Targeting of Porcine Pancreatic Phospholipase A₂ to Human Platelets

Abstract

In the present study we prepared by chemical modification a series of porcine pancreatic phospholipase A, (PLA) derivatives, that bind to the activated glycoprotein (GP) IIb/IIIa complex and hydrolyse phospholipids in the outer leaflet of the platelet membrane. To the native enzyme, an RGD-containing peptide was coupled to introduce affinity for GPIIb/IIIa in combination with lauric acid to improve binding to the membrane. As controls, derivatives containing only one of these modifications were prepared. Acylation of the enzyme improved the affinity for densely packed phospholipids, as deduced by kinetic analyses. After stimulation of platelets, the RGD-containing PLAs bound to GPIIblIlIa since GRGDS peptide and a monoclonal antibody against the complex interferred with binding. No binding was found with native PLA. The binding seen with lauric acid PLA was not mediated by GPIIb/IIIa. All modified PLAs induced 1 -3 % hydrolysis of ['Hlarachidonic-acid-labelled phospholipids in resting platelets. After activation with a-thrombin, hydrolysis increased to 17 %, corresponding to about 90% of ['Hlarachidonate-labelled phospholipids in the outer leaflet of the plasma membrane. RGD-containing PLAs were more active than lauroyl PLA, and their activity was mediated via GPIIb/IIIa since GRGDS inhibited release of ['Hlarachidonic acid. Acylation of the RGD-containing PLAs did not further improve the hydrolytic properties. We conclude that chemical modification of PLA leads to a targetted hydrolytic action and could be a basis for the design of enzymes that specifically destroy activated platelets.

Selection of peptide entry motifs by bacterial surface display

“…Another carrier peptide is the cell-binding Arg-Gly-Asp (RGD) motif, which is found in the adhesins of numerous viruses as well as in the adhesins of other pathogens [15]. The RGD motif is recognized by the integrin family of eukaryotic receptors that are expressed on numerous cell types and incorporation of the RGD sequence into recombinant proteins induces their uptake by eukaryotic cells [16].…”

A cell adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional enzyme

“…Several cell-binding ligands have been employed to modify the cell targeting properties of infectious recombinant viruses (Chambers et al, 1996;Nolan, 1997). Similarly, synthetic RGD-containing peptides have been used for the cell targeted delivery of attached enzymes (Bekkers et al, 1996). The results presented in this work show that short, RGD-containing cell-adhesion tags of viral origin, displayed at the surface of a recombinant protein, can be efficient tools for cell targeting of the whole chimera, with the construct remaining functional when attached to the cell surface as well as after its internalization.…”