The kunitz protease inhibitor domain of protease nexin-2 inhibits factor XIa and murine carotid artery and middle cerebral artery thrombosis

Abstract

Coagulation factor XI (FXI) plays an important part in both venous and arterial thrombosis, rendering FXIa a potential target for the development of antithrombotic therapy. The kunitz protease inhibitor (KPI) domain of protease nexin-2 (PN2) is a potent, highly specific inhibitor of FXIa, suggesting its possible role in the inhibition of FXI-dependent thrombosis in vivo. Therefore, we examined the effect of PN2KPI on thrombosis in the murine carotid artery and the middle cerebral artery. Intravenous administration of PN2KPI prolonged the clotting time of both human and murine plasma, and PN2KPI inhibited FXIa activity in both human and murine plasma in vitro. The intravenous administration of PN2KPI into WT mice dramatically decreased the progress of FeCl(3)-induced thrombus formation in the carotid artery. After a similar initial rate of thrombus formation with and without PN2KPI treatment, the propagation of thrombus formation after 10 minutes and the amount of thrombus formed were significantly decreased in mice treated with PN2KPI injection compared with untreated mice. In the middle cerebral artery occlusion model, the volume and fraction of ischemic brain tissue were significantly decreased in PN2KPI-treated compared with untreated mice. Thus, inhibition of FXIa by PN2KPI is a promising approach to antithrombotic therapy.

Figure 1

The inhibitory effect of PN2KPI in human and murine plasma. (A) The clotting time of mouse plasma is prolonged with externally added PN2KPI. (B) FXI activity in human plasma is inhibited by externally added PN2KPI and inhibitory activity is saturated at PN2KPI concentrations > 10μM. (C) Mouse FXI activity is ∼ 75% inhibited by human KPI and inhibitory activity is saturated at PN2KPI concentrations > 10μM.

Figure 2

Inhibitory effect of PN2KPI on thrombus development in the FeCl₃-induced mouse carotid artery thrombosis model. Thrombus formation is recorded by fluorescence microscopy as described in “Carotid artery thrombosis model.” The image of thrombus growth was captured from video clips recorded at different time points. Thrombus growth (A) in a representative untreated mouse and (B) in a PN2KPI-treated mouse was recorded. (C) Thrombus growth, represented by the artificial fluorescence intensity unit (AFU), was measured at various time points (1, 3, 5, 7,10,15, 20, 30, and 40 minutes) in 6 control (□) and 6 PN2KPI-treated (○) mice. Results shown are mean values ± SEM. *Significant differences with P < .05.

Figure 3

Pathologic sections of carotid artery thrombosis. (A) The histologic section of a normal carotid artery, stained with hematoxylin and eosin; (B-C) FeCl₃-treated vessels. Section of (B) a control mouse and (C) from a mouse treated with PN2KPI.

Figure 4

Coronal sections of the brains of mice treated either with vehicle or PN2KPI. The ischemic region of brain is indicated by the pale area within the right hemisphere of animals treated with vehicle (A) or PN2KPI (B) 24 hours after right middle cerebral artery occlusion. (C-D) The comparison of the stroke volume and stroke fraction between the 2 groups (n = 6 in each group, P < .05). The bars represent the mean ± SEM of stroke volume values. *Significant differences with P < .05 compared with the control group.