INTERACTION OF HEMORRHAGIC METALLOPROTEINASES WITH HUMAN ALPHA-2-MACROGLOBULIN

The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human α2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by α2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of α2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with α2-macroglobulin rapidly at 22 °C. Rate constants based on intrinsic fluorescence measurements were 0.62 × 105 M−1 s−1 for interaction of α2-macroglobulin with Ht-c and -d and 2.3 × 105 M−1 s−1 for the interaction of α2-macroglobulin with Ht-e. Ht-a interacted with α2-macroglobulin very slowly at 22 °C. Increasing the temperature to 37 °C and prolonging the time of interaction with α2-macroglobulin resulted in the formation of Mr 90 000 fragments and high molecular weight complexes (Mr <180000), in which Ht-a is covalently bound to the carboxy-terminal fragment of α2-M. The identification of the sites of specific proteolysis of α2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of α2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves α2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified. Ht-e cleaves α2-macroglobulin at two sites which are different from those observed with Ht-a, -c, and -d. With Ht-e the primary cleavage site is the Val689-Met690 peptide bond and the secondary site at Gly693-His694. Of the four toxins investigated, Ht-a is the most potent hemorrhagic toxin in vivo. We propose that reaction of Ht-a with the primary plasma proteinase inhibitor α2-M may partially explain the high hemorrhagic activity of this toxin. © 1990, American Chemical Society. All rights reserved.