Targeting anticoagulant protein S to achieve hemostasis in hemophilia

S. Calzavarini1, L. Bologna1, R. Prince1, M. Manetti2, D. Melchiorre2, I. Rosa2, N. Dewarrat1, P. Amini1, J.A. Fernandez3, C. Quarroz1, Y. Matsumura4, J.A. Kremer Hovinga1, J.H. Griffin3, H-U. Simon1, L. Ibba Maneschi2, F. Saller5, A. Angelillo-Scherrer1 (1Bern, Switzerland, 2Florence, Italy, 3La Jolla, USA, 4Chiba, Japan, 5Paris, France)

Date: 16.02.2017,
Time: 11:00 - 12:00

Objective: To rebalance hemostasis improving the bleeding phenotype in hemophilia A (HA) and B (HB) by protein S (PS) inactivation. PS is an anticoagulant acting as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation (TG).

Methods: Mouse matings were set to get F8-/-Pros1-/- and F9-/-Pros1-/-. Offspring was investigated with venous thrombosis (VTE), tail clipping (TC) and acute hemarthrosis (AH) models. Thrombin generation (TG) and joint immunostaining were performed in HA and HB patients.

Results: F8-/-Pros1-/- were viable and found at expected Mendelian frequency, while F9-/-Pros1-/- mice were less represented (14% vs 25%). Both hemophilic Pros1-/- mice did not display DIC neither an increased mortality in VTE model. TG showed that F8-/-Pros1-/- mice were APC and TFPI resistant and capable to generate a denser network of higlhy branched fibrin fibers compared to F8-/-. Comparable results were found in human HA plasma where blocking PS raised TG even in the presence of inhibitor. In mild and severe TC model, blood loss significantly decreased in F8-/-Pros1-/- compared to F8-/- mice (mild TC: 407±21 vs 636±36ul, p<0.0001; severe TC:173±14 vs 274.3±37ul, p<0.05). In addition, anti-hPS antibody infusion in F8-/-Pros1+/- mice reduced blood loss compared to F8-/-Pros1+/- mice which received an isotype IgG (196±10 vs 308±30ul, p=0.008). In AH model, F8-/-Pros1-/- mice displayed less joint swelling than F8-/- mice (0.11±0.03 vs 0.98±0.12mm, p<0.0001, n=10). These results were confirmed by PS-siRNA injection prior to AH challenge in F8-/- (0.35±0.08 vs 0.78±0.09mm siRNA control, p=0.03, n=3) mice. Similar results were obtained in F9-/-Pros1-/- mice. Immunostaining of joint sections showed PS and TFPI expression in the intra-articular space. A strong signal was found for TFPI and PS in the synovia of HA patients on demand. PS and TFPI stainings were remarkably decreased in HA under prophylaxis, and comparable to osteoarthritis patients. HB patients display less TFPI and PS synovial expression than HA patients.

Conclusion: Targeting PS ameliorates hemophilia as shown by in vivo limited bleeding in TC and AH models. In addition, the modulable presence of PS and TFPI in human joints is a novel pathophysiological aspect of hemarthrosis and constitutes a potential therapeutical target.