TARGETING OF PORCINE PANCREATIC PHOSPHOLIPASE A(2) TO HUMAN PLATELETS- INTRODUCTION OF AN RGD SEQUENCE BY GENETIC-ENGINEERING

The possibility to induce specific disruption of activated platelets b y binding of porcine pancreatic phospholipase A(2) (PLA(2)) was tested by constructing a set of PLA(2)-mutants containing an Arg-Gly-Asp (RG D) sequence. One mutant was made with RGD as part of a surface-exposed loop (RGD(loop)). Four mutants were made with RGD as part of a C-term inal extension: one with RGD directly coupled to the C-terminus (RGDc) and three mutants (CRSx) with x = 22, 42 and 82 hydrophylic non-charg ed amino acids between RGD and the enzyme. All mutants retained 20-80% activity of native PLA(2) and showed little binding to resting platel ets. The binding of the native enzyme and RGD(loop) was not increased following stimulation. In contrast, the mutants RGDc and CRSx showed s timulation-dependent binding to the platelet receptor GPIIb/IIIa, sinc e GRGDS-peptide and a monoclonal antibody against the complex interfer ed with binding. In alpha-thrombin-stimulated platelets, CRS42 and CRS 82 induced about 5% hydrolysis of [H-3]-arachidonic acid-labeled phosp holipids. Stimulation with a combination of alpha-thrombin and collage n (known to expose phosphatidylserine) increased hydrolysis to 11%. De spite the membrane disruption, the cells did not leak lactate dehydrog enase. We conclude that PLA(2) can be targeted to activated platelets by introducing RGD in a C-terminal extension with a minimum distance ( 42 amino acids) between RGD and the enzyme. However, more hydrolytic a ctivity is required to eliminate activated platelets among a suspensio n of resting platelets and other blood cells.