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 (RGDloop). 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 RGDloop 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.
