Page 215 - Haematologica - Vol. 105 n. 6 - June 2020

Cotl1 regulates shear-dependent thrombus formation
time, our results emphasize that reduced LTB4 production in Cotl1-/- platelets, which display no obvious activation defect per se, significantly contributed to the impaired aggregate formation in the presence of shear.
Cotl1 modulates thrombosis and hemostasis
To investigate whether the impaired shear-dependent aggregate formation translated into a phenotype in vivo, we subjected Cotl1-/- mice to an experimental model of arterial thrombosis. Since it is well documented that col- lagen is a main driver of thrombus formation in bigger vessels, particularly in models of mechanical injury, we chose a model where the abdominal aorta is mechanically injured. This procedure triggers rapid platelet adhesion to the injured vessel wall, followed by the development of a large occlusive thrombus associated with dynamic changes in both shear and biomechanical forces acting on adhering platelets in the growing thrombus. Strikingly, Cotl1-/- mice were profoundly protected from occlusive thrombus formation in this model (Figure 5A and B). In
A
WT mice, irreversible vessel occlusion was observed within 7 minutes (min) after injury (mean occlusion time 3.37±0.72 min). In sharp contrast, while a progressive reduction in blood flow occurred during the first minutes after injury in Cotl1-/- mice, indicative of beginning throm- bus formation and increasing stenosis, blood flow after- wards normalized and 9 of 11 mice displayed normal blood flow through the injured vessel at the end of the observation period (30 min) (***P<0.001). These results demonstrated that Cotl1 is essential for occlusive arterial thrombus formation in vivo.
To assess the hemostatic function of Cotl1-/- platelets, we performed a tail bleeding assay. Notably, while tail bleeding times were overall significantly increased in Cotl1-/- mice (7.8±12.2 min in Cotl1-/- mice vs. 3.3±1.8 min in WT; *P<0.05) the hemostatic defect was rather mild given the profound protection in the arterial thrombosis model (Figure 5C), indicating that Cotl1 may be particu- larly important in settings of pathological thrombus for- mation.
BC
Figure 4. Defective shear-dependent thrombus formation in Cotl1-deficient mice can be res- cued by exogenous addition of leukotriene B4. (A-C) Assessment of platelet adhesion (A and B) and aggregate formation in heparinized blood (A and C) on Horm collagen (70 μg mL-1) under flow (1700 s-1). WT and Cotl1-/- samples were either left untreated or were pre-incubated for 5 minutes (min) with LTB4 [0.25 nM]. Images are representatives of at least 12 mice per group. Values are mean±standard deviation. Scale bar, 50 μm. *P<0.1; **P<0.01.
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