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. (Blood. 2012;120(3):671-677) IntroductionCoagulation factor XI (FXI) has an essential role both in maintaining normal hemostasis and in the pathogenesis of thrombosis. 1 Because deficiencies of factor XII (FXII), prekallikrein, and high M r kininogen are not associated with hemostatic abnormalities but FXI deficiency produces relatively mild, posttraumatic bleeding complications in ϳ 50% of affected individuals, 2-4 the more relevant pathway for activation of plasma FXI might be via feedback activation by thrombin or possibly autoactivation by FXIa. 5,6 High levels of FXI constitute a risk factor for deep venous thrombosis (DVT) 7 and cardiovascular disease in women. 8 Although inherited FXI deficiency confers no protection against acute myocardial infarction (AMI), 9 a large clinical study showed that the risk of DVT in the 10% of individuals in a population with the highest levels of FXI was Ͼ 2-fold greater than that for the remaining 90% of the population, supporting the conclusion that elevated FXI levels constitute an important risk factor for venous thrombosis. 7,8,10 There is also a reduced incidence of ischemic stroke in patients with severe FXI deficiency. 11 One study followed 600 patients with a first episode of venous thromboembolism and found an increased risk of recurrence, especially among those with increased levels of thrombin-activatable fibrinolysis inhibitor (TAFI) and FXI, 12 whereas another study showed that increased levels of TAFI, but not FXI, increased the risk of venous thromboembolism in patients with FV Leiden carrier status. 10 These studies are consistent with the results of studies demonstrating increased rabbit jugular vein thrombolysis by FXI Ab neutrali...

Role of prostacyclin signaling in endothelial production of soluble amyloid precursor protein-α in cerebral microvessels

“…12,14 Our studies are the first to demonstrate that in human BMECs, activation of the IP-cAMP signaling pathway stimulates expression of APP and production of sAPPa, an anticoagulant and neuroprotective molecule derived from non-amyloidogenic processing of APP. In contrast, deletion of IP receptor in mouse reduces APP protein levels in cerebral microvessels, indicating an important in vivo role of IP-cAMP signaling in regulation of cerebrovascular expression of APP.…”

“…12,[14][15][16] Thus, both PGI 2 and APP protect cerebral vasculature against thrombosis; however, to date the functional relationship between these two molecules has not been studied. In the present study we tested the hypothesis that activation of the IP receptor signal transduction pathway in cerebral microvessels plays an important role in metabolism of APP.…”

Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

“…Reduced TFPI levels reversed the hemorrhagic defect and prolonged survival of TF-null mice expressing a low level of human TF. 14 On the basis of the observation that members of the Kunitz-type class of inhibitors, such as protease nexin 2 and bovine pancreatic trypsin inhibitor (aprotinin), inhibit FXIa activity, 15,16 and the fact that aprotinin is a close analog of TFPI, we hypothesized that TFPI interacts with FXIa. Here we demonstrate that TFPI binds FXIa and that FXIa proteolyzes recombinant TFPI and TFPI derived from platelets and on endothelial cells.…”

Selection and Characterization of a DNA Aptamer Inhibiting Coagulation Factor XIa

“…One way to achieve this goal might be through its derivatization with bulky groups that would further block access to the active site for other substrates. Such optimization will likely be necessary before the FXIa aptamer can be tested in animal models of thrombosis and bleeding; doses of 3.6 mg/kg KPI were required to halve thrombus size in the carotid arteries of mice subjected to ferric chloride injury, and that protein demonstrated a K i four to five orders of magnitude lower for FXIa than FELIAP 43 .…”