Demonstration of cathepsins K, L, and S fibrinolytic activity leads to further investigation of contributory roles in disrupting vascular hemostasis, or breakdown of fibrin-based engineered vascular constructs where non-plasmin mediated fibrinolysis must be considered.

Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin-based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis. To further explore this, we used two independent computational technqiues, molecular docking and bioinformatics sequence analysis (PACMANS), to predict potential binding interactions and sites of hydrolysis between cathepsins K, L, and S and fibrinogen. By comparing the results from these two objective, computational methods, it was determined that cathepsins K, L, and S do bind and cleave fibrinogen α, β, and γ chains at similar and unique sites. These differences were visualized experimentally by the unique cleaved fibrinogen banding patterns after incubation with each of the cathepsins, separately. In conclusion, human cysteine cathepsins K, L, and S are a new class of proteases that should be considered during fibrin(ogen) degradation studies both for disease processes where coagulation is a concern, and also in the implementation and design of bioengineered systems.

Sickle cell disease (SCD) is the most common hereditary blood disorder in the United States. SCD is frequently associated with osteonecrosis, osteoporosis and osteopenia and other bone related complications such as vaso-occlusive pain, ischemic damage, osteomyelitis, and bone marrow hyperplasia known as sickle bone disease (SBD)1,2. Previous SBD models have failed to distinguish the age- and sex-specific characteristics of bone morphometry. In this study, we use the Townes mouse model of SCD to study the pathophysiological complications of SBD in both SCD and sickle cell trait. Changes in bone microarchitecture and bone development were assessed by high-resolution quantitative micro-computed tomography (microCT) and the 3D reconstruction of femurs from male and female mice. Our results indicate that SCD causes bone loss and sex-dependent anatomical changes in bone. Particularly, SCD female mice are prone to trabecular bone loss while cortical bone degradation occurs in both sexes. Additionally, we describe the impact of genetic knockdown of cathepsin K and E-64 mediated cathepsin inhibition on SBD.