Some cytologic characteristics of the hemocytes of Limulus during clotting

The hemolymph of the horseshoe crab, Limulus (Xiphosura) polyphemus , contains a single cell type. The hemocytes are ovoid and contain many refractile granules. One‐half to one minute after the onset of clotting the hemocytes swell and a hyaline cytoplasmic ring, essentially devoid of granules, appears about their circumference. During this time the granules disperse and the nucleus becomes visible. Three to five minutes following the initiation of clotting, the cell extends long pseudopodial processes. Phase contrast time‐lapse cinematography reveals that the cells are extremely motile during this phase. Concomitant with these changes, many of the granules lose their refractility and one by one disappear from the cytoplasm leaving what appears to be a vacuole. Electron micrographs of native (un‐clotted) hemocytes and of clots fixed in glutaraldehyde and post‐fixed in osmium reveal that the membrane‐bounded granules of native cells are very dense and homogeneous with no evidence of an internal structure. One‐half to one minute after clotting, however, they become less dense and 250 Å microtubules spaced at ca. 500 Å intervals appear parallel to the long axis of the granule. Further degradation of the granule ensues and involves (a) change to a spherical shape, and (b) further decrease in density and better resolution of the microtubules. The microtubular component subsequently disintegrates leaving a membrane‐bounded granule containing particulate material. Finally, the membrane of the granule fuses with the plasma membrane and the components of the granule are dispersed in the plasma where they presumably contribute to the formation of the gelatinous clot.