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

[heparin-induced thrombocytopenia]

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 (PF 4 )-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-PF 4 /heparin complexes . The integrity of the PF 4 /heparin-complexes was shown by binding of anti-PF 4 /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 PF 4 /heparin-IgG complexes . With the smallest micropatterns (0 .5 x 0 .5 µm ) we show that single platelets become strongly activated by binding to surface-immobilised PF 4 /heparin-IgG , while on larger micropatterns (10 x 10 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 .