Chymase Inhibition Resolves and Prevents Deep Vein Thrombosis Without Increasing Bleeding Time in the Mouse Model

Abstract

Background Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life-threatening cardiovascular disease in North America. Post-DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT. It has recently been shown that mast cells play a crucial role in a DVT murine model. The underlying mechanism involved in the prothrombotic properties of mast cells, however, has yet to be identified. Methods and Results C57BL/6 mice and mouse mast cell protease-4 (mMCP-4) genetically depleted mice (mMCP-4 knockout) were used in 2 mouse models of DVT, partial ligation (stenosis) and ferric chloride-endothelial injury model of the inferior vena cava. Thrombus formation and impact of genetically repressed or pharmacologically (specific inhibitor TY-51469) inhibited mMCP-4 were evaluated by morphometric measurements of thrombi immunochemistry (mouse and human DVT), color Doppler ultrasound, bleeding times, and enzymatic activity assays ex vivo. Recombinant chymases, mMCP-4 (mouse) and CMA-1 (human), were used to characterize the interaction with murine and human plasmin, respectively, by mass spectrometry and enzymatic activity assays. Inhibiting mast cell-generated mMCP-4, genetically or pharmacologically, resolves and prevents venous thrombus formation in both DVT models. Inferior vena cava blood flow obstruction was observed in the stenosis model after 6 hours of ligation, in control- but not in TY-51469-treated mice. In addition, chymase inhibition had no impact on bleeding times of healthy or DVT mice. Furthermore, endogenous chymase limits plasmin activity in thrombi ex vivo. Recombinant mouse or human chymase degrades/inactivates purified plasmin in vitro. Finally, mast cell-containing immunoreactive chymase was identified in human DVT. Conclusions This study identified a major role for mMCP-4, a granule-localized protease of chymase type, in DVT formation. These findings support a novel pharmacological strategy to resolve or prevent DVT without affecting the coagulation cascade through the inhibition of chymase activity.

Keywords: TY‐51469; chymase; deep vein thrombosis; mouse; mouse mast cell protease‐4 knockout; plasmin.

Figure 1. Short‐term single dose of TY‐51469 protects against deep vein thrombosis (DVT) formation in a dose‐dependent manner without bleeding adverse effects.

A, Dose‐dependent effect on the weight (mg; i) and length (mm; ii) of thrombi obtained 24 hours after DVT from inferior vena cava–ligated male wild‐type (WT) mice treated with 0.1, 1, and 10 mg/kg chymase inhibitor TY‐51469 1 hour post‐ligation. B, Dose‐dependent impact of chymase inhibitor TY‐51469 on the thrombus incidence in WT mice. C, Bleeding times in male mice, as assessed by a tail‐bleeding assay, reveal no difference between WT mice with or without 10 mg/kg TY‐51469, in mouse mast cell protease‐4 (mMCP‐4) knockout (KO) mice, or DVT WT mice with or without 10 mg/kg TY‐51469. The 300 U/kg heparin‐treated WT mice were used as positive controls. Each group of mice represents the mean±SEM (n=6–13). *P<0.05, **P<0.01, and ***P<0.001 compared with WT control group (asterisks directly placed over the error bar of each column); comparisons were also made between treated groups.

Figure 2. Short‐term chymase inhibition reinstates vena cava blood flow in deep vein thrombosis (DVT) mice.

Doppler color blood flow mapping of the inferior vena cava (IVC) and adjacent abdominal aorta (AA) in 8‐week‐old female mice. TY‐51469 (10 mg/kg) was administered intraperitoneally 1 hour after ligation. The image illustrates the apical view of the IVC and AA with the ultra‐high‐frequency linear array transducer (or scan head) tilted at an angle of 35±10 degrees from the posterior end of the mouse. DVT‐induced IVC blood stasis in control WT mice 6 hours after ligation. WT mice treated with TY‐51469, 1 hour after ligation, show no variation in IVC and AA blood velocities when compared with the same animals before ligation. Control WT, unlike TY‐51469–treated DVT mice, also showed compensatory increases in AA blood flow, 6 and 24 hours after ligation. The images are representative of 6 to 8 independent experiments. Bar=1 mm. WT indicates wild type.

