Increased Hypercoagulation Markers in a Humanized Sickle Cell Mouse Model Caused By Activated Cathepsin-Mediated Fibrinogenolysis

Sickle cell disease (SCD) affects approximately 100,000 people in the United States. Globally 300,000 babies are born with SCD. Coagulation is chronically activated in SCD, and etiology is not understood, but can contribute to sickle pain crisis. We need to understand mechanisms underlying chronic coagulation in SCD to develop anticoagulant therapies for the SCD population. During coagulation, thrombin cleaves fibrinopeptide A (FpA) and B from fibrinogen. People with SCD have increased FpA in plasma, possibly contributing to chronic coagulation activation. Computational docking identified cathepsin K (catK) has cleavage sites on fibrinogen near a Gly-Pro-Arg sequence, releasing FpA. Inflammation of SCD causes increased catK production by vascular cells, and we hypothesize that increased plasma levels of FpA in SCD is due to catK activity. We assessed amounts of active cathepsins and FpA in plasma of humanized sickle cell mice: non-sickle (AA), sickle cell trait (AS), and sickle cell anemia (SS) genotypes. To determine if cysteine cathepsins were causal agents of increased FpA, mice were injected with E64, cathepsin inhibitor, from ages 1 to 3 months, hypothesizing that E64 will reduce catK activity, and generate less FpA. From zymography, untreated 1 month SS mice had twice as much active cathepsins compared to 1 month AA and AS mice. Plasma was elevated in 3 month SS mice compared to 1 month SS mice. SS male mice had more active cathepsins than SS females. Preliminary FpA ELISAs show untreated SS mice may contain higher amounts of circulating FpA compared to AA and AS mice, but E64 treatment reduced amounts of active cathepsins and FpA. These data support that SCD mediates increased levels of active catK in the plasma which cleaves FpA from fibrinogen. We are among the first to show this in an animal model, which helps us understand chronic coagulation activation in SCD.