Targeting of Porcine Pancreatic Phospholipase A₂ to Human Platelets: Introduction of an RGD Sequence by Genetic Engineering

Summary

The possibility to induce specific disruption of activated platelets by binding of porcine pancreatic phospholipase A₂ (PLA₂) was tested by constructing a set of PLA₂-mutants containing an Arg-Gly-Asp (RGD) 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-terminal extension: one with RGD directly coupled to the C-terminus (RGDc) and three mutants (CRSx) with x = 22,42 and 82 hydrophylic non-charged amino acids between RGD and the enzyme. All mutants retained 20-80% activity of native PLA₂ and showed little binding to resting platelets. The binding of the native enzyme and RGD_loop was not increased following stimulation. In contrast, the mutants RGDc and CRSx showed stimulation-dependent binding to the platelet receptor GPIIb/IIIa, since GRGDS-peptide and a monoclonal antibody against the complex interfered with binding. In α-thrombin-stimulated platelets, CRS42 and CRS82 induced about 5% hydrolysis of [³H]-arachidonic acid-labeled phospholipids. Stimulation with a combination of a-thrombin and collagen (known to expose phosphatidylserine) increased hydrolysis to 11%. Despite the membrane disruption, the cells did not leak lactate dehydrogenase. We conclude that PLA₂ 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 activity is required to eliminate activated platelets among a suspension of resting platelets and other blood cells.

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