Blocking VEGF-A using tinzaparin reduces VWF fiber formation in tumor microvasculature. Immunofluorescence stainings for VWF (green) and anti-CD31 (red) in cryosections of ret transgenic tumors were performed. Nuclei were stained with DAPI. Tumor microvessels of control mice showed formation of ULVWF fibers in the vessel lumen (A, arrows). By contrast, microvessels of tinzaparin-treated mice showed almost no ULVWF fiber formation and a punctual pattern of VWF within the vessel wall (B, arrowheads), indicative of reduced endothelial cell activation. Representative pictures of tumor microvessels are shown (n = 10 animals of 2 independent experiments; scale bars = 20 µm). Tinzaparin treatment correlated with a significant reduction of vessels with intraluminal VWF fibers (C). Tumor vessels were analyzed for platelet aggregation using VWF (green) and GPIb (red) staining. Quantification showed a significant increase in platelet-covered area in the lumen of tumor vessels compared with control. This effect was abolished by treatment with tinzaparin (D-E). Panel Ei shows a single platelet in the lumen of a tumor blood vessel. In addition, the treatment of ret transgenic mice with tinzaparin (gray) reduced the appearance of middle (ii) and big aggregates (iii) to healthy control skin levels (white) compared with vehicle-treated control tumors (black). Plot shows mean ± SD (n = 5-10; *P < .05; **P < .005; ***P < .001; scale bars = 5 µm).