Figure 3. Chymase inhibition protects against and resolves deep vein thrombosis in male mice in 2 murine models.

Male wild‐type (WT) mice were treated with 10 mg/kg TY‐51469 (intraperitoneally) (48 hours before and 6 or 24 hours after ligation), and mouse mast cell protease‐4 (mMCP‐4) knockout (KO) mice were subjected to an inferior vena cava stenosis for 24 (A) or 48 (B) hours. Morphometric parameters of thrombi were measured as weight (mg; i) and length (mm; ii). C, Weight of thrombi obtained after the ferric chloride insult model, 30 minutes after damage, from male WT mice treated with 10 mg/kg TY‐51469 and mMCP‐4 KO mice. Each group represents the mean±SEM (n=6–12). ***P<0.001 compared with the WT control group.

Figure 4. Mouse mast cell protease‐4 (mMCP‐4) immunoreactivity and toluidine blue (TB) mast cell staining in the inferior vena cava (IVC) of deep vein thrombosis mice.

A, TB staining of a nonligated IVC from a wild‐type (WT) mouse. B, mMCP‐4 immunoreactivity of a nonligated IVC from a WT mouse. C, TB staining of a ligated vein from a WT mouse. D, mMCP‐4 immunoreactivity in a ligated IVC from a WT mouse (vessel lumen). E, TB staining of a ligated IVC from an mMCP‐4 knockout mouse. F, Absence of mMCP‐4 immunoreactivity in the same ligated IVC as in E. Dilution of the mMCP‐4 antibody: 1×400. Red arrows: mast cells; black arrows: mMCP‐4 immunoreactivity. The images are representative of 3 independent experiments. Bar=50 μm.

Figure 5. Mouse mast cell protease‐4 (mMCP‐4) reduces plasmin activity within thrombi of deep vein thrombosis mice.

A, Plasmin‐like activity was measured in vitro using mouse purified plasmin incubated for 24 hours at 37°C with recombinant mMCP‐4 with or without increasing concentrations of the chymase inhibitor TY‐51469. B and C, Ex vivo enzymatic activity in thrombi harvested 24 or 48 hours after ligation from wild‐type (WT) mice. WT thrombi were treated with vehicle or 10 μM TY‐51469 before the reading. B, Plasmin activity was measured as 7‐amino‐4‐methylcoumarin (AMC)–specific cleavage (nM) of the fluorogenic substrate D‐Ala‐Leu‐Lys‐AMC. C, Chymase activity was measured as AMC‐specific cleavage of the fluorogenic substrate Suc‐Leu‐Leu‐Val‐Tyr‐AMC. Each bar represents the mean±SEM (n=6–8 independent experiments). **P<0.01, ***P<0.001 compared with the plasmin only group or WT nontreated group. Max. indicates maximum.

Figure 6. Specific chymase immunostaining is found within human venous thrombi, and human recombinant chymase (rCMA‐1) cleaves/inactivates human plasmin.

A, Human deep vein thrombosis (DVT) samples show chymase immunoreactivity (brown) and chymase‐positive mast cells inside the thrombi. Dilution of the CMA‐1 antibody: 1×200; black arrows: CMA‐1 immunoreactivity. The image is representative of independent experiments performed with venous thrombi extracted from 3 patients. Bar=20 μm. B, Plasmin‐like activity was measured in vitro with purified human plasmin incubated for 24 hours at 37°C with rCMA‐1 with or without increasing concentrations of the chymase inhibitor TY‐51469. Each bar represents the mean±SEM (n=8–13). *P<0.05, ***P<0.001. C, Identification of rCMA‐1 cleavage sites in human plasmin using trypsin digestion and cleavage product identification by liquid chromatography–tandem mass spectrometry, represented as arbitrary intensity (4 samples tested). D, Schematic representation of human plasmin/plasminogen. rCMA‐1 cleavage sites are indicated by black arrows. Created with BioRender.com. AMC indicates 7‐amino‐4‐methylcoumarin; AU, arbitrary unit; and Max., maximum.