CONTIGUOUS BINDING AND INHIBITORY SITES ON KININOGENS REQUIRED FOR THE INHIBITION OF PLATELET CALPAIN

Both high molecular weight kininogen (HK) and low molecular weight kin inogens (LK) are potent tight binding inhibitors of platelet calpain ( K(i) = 2 nM), but the molecular basis for the inhibitory function is n ot well delineated. The amino acid sequences of the calpain inhibitory domain 2 from human and rat HK were compared for homology with the no ninhibitory domains from human and rat domain 3 and from domain 2 of r at T-kininogen, and two areas of nonconserved differences were detecte d. Computer three-dimensional models were constructed on a template bu ilt using the x-ray crystallographic data for cystatin, an evolutionar y precursor of HK. Two nonconserved regions in the calpain inhibitory domains flank the highly conserved motif QVVAG to form a continuous su rface for interaction with cysteine proteases. Three peptide sequences , components of the modeled surface, were chosen for synthesis from HK D-2: VHPISTQSPDLE (peptide 146-156, NH2-terminal), CTDNAYIDIQLRIASFSQ NC (peptide 229-248, COOH-terminal), and CQRQVVAGLNFRIC (185-189, cent ral) containing QVVAG. This last peptide differs from the natural sequ ence by substitutions of A185C and T195C. Peptides 185-198 and 229-248 were folded by air oxidation of their cysteine residues and then test ed for their ability to inhibit calpain and papain. The folded peptide 229-248 inhibited calpain with an IC50 35 muM and unfolding reduced t his effect. The folded peptide 185-198 did not inhibit calpain, but wh en preincubated with calpain, could block the inhibition by HK indicat ing a probable enzyme binding site. Peptide 146-157 did not inhibit ca lpain but could inhibit papain with an IC50 of 20 muM. We have thus de fined separate binding and inhibitory sequences on HK which form a con tiguous surface for thiol protease interactions.