Diminished Multi-Organ Pathologies and Inflammation Associated With Sickle Cell Disease In Mice With Genetically Limited Prothrombin Levels
Authors: Arumugam Paritha , Mullins Eric S. , Shaw Maureen A , Shanmukhappa Kumar , Loberg Anastacia , Rizvi Tilat , Wansapura Janaka , Witte David , Malik Punam , Degen Jay L. 
Published: October 2019

Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusions, chronic inflammation and an oxidative state, with progressive and widespread organ dysfunction and reduced life expectancy. Systemic coagulation system activation has been well documented in both SCD patients and animal models of SCD. However, the precise causal relationship, if any, between hemostatic factors and SCD pathogenesis has remained uncertain. Here, we test the hypothesis that thrombin-mediated proteolysis is a key mediator of the end-organ pathologies in SCD by evaluating the phenotypic consequences of imposed deficits in prothrombin in the well-characterized humanized murine model of SCD, Berkeley sickle mice (HbS mice). Prothrombin (fII) specific anti-sense oligonucleotide (ASO) gapmers (ISIS Pharmaceutics) were used to pharmacologically suppress circulating plasma fII levels to less than 10% of normal in cohorts of HbS mice starting at 3 weeks of age. Kaplan-Meier analysis revealed a major survival benefit in HbS mice when hepatic fII expression and circulating fII levels were selectively lowered, with ∼90% of fIILow mice surviving to 15 weeks of age as compared to just 60% of HbS mice treated with control ASO gapmer (P<0.04). As a complementary approach to better define the importance of fII in SCD pathobiology, we extended these studies to explore SCD in previously described fIIlox/- mice with a life-long, genetic deficit in circulating prothrombin. These animals carry ∼10% the normal level of fII with a resulting INR of 1.7, yet exhibit normal developmental and reproductive success, and never develop spontaneous bleeding events. Here, we utilized a hematopoietic stem cell transplantation (HSCT) approach using HbS stem cell donors to generate HbS/fIIlox/- and HbS/fIIWT cohorts. These transplanted mice were followed for a year after HSCT to examine the inflammatory and hemostasis parameters and organ pathologies and function. There was no significant difference in the RBC indices between HbS/fIIWT and HbS/fIIlox/- mice. However, HbS/fIIlox/- mice exhibited significantly reduced neutrophil counts (4.9 ± 0.3 vs.8.7 ± 1.2, P<0.01) and platelet counts (984 ± 66 vs. 1208 ± 55, P<0.01) compared to HbS/fIIWT mice. Furthermore, circulating IL-6, sVCAM, and D-dimer levels were significantly lower in HbS/fIIlox/- mice. Taken together, these data indicate that baseline inflammation was decreased as a consequence of reduced fII. Lower fII levels in HbS/fIIlox/- mice also resulted in significantly improved organ pathologies, including reduced infarctions in the liver, reduced inflammation in the lungs, remarkably improved kidney glomerular and tubular pathology, and decreased right ventricular (RV) hypertrophy. This was accompanied with a much improvement in organ function. Here, there was significantly improved renal function in HbS/fIIlox/- mice relative to HbS/fIIWT animals was reflected by a markedly reduced albumin (477 ± 96 vs. 1694 ± 118, P<0.0001) and improved urine concentrating ability (urine osmolality 1731 ± 185 vs. 1208 ± 169; P<0.01). Similarly, diminished hepatic damage was reflected in reduced transaminitis. Pulmonary hypertension also appeared to be significantly ameliorated in the HbS/fIIlox/- animals relative to HbS/fIIWT mice as reflected in decreased pulmonary artery smooth muscle hyperplasia in the lungs, and reduced RV hypertrophy by cardiac MR. Left ventricular diastolic dysfunction was also significantly ameliorated as reflected in reduced end diastolic volumes by cardiac MR. Notably, the median INR values in HbS/fIIlox/- and HbS/fIIWTmice, were 2.0 and 1.2, respectively, and not associated with any spontaneous hemorrhage in either group. Therefore, these data suggest that long-term therapeutic intervention with direct thrombin inhibitors, fXa inhibitors, or warfarin, even at doses that would only modestly increase INRs, could significantly reduce organ dysfunction and favorably affect the long term outcome in patients with SCD. In summary, our data supports the hypothesis that thrombin significantly contributes to inflammation and end organ pathologies in SCD, and may represent a novel therapeutic target to improve renal, cardiac and pulmonary functions, thereby reducing the morbidity and mortality in this patient population.

Arumugam P and Mullins E contributed equally. Malik P and Degen J are Co-corresponding authors.

Disclosures:

Mullins: Baxter : Consultancy.