Blocking Protein S To Treat Hemophilia A
Authors: Bologna Luca , Calzavarini Sara , Saller François , Angelillo-Scherrer Anne 
Published: October 2019

Abstract

Introduction
Protein S (ProS) is a natural anticoagulant, acting as non-enzymatic cofactor for activated Protein C (APC) in the inactivation of activated factor V (FV) and factor VIII (FVIII), and for Tissue Factor Pathway Inhibitor (TFPI) in the inhibition of activated factor X (FXa). This dual role makes ProS a key regulator of the inhibition of thrombin generation as suggested by the phenotypes of ProS-deficient patients: venous thromboembolism at adult age for heterozygous patients and massive disseminated intravascular coagulation (DIC) incompatible with life for homozygous.
Hemophilia A (HA) is an X-linked disorder caused by mutations in the F8gene, leading to deficiency or absence of FVIII, an essential component of the intrinsic pathway of blood coagulation.
HA patients suffer from spontaneous excessive bleeding episodes affecting soft tissue, joints and muscles. Currently, recombinant factor FVIII (rFVIII) is the first line of HA therapy. Unfortunately, a major drawback observed in 30% of HA patients, is the generation of inhibitory alloantibodies that abrogate rFVIII activity, rendering the therapy ineffective. Another disadvantage is the short half-life of rFVIII.

Aims
To enhance thrombin production in HA by specific blockage of ProS, thus overcoming the problems of rFVIII therapy. Lack of ProS cofactor activities on APC and TFPI might increase the generation of FXa, thus compensating for FVIII deficiency.

Methods & Results
HA mice are useful models to probe the effectiveness of procoagulant therapeutics as they recapitulate aspects of the human disease. Taking advantage from the availability of HA and ProS+/- mice, we aimed at generating ProS and FVIII double knock-out mice (HA-ProS-/-). To date ProS-/- mice were not viable and died in utero between E15.5-17.5 due to severe intracranial hemorrhages and consumptive coagulopathy. Surprisingly, by mating HA-ProS+/- mice we were able to rescue the lethal ProS-/- embryonic phenotype: HA-ProS-/-mice were viable and were found at the expected Mendelian frequency (45/192 neonates, 25%) with no increased mortality compared to HA mice.
Plasma levels of FVIII and ProS further confirmed that HA-ProS-/- mice had comparable low FVIII and extremely reduced ProS compared to HA mice (1.8±0.5 vs 93.8±15.7 % respectively P=0.001) (mean±SEM). Analysis of complete blood counts established that these animals had a normal blood cell profile. Moreover, no differences were found in FV or FIX activity and in APTT and PT between HA and HA-ProS-/- mice. A decrease in fibrinogen levels in HA-ProS-/-when compared to HA mice (1.3±0.1 vs 1.7±0.1 g/L respectively, P< 0.001) was observed.
To assess the in vivo effect of ProS-/- on HA mice hemostasis, a tail-clipping assay was used. In particular, blood loss assayed as blood volume lost over 10 min following a 4-mm tail transection significantly decreased to 158±11 uL (n=6) in HA-ProS-/- mice compared to HA and HA-ProS+/- (247 ±19 uL, n=10 and 239±11 uL, n=8 respectively, P<0.006). To further confirm that blocking ProS could improve the bleeding phenotype of HA mice, we infused 2.1 mg/kg of Ab against hProS on HA-ProS+/-mice, 2 minutes before tail clip. In line with our previous data, the reduction of ProS levels shortened blood loss to 196±10 uL (n=5, P<0.02).
As recurrent joint bleeding is the most common manifestation of HA resulting in arthropathy, we challenged HA-ProS-/- in an acute hemarthrosis model. Joint swelling assessed as the ratio between punctured and non-punctured knees showed that HA-ProS-/- reached the biggest swelling at 24h (1.09±0.03, n=7 vs 1.28±0.08, n=7 HA) but remained stable until 72h in contrast to HA mice (1.11±0.02, n=7 vs 1.48±0.13, n=7 HA; P=0.018). Moreover, a relevant decrease of joint bleeding in HA-ProS-/- mice (0.5±0.2 vs 2.67±0.17; P<0.001) as assessed by visual bleeding score, was observed.

Conclusion
These data provide the first evidence that blocking ProS has the ability to ameliorate HA as judged by in vivo improvement of bleeding phenotype in the tail clipping assay as well as in the acute hemarthrosis model. Strikingly, total FVIII deficiency does rescue the embryonic lethal phenotype of ProS-/- embryos and allows their survival without overt DIC. Together these results indicate that specific targeting of ProS favors the extrinsic pathway overcoming the intrinsic pathway deficiency, suggesting a new valuable tool for hemophilia therapy.

Disclosures:
No relevant conflicts of interest to declare.