TARGETING OF PORCINE PANCREATIC PHOSPHOLIPASE A(2) TO HUMAN PLATELETS- INTRODUCTION OF AN RGD SEQUENCE AND ACYL-GROUP BY CHEMICAL MODIFICATION

In the present study we prepared by chemical modification a series of porcine pancreatic phospholipase A(2) (PLA) derivatives, that bind to the activated glycoprotein (GP) IIb/IIIa complex and hydrolyse phospho lipids in the outer leaflet of the platelet membrane, To the native en zyme, an RGD-containing peptide was coupled to introduce affinity for GPIIb/IIIa in combination with lauric acid to improve binding to the m embrane, As controls, derivatives containing only one of these modific ations were prepared, Acylation of the enzyme improved the affinity fo r densely packed phospholipids, as deduced by kinetic analyses. After stimulation of platelets, the RGD-containing PLAs bound to GPIIb/IIIa since GRGDS peptide and a monoclonal antibody against the complex inte rferred with binding. To binding was found with native PLA, The bindin g seen with lauric acid PLA was not mediated by GPIIb/IIIa. All modifi ed PLAs induced 1-3% hydrolysis of [H-3]arachidonic-acid-labelled phos pholipids in resting platelets; After activation with alpha-thrombin h ydrolysis increased to 17%, corresponding to about 90% of [H-3]arachid onate, labelled phospholipids in the outer leaflet of the plasma membr ane. RGD-containing PLAs were more active than lauroyl PLA, and their activity was mediated via GPIIb/IIIa since GRGDS inhibited release of [H-3]arachidonic acid. Acylation of the RGD-containing PLAs did not fu rther improve the hydrolytic properties. We conclude that chemical mod ification of PLA leads to a targetted hydrolytic action and could be a basis fur the design of enzymes that specifically destroy activated p latelets.