Reversible inhibition by protamine of human synovial and rabbit platelet secretory phospholipase A2.

ABSTRACT

We investigated the effects of protamine on the release and the activity of 14 kDa type II phospholipase A2 (sPLA2). Protamine blocks both release and activity of sPLA2 from thrombin-stimulated platelets in a concentration-dependent manner. Heparin, an anionic sulfate polysaccharide which has a high affinity for this enzyme, has no inhibitory effect on sPLA2 by itself but it is able to reverse the inhibitory effect of protamine. The liberation by thrombin of platelet factor 4, an alpha-granule constituent, unlike to that of ATP stored in dense bodies, was suppressed by protamine. Platelet aggregation, determined in parallel, was not affected by protamine. Also, protamine did not inhibit platelet arachidonic acid liberation, which is mainly produced by cytosolic PLA2. The non-proteinaceous polycationic hexadimethrine and acidic protein casein failed to inhibit platelet sPLA2 activity. By contrast, the basic polypeptides poly(L-arginine) and poly(L-lysine) potently inhibited sPLA2 activity, indicating the important role of basic amino acids in the inhibitory effect evoked by protamine. Activities of the human recombinant sPLA2 and the unpurified synovial enzyme of patients with rheumatoid arthritis were also inhibited by the same range of protamine, poly(L-arginine) and poly(L-lysine) concentrations. Our results demonstrate that protamine, unlike heparin, blocks platelet sPLA2 release and exerts a reversible inhibitory effect on its activity, probably through the interaction of basic amino acids with the enzyme.