CAN12 delivered 15 minutes after injury was able to prolong the time to complete occlusion to 84 moments (Fig. PAR4 cleavage. In addition, CAN12 inhibits thrombin induced human platelet aggregation and secretion in a dose dependent manner. We next decided that this specificity of CAN12 is usually agonist dependent. in the Rose Bengal model. The time to thrombosis was delayed to more than 90 moments when CAN12, 1.0 mg/kg (~14 g/ml plasma concentration), was injected 10 minutes prior to injury (Fig. 6A). We next wanted to determine the minimal dose of CAN12 required to influence the time to thrombosis. The intermediate doses of 0.5 mg/kg and 0.25 mg/kg LAMC2 had a time to thrombosis of 82 minutes and 60 min, respectively. At 0.125 mg/kg CAN12, the time to occlusion was 37 minutes; the same time as the controls (saline and IgG) (Fig 6A). We verified that the delay in thrombosis was not due to a decrease in the platelet number (Fig. 6B). Next we investigated whether CAN12 prolonged the time to thrombosis when administered after initiation of the injury. For these studies we used the lowest dose of CAN12 (0.5 mg/kg) that significantly prolonged the time to (S)-Mapracorat occlusion (observe Fig. 6A). CAN12 delivered 15 minutes after injury was able to prolong the time to total occlusion to 84 moments (Fig. 6C). CAN12 also did not reduce platelet figures when administered after the injury (Fig. 6D). Similarly, there was no difference in platelet number between IgG and CAN12 treatment when injury was not initiated (425 106 56 platelets/ml vs. 462 106 90 platelets/ml, respectively). Overall, CAN12 treatment is able to delay arterial thrombosis when delivered either before or after injury. Open in a separate window Physique 6 CAN12 inhibits arterial thrombosis(A) C57BL/6 mice (S)-Mapracorat were pretreated with saline, goat IgG (2 mg/kg), or CAN12 (1, 0.5, 0.25, 0.125 mg/kg) for 10 (S)-Mapracorat min and then subjected to the Rose Bengal carotid artery thrombosis model. Time to total occlusion is usually indicated or the experiment was terminated at 90 min. (B) The concentration of platelets in the blood at termination of the experiment was decided. (C) 15 minutes after the initiation of carotid artery thrombosis, C57BL/6 mice were injected with goat IgG (2 mg/kg) or CAN12 (0.5 mg/kg) and the time to complete arterial occlusion was determined. The experiment was terminated at 90 min. (D) The concentration of platelets in the blood at termination of the experiment (S)-Mapracorat was decided. **p 0.01 CAN12 does not affect bleeding time Finally, we wanted to examine if CAN12 treatment influences hemostasis using two assays. The first was the tail clip assay. C57BL/6 mice were injected with IgG (2 mg/kg) or a high dose of CAN12 (2 mg/kg) 10 minutes before the process. There was no difference in time to cessation of bleeding or total blood loss between IgG or CAN12 treated mice (Fig. 7A, B). PAR4?/? mice have a prolonged bleeding phenotype and were used as controls. An alternative method for examining the effect of CAN12 on hemostasis was the saphenous vein model. CAN12 (2 mg/kg) experienced no effect on the bleeding time or quantity of clot formations compared to the IgG (2 mg/kg) control (Fig. 7C, D). Similar to the tail clip model, PAR4?/? mice experienced a prolonged bleeding time and fewer clot formations. Using two impartial methods, we exhibited that CAN12 treatment does not delay hemostasis in mice. Open in a separate window Physique 7 CAN12 does not impact bleeding time(A) C57BL/6 mice or PAR4?/? mice (S)-Mapracorat were anesthetized and 3 mm of the tail was slice. The time to cessation of bleeding was decided or the experiment was terminated at 10 min. (B) The total amount of blood loss.