AGGREGATION OF HORSESHOE CRAB (LIMULUS POLYPHEMUS) AMEBOCYTES AND REVERSIBLE INHIBITION OF AGGREGATION BY EDTA

¹ Boston University Graduate School, Boston, Massachusetts and Marine Biological Laboratory, Woods Hole, Massachusetts

1. The photometric method for measuring cell aggregation was adapted to study the aggregation of the amebocytes of the horseshoe crab, Limulus polyphemus.

2. At 15° C and a constant strring speed, amebocyte aggregation showed reproducible characteristics. Lowering the temperature below 15° C decreased both the rate and extent of aggregation but some slight aggregation still occurred at 10° C.

3. Aggregation was markedly retarded by buffered EDTA. The retardation of aggregation was dependent on the concentration of EDTA. Equimolar magnesium restored full aggregation to amebocytes in EDTA, whereas, similar concentrations of calcium caused only partial aggregation of EDTA treated amebocytes.

4. Dilute quantities of the serum supernatant of aggregated amebocytes and the supernatant of homogenates of amebocytes isolated at 0° C caused immediate aggregation of ameboyctes in EDTA. No significant aggregation was noted when EDTA treated amebocytes were added to the cell free plasma of hemolymph withdrawn at 0° C.

5. Serum or amebocyte homogenate supernatants did not require additional calcium and/or magnesium to induce aggregation of amebocytes in EDTA.

6. The aggregating activity of serum and amebocyte homogenate supernatants was essentially non-dialyzable and heat labile.

7. The data indicate the presence of an aggregation promoting material within amebocytes that is released from the cells during aggregation.

8. The possible relationship between this aggregation promoting material and EDTA induced aggregation inhibition is discussed.