Blocking Protein S Improves Hemostasis in Hemophilia a and B

Introduction&Aim

Hemophilia A and B are X-linked disorders caused by an absence or a reduction in coagulation FVIII and respectively, FIX. Patients with hemophilia often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis.

Here, we investigated whether targeting protein S (PS) could promote hemostasis in hemophilia by re-balancing coagulation. PS is a natural anticoagulant, acting as non-enzymatic cofactor for activated protein C (APC) in the inactivation of FVa and FVIIIa, and for tissue factor pathway inhibitor (TFPI) in the inhibition of FXa. This dual role makes PS a key regulator of the inhibition of thrombin generation (TG).

Methods & Results

Hemophilic (F8-/- or F9-/-) Pros1+/- mice were intercrossed. F8-/-Pros1-/- and F9-/- Pros1-/- mice were viable and found at the expected Mendelian frequency with no increased mortality compared to hemophilic mice.

F8-/-Pros1-/- mice did not show DIC onset neither an increased mortality once challenge with tissue factor (TF). Ex vivo evaluation of TG potential showed that F8-/-Pros1-/- mice were APC-resistant, they had an improved low TF induced-TG and a 20% more dense fibrin network with larger fibrin fibers diameter as compared to F8-/-mice. Comparable results were found in human HA plasma where blocking PS raised TG even in the presence of high inhibitor titer.

To assess the in vivo effect of PS inhibition on HA mice hemostasis, two tail- clipping (TC) assays were used. In both, mild TC model (2 mm cut) and severe TC (4 mm cut), blood loss significantly decreased in F8-/-Pros1-/- compared to F8- /- mice (mild TC: 407±21 vs 661±29ul, p<0.0001; severe TC:173±14 vs 249±24ul, p<0.05). In addition, the infusion of anti-hPS antibody on F8-/-Pros1+/- mice, reduced the blood loss compared to F8-/-Pros1+/- mice infused with an isotype IgG (196±10 vs 302±25ul, p=0.005).

As recurrent joint bleeding is the most common manifestation of HA, we challenged F8-/-Pros1-/- mice in an acute hemarthrosis (AH) model. Joint swelling 72 hours after injury was reduced in F8-/-Pros1-/- compared to F8-/- (0.11±0.03 vs 1.02±0.07mm, p<0.0001, n=10). These results were also confirmed by s.c. injection of anti-hPS antibody (0.46±0.0 vs 0.78±0.09mm isotype IgG, p=0.02, n=9) and by i.v. injection of PS-siRNA prior to AH challenge in F8-/-Pros1+/- (0.29±0.09 vs 0.92±0.12mm siRNA control, p=0.03, n=5) and F8-/- mice(0.35±0.08 vs 0.78±0.09mm siRNA control, p=0.05, n=5). Similar results were obtained in F9-/-Pros1-/- mice. Histological analysis of joint showed joint bleeding reduction in F8-/-Pros1-/- compared to F8-/- and an increased fibrin staining comparable to F8+/+ mice.To understand the intra-articular hemostatic effect of blocking PS, joint sections were stained for TFPI. Preliminary results indicate a massive staining in the synovia of F8-/- mice, while F8-/-Pros1-/- and F8+/+ mice present a less intense signal. These data suggest that the intra-articular space is a modulable anticoagulant environment. Human HA joint tissues were then analyzed for both PS and TFPI. A strong signal was found for TFPI and PS in the synovial lining and sublining layers of HA patients on demand (n=7). Interestingly, PS and TFPI stainings were remarkably decreased in HA patients under prophylaxis (n=5). Joint section from osteoarthritis patients (n=7) did not show an intense staining for TFPI and PS similarly to hemophilic patients under prophylaxis.

To understand the improved phenotype of F8-/-Pros1-/- after AH, the function of macrophages were investigated. At the steady state, F8-/-Pros1-/- presented significantly 2-fold increased levels of inflammatory macrophages (M1) than in F8-/- mice. In addition bone marrow derived macrophages from F8-/-Pros1-/- exhibit 10-fold higher RBC phagocytic activity than F8-/- . Preliminary results indicate an increase of a monocyte attractant MCP-1 level, in knee lavage after AH in F8-/-Pros1-/- than F8-/- mice.

Conclusion

These data provide the first evidence that blocking PS has the ability to ameliorate hemophilia as judged by in vivo improvement of bleeding phenotype in the TC assay as well as in the AH model, suggesting a new valuable tool for hemophilia therapy. In addition, the modulable presence of PS and TFPI in human and mice joints is a novel pathophysiological aspect of hemarthrosis and constitutes a potential therapeutical target.