horseshoe crab against invading micro-organisms. To elucidate the overall molecular events of the LPS induced activation of the Limulus clotting system, the chapter isolates the clotting factors from the hemocyte lysate. The Limulus hemocytes contain two types of granules, large but less dense granules and smaller but denser ones. Immunohistochemical studies show that the proclotting enzyme is localized exclusively in the large granules, and is apparently released through exocytosis on LPS stimulation of the hemocytes. The amidase activity of the clotting enzyme is inhibited by antithrombin III in the presence of heparin and α₂-plasmin inhibitor, but is not inhibited by hirudin, aprotinin, and soybean trypsin inhibitor.