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Thromb Haemost 1995; 74(04): 1138-1144
DOI: 10.1055/s-0038-1649894
Original Article
Platelets
Schattauer GmbH Stuttgart

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

A C A P A Bekkers
1   The Department of Enzymology and Protein Engineering, Utrecht, The Netherlands
,
H van der Vuurst
2   The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
G van Willigen
2   The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
J W N Akkerman
2   The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
H M Verheij
1   The Department of Enzymology and Protein Engineering, Utrecht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 18 January 1995

Accepted after revision 29 June 1995

Publication Date:
09 July 2018 (online)

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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.

 

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