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Updated: May 16, 2026

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors
Published on: July 17, 2020
P1 and P2' site mutations convert protease nexin-2 from a factor XIa inhibitor to a plasmin inhibitor
Duraiswamy Navaneetham1, Wenman Wu, Hongbo Li
1The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Abstract:
The kunitz protease inhibitor domain of PN2 (PN2KPI) is a potent and specific inhibitor (K(i) 0.5-2 nM) of factor XIa (FXIa) and inhibits cerebrovascular thrombosis in mice. To determine whether the antithrombotic properties of PN2KPI arise from its FXIa-inhibitory activity, we have now prepared mutant forms of PN2KPI. Mutations at the P1 (Arg(15)) site in combination with P2' (Met(17)) mutations profoundly affect inhibition of FXIa, plasmin, kallikrein, factor Xa and thrombin. The mutant proteins PN2KPI-R(15)K, -M(17)K, -R(15)K,M(17)K and -R(15)K,M(17)R lost inhibitory activity against FXIa (K(i) 34, 94, 3081 and 707 nM, respectively) and kallikrein (no inhibition) and gained inhibitory activity against plasmin (K(i) 108, 7, 8 and 8 nM, respectively). The intravenous administration of rPN2KPI into mice dramatically decreased thrombus formation in a murine model of FeCl(3)-induced carotid injury, whereas rPN2KPI-R(15)K,M(17)K failed to inhibit thrombus formation. Molecular modelling studies showed that fine structural variations explain the observed functional differences in FXIa and plasmin inhibition. PN2KPI has potent antithrombotic activity due to its specific FXIa anticoagulant activity, whereas PN2KPI-R(15)K,M(17)K and PN2KPI-R(15)K,M(17)R have potent antifibrinolytic (antiplasmin) activity without anticoagulant or antithrombotic activity.
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