Isolation and studies of the granules of the amebocytes of Limulus polyphemus,the horseshoe crab

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water. Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin. The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.