Micropatterned array to assess the interaction of single platelets with platelet factor 4-heparin-IgG complexes

Nikolay Medvedev; Raghavendra Palankar; Krystin Krauel; Andreas Greinacher; Mihaela Delcea

doi:10.1160/TH13-09-0752

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 111(05): 862-872
DOI: 10.1160/TH13-09-0752

Platelets and Blood Cells

Schattauer GmbH

Micropatterned array to assess the interaction of single platelets with platelet factor 4-heparin-IgG complexes

Nikolay Medvedev^#

¹ZIK HIKE-Zentrum für Innovationskompetenz ˵Humorale Immunreaktionen bei kardiovaskulären Erkrankungen”, Greifswald, Germany

,

Raghavendra Palankar^#

¹ZIK HIKE-Zentrum für Innovationskompetenz ˵Humorale Immunreaktionen bei kardiovaskulären Erkrankungen”, Greifswald, Germany

,

Krystin Krauel

² Institut für Immunologie und Transfusionsmedizin, Greifswald, Germany

,

Andreas Greinacher^*

² Institut für Immunologie und Transfusionsmedizin, Greifswald, Germany

,

Mihaela Delcea^*

¹ZIK HIKE-Zentrum für Innovationskompetenz ˵Humorale Immunreaktionen bei kardiovaskulären Erkrankungen”, Greifswald, Germany

› Author Affiliations

Abstract

Summary

We report a strategy to generate by electron beam lithography high fidelity micropatterned arrays to assess the interaction of single platelets with immobilised ligands. As a proof-of-principle we functionalised the microarrays with platelet factor 4 (PF4)-heparin-IgG complexes. We embedded biotinylated water-soluble quantum dots into polyethylene glycol (PEG)-coated micropatterned arrays and functionalised them via streptavidin to bind biotinylated ligands, here biotinylated-PF4/heparin complexes. The integrity of the PF4/heparin-complexes was shown by binding of anti-PF4/heparin antibodies. Ligand density was quantified by immunofluorescence and immunogold antibody labelling. Real-time calcium imaging was employed for read-out of single platelets activated on micropatterned surfaces functionalised with PF4/heparin-IgG complexes. With the smallest micropatterns (0.5x0.5 µm) we show that single platelets become strongly activated by binding to surface-immobilised PF4/heparin-IgG, while on larger micropatterns (10x10 µm), platelet aggregates formed. These findings that HIT antibodies can cause platelet activation on microarrays illustrate how this novel method opens new avenues to study platelet function at single cell level. Generating functionalized microarray surfaces to which highly complex ligands can be bound and quantified has the potential for platelet and other cell function assays integrated into high-throughput microfluidic microdevices.

Keywords

Heparin-induced thrombocytopenia - platelet factor 4 - micropatterned arrays - electron beam lithography - platelet activation

Full Text

References

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