Recombinant chymase inhibits fibrinolysis induced by endogenous plasmin in clotted human blood

Laurence Vincent ¹ Catherine Lapointe ² Patrick P McDonald ³ Christos Rammos ⁴ Tienush Rassaf ⁴ Maria Kollenberger ⁵ Hanna Tinel ⁵ Stefan Heitmeier ⁵ Pedro D'Orléans-Juste ^1*

• ¹ Université de Sherbrooke, Sherbrooke, Canada
• ² University of North Carolina Hospitals, Chapel Hill, North Carolina, United States
• ³ Insmed Inc., Bridgewater, New Jersey, United States
• ⁴ Essen University Hospital, Essen, North Rhine-Westphalia, Germany
• ⁵ Bayer (Germany), Wuppertal, North Rhine-Westphalia, Germany

Background: Our group recently reported that chymase, a serine protease synthetized and released by mast cells, plays a pivotal role in thrombi stabilization in murine deep vein thrombosis (DVT) models, by interfering with the fibrinolytic properties of endogenous plasmin within the thrombi. However, the impact of mast cell-derived chymase on endogenous plasmin activity in human blood clots has yet to be explored.The antifibrinolytic properties of human recombinant chymase (rCMA-1) were investigated using an in vitro thrombolysis assay based on halo-shaped human blood clots. In addition, a fluorogenic assay was used to detect chymase activity in human thromboembolic biopsies. In both assays, the activity of human chymase was validated using a specific chymase inhibitor, fulacimstat (BAY 1142524). Results: Although rapidly neutralized in plasma, rCMA-1 remains active within the local microenvironment of a blood clot, inducing resistance to endogenous plasmin-mediated fibrinolysis in the presence of recombinant tissue plasminogen activator (tPA). This leads to a concentration-Supprimé: dependent decrease in clot lysis. The plasmin-inactivating properties of rCMA-1 were inhibited by fulacimstat, resulting in an accelerated clot dissolution. The pro-fibrinolytic effects of fulacimstat in human blood clots were reversed by a plasminogen/plasmin inhibitor, BAY 1214237. Finally, fulacimstat-sensitive chymase activity was identified in human thrombus clots, emphasizing the potential role the mast cell-derived serine protease in human blot clot stabilization under pathological conditions.These in vitro experiments with human whole blood suggest that mast cell-derived chymase impairs blood clot resolution by interfering with the fibrinolytic activity of endogenous intraclot plasmin. Our findings provide evidence that recombinant mast cell chymase interferes with endogenous plasmin activity in human whole blood clots in vitro and support the potential of chymase inhibitors, such as fulacimstat, as fibrinolytic agents for thrombotic and thromboembolic disorders.