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Effect of Plasmepsin on Venous Thrombosis in Sickle Cell Disease Jintao Wang, Paul Silaghi, Daniel T.Eitzman. University of Michigan, Ann Arbor, MI, USA

Abstract

Background Sickle cell disease (SCD) is characterized by hemolytic anemia and widespread vaso-occlusive events. Deoxygenated sickle hemoglobin leads to formation of insoluble fibers causing erythrocytes to become rigid and assume a “sickled” shape. Subsequent trapping of erythrocytes in microvasculature leads to tissue hypoxia and premature lysis of erythrocytes. SCD is associated with increased incidence of deep venous thrombosis (DVT). Mechanism(s) for this increased risk remain unclear.  Objectives This objective of this study was to determine the causal role of SCD in venous thrombosis and the effect of a hemoglobinase (plasmepsin I). Methods A femoral vein ligation model was established in mice with SCD. Morphology analysis was performed with scanning electron microscopy (SEM). Changes of free sickle hemoglobin during coagulation were monitored. A potential treatment targeting free hemoglobin in SCD using plasmepsin, a hemoglobinase, was also investigated. Results We established a femoral vein ligation model (Figure 1A). 3 days following femoral vein and branch ligation, an RBC-rich thrombus was observed in the majority of WT mice. A thrombus was observed in all SCD mice with no mortality. Scanning electron microscopy (SEM) of the thrombus showed that polyhedrocytes were compressed by fibrin and platelets in WT mice (Figure 1B), while in SCD mice, rigid elongated RBCs were intertwined together to form unique structures (Figure 1 C, D). Additionally, protruding fiber-like structures connecting sickle RBCs were common (Figure 1C). Some RBCs in SCD thrombi appeared damaged with membrane disruption (Figure 1D). Recombinant Plasmodium falciparum Plasmepsin I (PMI), a hemoglobinase, was used to digest hemoglobin and the efficacy of PMI was confirmed in SCD mouse plasma with reduction of free hemoglobin (Figure 1E). The thrombus was larger in SCD mice compared to WT mice (Figure 1F, G) and was reduced in SCD mice after PMI treatment, suggesting the contribution of sickle hemoglobin to thrombosis in SCD (Figure 1F, G). To determine the effect of sickle hemoglobin on coagulationplasma was isolated from whole blood immediately or 2 hours following a blood draw. Free plasma hemoglobin increased with the 2-hour incubation indicating release of hemoglobin from static SCD blood (Figure 2A). SCD whole blood was divided into two groups to collect plasma or serum. Free hemoglobin concentration was reduced in serum compared with plasma, indicating consumption of free sickle hemoglobin during coagulation (Figure 2B). In SCD mice, free plasma hemoglobin was also reduced after DVT (Figure 2C). To determine if sickle hemoglobin affects coagulation, normal or sickle hemoglobin samples were spiked into normal blood, and an in vitro coagulation assay was performed. Coagulation was triggered with addition of CaCl2 to EDTA anticoagulated mouse whole blood (Figure 2D). Sickle hemoglobin shortened the clotting time compared with normal hemoglobin (Figure 2E). Conclusions These findings indicate a prothrombotic effect of sickle hemoglobin that may promote venous thrombosis risk in SCD. Targeting sickle hemoglobin may be useful in the treatment of thrombotic complications of SCD.  

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