ALPHA(2)-MACROGLOBULIN-MEDIATED CLEARANCE OF PROTEASES FROM THE PLASMA OF THE AMERICAN HORSESHOE-CRAB, LIMULUS-POLYPHEMUS

Because proteases free in the body are damaging to the tissues, animals have evolved various agents for their inactivation and clearance. Mammals, for instance, have a diverse array of active site protease inhibitors in the plasma, in addition, mammals have alpha(2)-macroglobulin (alpha(2)M), which binds active proteases, and the alpha(2)M-protease complex is then cleared from the plasma by a receptor-mediated endocytotic process. (alpha(2)M is also present in the plasma of many invertebrates, and in the American horseshoe crab, Limulus polyphemus, it is the only protease inhibitor in the plasma. To search for a clearance process for proteases in Limulus, fluerescein isothiocyanate (FITC)-labeled proteins were injected into the blood, and the fluorescence in the plasma and associated with the blood cells was determined. FITC-labeled trypsin was cleared with an initial mixing period (0-10 min) and a rapid clearance period (10-30 min), followed by the reappearance of FITC in the plasma (45-90 min). Before and during the clearance process, the labeled trypsin was associated with a complex having a molecular mass identical to that of Limubus alpha(2)M, and that was precipitated by antibodies directed against Limubus alpha(2)M. The fluoresceinated material that reappeared in the plasma after 45 min was of low molecular mass (<10 kDa) and thus appears to have experienced degradation. The clearance of trypsin requires its protease activity, since phenylmethylsulfonyl fluoride-inactivated, FITC-labeled trypsin was cleared only very slowly if at all (t1/2 > 180 min). FITC-labeled, trypsin-reacted Limubus alpha(2)M was cleared rapidly from the plasma of Limubus, whereas FITC-labeled, native Limubus alpha(2)M persisted undiminished in excess of 400 min. The blood cells of Limubus bound FITC-labeled trypsin-reacted Limubus alpha(2)M, and the peak of recovery from the blood cells coincided with the minimum concentration of FITC-labeled protein in the plasma, suggesting that the blood cells participate in the clearance of alpha(2)M-protease complex from the plasma. Thus, we have demonstrated the existence of a clearance pathway in Limubus that operates selectively on enzymatically active proteases and have shown that Limulus alpha(2)M is the probable agent for protease clearance. This is the first documentation of a protease clearance pathway in invertebrates and represents the first identified physiological function for alpha(2)M in invertebrates.