In vitro microfluidic thrombosis model of HIT. (A) Kinetic quantitative fluorescence analysis of platelet accretion to VWF-coated surfaces after perfusion of citrated whole blood from transgenic mice. Data are expressed as relative platelet accretion calculated from area under the curve (AUC) of percent area covered by platelets (AUC of sample stimulated with KKO divided by AUC of sample stimulated with RTO). Dotted line represents no additional activation with KKO compared with RTO. Mean ± SEM is shown. N = 8, 6, and 5 for each transgenic line, respectively. (B) Same studies analyzed for relative platelet aggregate size. (C) Kinetic quantitative fluorescence analysis of platelet accretion to VWF-coated surfaces after perfusion of citrated whole human blood. Data are expressed as in (A). N = 7 with each experiment performed in duplicate. (D) Same as (B), using human blood. N = 7 with each experiment performed in duplicate. (E) Similar studies as in (C) conducted with isolated IgG from patients with clinically and serologically documented HIT compared with normal hIgG. Each HIT-IgG was tested with blood from 3 healthy donors, each done in 3 independent experiments. Dotted line represents no activation with the HIT IgG compared with normal control IgG. Two-tailed Student t tests were used to compare KKO-induced accretion and aggregate size vs the paired RTO control. (F) Representative confocal microscopy images of fixed thrombi formed in microfluidic channels. Platelet aggregates are shown in green, fibrin fibers visualized by adding of Alexa 647-labeled fibrinogen are purple, and white blood cells are cyan (overlap of blue nuclear dye is Hoechst and green calcein AM). The attachment points of fibrin to platelets are white because of superposition of purple and green colors. Images were acquired using a Zeiss LSM 710 AxioObserver inverted microscope (Germany) with a 40× 1.1 NA water immersion objective. n.s., not statistically significant following KKO vs RTO exposure.