Platelet functional effects induced by fluorescent labeling via transgenic‐ and antibody‐based approaches

Background ROSA26‐Enhanced Yellow Fluorescent Protein (R26R‐EYFP) mice have been widely used for cell lineage tracing and tracking specific cells in vivo. To visualize platelets in vivo, we bred R26R‐EYFP mice with Platelet Factor 4‐Cre (PF4‐Cre) mice to generate mice that specifically expressed EYFP in platelets. To test possible effects of genetic manipulation and EYFP expression on platelet function, we compared platelet aggregation and adhesion under flow of platelets expressing EYFP from these mice (Cre+, or R26R‐EYFP/PF4‐Cre(+)) with that of littermate controls (Cre‐, or R26R‐EYFP‐Cre/(−)) and C57BL/6 mice. We also assessed platelet function changes induced by antibody labeling of platelets from C57BL/6 mice with DyLight 488 anti‐Glycoprotein Ibβ (x‐488). Methods Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). We studied platelet aggregation and adhesion under flow from transgenic mice with either fluorescent platelets (Cre+) or littermate controls with non‐fluorescent platelets (Cre−). C57BL/6 (BL/6) mice were included as an additional control. Blood was collected into either Ethylenediaminetetraacetic acid for imaging flow cytometry, Acid Citrate Dextrose Solution for platelet aggregometry, or D‐Phenylalanyl‐prolyl‐arginyl Chloromethyl Ketone for platelet adhesion under flow. Washed platelets were subjected to thrombin or collagen mediated aggregation. Platelet adhesion under flow was assayed by perfusing anticoagulated blood over a Type III collagen‐coated surface in a microfluidic parallel‐plate flow system, and platelet adhesion was quantified under bright field illumination. Statistical comparisons were performed with paired or non‐paired Student's t‐ test, as appropriate. Results Imaging flow cytometry of blood derived from Cre+ mice confirmed that EYFP expression was specific for platelets. Platelet aggregometry demonstrated that platelets from Cre+ mice had enhanced aggregation responses to low concentrations of thrombin and collagen when compared with platelets from BL/6 mice (50±14% vs. 34±18% for 0.01U/mL thrombin, p<0.05; 42±19% vs. 10±6.7% for 0.5μg/mL collagen, p<0.01). At higher concentrations of thrombin (0.02U/mL) and collagen (1μg/mL), platelet aggregation was comparable (58±12% vs. 60±14%, and 65±8% vs. 65±9%, respectively, N.S.). Both strains of transgenic mice (Cre+ and Cre−) demonstrated enhanced platelet adhesion under flow as compared to BL/6 mice. In contrast to the genetic fluorescent protein labeling method, x‐488 antibody labeling of platelets from BL/6 mice did not induce significant differences in platelet adhesion under these conditions. Conclusions Genetic fluorescent protein labeling of platelets with ROSA26‐EYFP induces greater alterations in platelet function ex vivo than antibody labeling with x‐488. Support or Funding Information Support: VA/BLR&D Merit Review Grant 1I01BX002551 & NIH/NHLBI R01 HL116524