Targeting of porcine pancreatic phospholipase A2 to human platelets: introduction of an RGD sequence by genetic engineering
Bekkers, A.C.; van der Vuurst, H.; van Willigen, G.; Akkerman, J.W.; Verheij, H.M.
Thrombosis and Haemostasis 74(4): 1138-1144
1995
ISSN/ISBN: 0340-6245 PMID: 8560426 Document Number: 452728
The possibility to induce specific disruption of activated platelets by binding of porcine pancreatic phospholipase A-2 (PLA-2) was tested by constructing a set of PLA-2-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 PLA-2 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 alpha-thrombin-stimulated platelets, CRS42 and CRS82 induced about 5% hydrolysis of (3H)-arachidonic acid-labeled phospholipids. Stimulation with a combination of alpha-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 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 activity is required to eliminate activated platelets among a suspension of resting platelets and other blood cells.