Inhibition of human secretory class II phospholipase A2 by heparin

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Abstract
By means of kinetic analyses using Triton X‐100/deoxycholic acid/dilauroylglycerophosphoethanolamine (4 : 2 : 1, molar ratio) mixed micelles we examined the effects of heparin on the activity of several phospholipases A2 (PLA2). Heparin avidly bound cationic PLA2s including human secretory class II PLA2 and thereby inhibited their hydrolysis of phospholipids in the mixed micelles. Initial velocity measurements indicated that heparin behaved as a competitive inhibitor for human secretory class II PLA2 and closely related A.h. blomhoffii PLA2 and A.p. piscivorus PLA2. In particular, heparin showed the highest specificity for human secretory class II PLA2. In the absence of deoxycholic acid in mixed micelles, A.h. blomhoffii PLA2 was also strongly inhibited by heparin. The observed inhibition was not due to the interaction of heparin with the active site of PLA2 because heparin did not inhibit the hydrolysis of monomeric substrates by PLA2s. Both kinetic measurements and fluorescence measurements of PLA2‐bound 8‐anilino‐1‐naphthalene sulfonate in the presence of varying amounts of heparin showed that a heparin molecule bound about seven molecules of PLA2. When positive charges of four lysines in the amino‐terminal region of A.h. blomhoffii PLA2 were neutralized by limited carbamoylation, heparin neither bound the carbamoylated A.h. blomhoffii PLA2 nor inhibited the hydrolysis of Triton X‐100/dilauroylglycerophosphocholine mixed micelles by the carbamoylated A.h. blomhoffii PLA2 that retained 50% activity of native A.h. blomhoffii PLA2. Also, heparin did not inhibit the hydrolysis of mixed micelles by 7,10‐bis(octanoyl)ated A.p. piscivorus PLA2 in which two lysines in the amino‐terminal α‐helix are acylated. These results indicate that the inhibition of human secretory class II PLA2 and related cationic PLA2s by heparin originates from the interaction of heparin with cationic residues in the amino‐terminal region that forms a part of interfacial binding site. In addition, unique structural features of human secretory class II PLA2, together with its unique mode of interaction with heparin, suggest that this PLA2 might have an additional heparin‐binding site. Although the heparin‐PLA2 binding diminished as the ionic strength of reaction medium increased, the inhibition of human secretory class II PLA2 by heparin remained significant at the physiological ionic strength. An estimated value of inhibition constant (Ki) was 0.1 μM under physiological conditions, which suggests that a normal pharmaceutical dose of heparin might inhibit human secretory class II PLA2 and regulate its biological effects.

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