Specific inhibition of extracellular CyPA affects platelet and monocyte functions in vitro and in vivo

S. von Ungern-Sternberg1, B. Walker-Allgaier1, S. Vogel1, E. Kremmer2, O. Borst1, T. Billar3, A. May1, M. Gawaz1, P. Seizer1 (1Tubingen, Germany, 2Munich, Germany, 3Pittsburgh, USA)

Date: 18.02.2017,
Time: 10:00 - 11:15

Objective: Cyclophilin A (CyPA) is an ubiquitously expressed intracellular chaperon protein and. Under inflammatory conditions CyPA is secreted and binds to its receptor EMMPRIN (CD147). Due to the binding between CyPA and EMMPRIN platelets get activated. CyPA is involved in the pathophysiology of several inflammatory diseases, such as myocarditis, myocardial infarction and atherosclerosis. At present there is no specific inhibitor targeting extracellular CyPA without affecting other extracellular cyclophilins or the intracellular CyPA. In this study we developed an antibody-based inhibitor of CyPA that specifically neutralizes extracellular CyPA in vitro and in vivo.

Methods: Mice and rats were immunized with a peptide containing the EMMPRIN binding side and various antibody clones were selected and purified. At first all antibodies were tested for their binding capacity on recombinant and cell-derived CyPA in Western Blot and their functional activity in a migration assay. The best antibody, 8H7-mAb, was chosen for further experiments. At first 8H7-mAb was tested in CyPA-derived modified Boyden chamber migration assays in vitro and in vivo in a CyPA-induced peritonitis model. Then the anti-thrombotic functions of 8H7-mAb were analyzed by measuring p-selectin expression on platelets by flow cytometry and thrombus formation in vitro and in vivo in a FeCl3 model. Next, we analyzed the functional effects of 8H7-mAb is a trauma/hemorrhagic shock mice model.

Results: According to the results of the Western Blot and migration assay the clone 8H7-mAb was chosen for further experiments. In a binding assay we could show that there is a specific binding of 8H7-mAb on recombinant CyPA compared to IgG control and that 8H7-mAb is only detecting CyPA in Western blot assay and not CyPB. We found that 8H7-mAb reduced the CyPA-induced migration of monocytes/macrophages in vitro and in vivo vivo (CyPA-induced peritonitis). 8H7-mAb is able to reduce the thrombus formation on a collagen matrix in vitro and in a FeCl3 model in vivo. Interestingly, 8H7-mAb treated shock mice had significantly less platelet aggregation of circulating platelets and less platelet and monocyte infiltrates in the liver compared to IgG control treated mice.

Conclusion: Our study provides evidence that the novel CyPA-inhibitor 8H7-mAb affects CyPA-induced thrombosis and thrombo-inflammation in vitro and in vivo.