The primary structure of kaouthiagin, a metalloproteinase from the venom of the cobra snake Naja kaouthia which specifically cleaves human von Willebrand factor (VWF), was determined by amino acid sequencing. Kaouthiagin is composed of 401 amino acid residues and one Asn-linked sugar chain. The sequence is highly similar to those of high-molecular mass snake venom metalloproteinases from viperid and crotalid venoms comprised of metalloproteinase, disintegrin-like, and Cys-rich domains. The metalloproteinase domain had a zinc-binding motif (HEXXHXXGXXH), which is highly conserved in the metzincin family. Kaouthiagin had an HDCD sequence in the disintegrin-like domain and uniquely had an RGD sequence in the Cys-rich domain. Metalloproteinase-inactivated kaouthiagin had no effect on VWF-induced platelet aggregation but still had an inhibitory effect on the collagen-induced platelet aggregation with an IC50 of 0.2 μM, suggesting the presence of disintegrin-like activity in kaouthiagin. To examine the effects of these HDCD and RGD sequences, we prepared synthetic peptides cyclized by an S - S linkage. Both the synthetic cyclized peptides (RAAKHDCDLPELC from the disintegrin-like domain and CFDLNMRGDDGSFC from the Cys-rich domain) had an inhibitory effect on collagen-induced platelet aggregation with IC50 values of ∼90 and ∼4.5 μM, respectively. The linear peptide (RAAKHDCDLPELC) and the cyclized peptide (CFDLNMRGEDGSFC) had little effect on collagen-induced platelet aggregation. These results suggest that kaouthiagin not only inhibits VWF-induced platelet aggregation by cleaving VWF but also disturbs the agonist-induced platelet aggregation by both the disintegrin-like domain and the RGD sequence in the Cys-rich domain. Furthermore, our results imply that the corresponding part of the Cys-rich domain in other snake venom metalloproteinases also has a synergistic disturbing effect on platelet aggregation, serving as a second disintegrin-like domain. This is the first report of an elapid venom metalloproteinase with two disintegrin-like sequences.
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