Nanobodies Nd4 and Nd6 inhibit the activity of human VWF. (A) Ristocetin-induced platelet aggregation traces with or without the addition of 1 μM Nd4 or Nd6. Human platelet-rich plasma (PRP) was gently stirred with addition of each nanobody at 30 seconds, followed by addition of 1.5 mg/mL ristocetin at 60 seconds. (B) Representative flow chamber surface images at 120 seconds of human whole blood perfusion on collagen. Recalcified whole blood labeled with DIOC-6 was mixed with noted nanobody or aptamer inhibitors and then perfused over a collagen surface at given shear rates. The scale bar is 100 μm. Platelet adhesion was measured by thresholding pixel densities to calculate the area covered by adherent platelets. (C) Quantitative plots of platelet adhesion on collagen under noted shear rates and with treatment of various inhibitors (Nd4 in blue, Nd6 in green, VHH81 in pink, and ARC1172 in purple). Comparison between platelet adhesion was analyzed with a 2-way ANOVA with mixed effects with Tukey multiple comparison correction. Asterisks in given colors represent statistically significant values (P < .0001) between control blood and individual treatments at a given shear rate after multiple comparison corrections. Data are means ± SD (n = 8-12 fields analyzed per condition). Interaction F (8, 142) = 26.31; P < .0001; shear rate F (2, 142) = 7.47; P = .0008; treatment F (4, 142) = 141.7; P < .0001. ANOVA, analysis of variance.