Fig. 6.
CLEC-2 inhibition by F(ab) 2 does not alter thrombus formation or leukocyte recruitment over activated endothelial cells. ( A ) Whole blood in the presence of an IgG control to F(ab) 2 was perfused in a µ-Slide VI 0.4 flow chamber over 24 h-TNF-α activated (10 ng/mL) or unstimulated, human umbilical vein endothelial cells (HUVEC) for 3 minutes. ( Ai ) Thrombus surface coverage over 3 minutes, measured using DiOC6 fluorescence (2 µM); ( Aii ) subsequent area under the curve (AUC) was calculated (a.u. = arbitrary units). ( B ) Whole blood was perfused at venous shear stress (5 dyn.s/cm 2 ) over 24 h-TNF-α activated (10 ng/mL) HUVEC for 3 minutes in the presence or absence of F(ab) 2 (10 µg/mL) for 10 minutes prior to perfusion. ( Bi ) Leukocyte recruitment was analyzed by manual identification from five fields of view per donor and ( Bii ) representative images were taken postperfusion. ( Biii ) Thrombus surface coverage over 3 minutes, measured using DiOC6 fluorescence (2 µM) and ( Biv ) subsequent area under the curve (AUC) was calculated (a.u. = arbitrary units). ( Bv ) Representative images presented after 4 minutes ( n = 8 donors; arrow indicates direction of flow). ( C ) Whole blood was perfused at arterial shear stress (10 dyn.s/cm 2 ) over 24 h-TNF-α activated (10 ng/mL) HUVEC for 3 minutes in the presence or absence of F(ab) 2 (10 µg/mL) for 10 minutes prior to perfusion. ( Ci ) Thrombus surface coverage over 3 minutes, measured using DiOC6 fluorescence (2 µM) and ( Cii ) subsequent AUC was calculated. ( Ciii ) Representative images shown after 4 minutes ( n = 8 donors; arrow indicates direction of flow). The statistical significance between two groups was analyzed using a paired t-test. *** p < 0.001. IgG, immunoglobulin G.