Thrombomodulin and Endothelial Dysfunction in Sickle Cell Anemia
Authors: Ruiz Maria Armila , Shah Binal N. , Han Jin , Raslan Rasha , Gordeuk Victor R , Saraf Santosh L. 
Published: November 2019

Thrombomodulin (THBD) is a transmembrane protein that regulates endothelial function by 1) binding thrombin and inhibiting its interaction with fibrinogen, 2) augmenting protein C activation, and 3) down-regulating complement activation by inactivating C3a and C5a (PMID 29866818). Reduced THBD function has been implicated in several vasculopathies (PMID 29866818). Oxidative damage increases cleaved non-functional THBD in circulation and increased circulating non-functional THBD predicts the severity of organ damage in thrombotic microangiopathies (PMID 11190905). Vascular endothelial dysfunction is a hallmark feature of sickle cell disease (SCD) that leads to acute and chronic organ damage and may, in part, be mediated by hemolysis (PMID 28248201). Hemolysis leads to release of cell-free hemoglobin which may lead to vasculopathy through consumption of NO, activation of TLR4 pathways, and direct oxidative damage. We investigated whether decreased THBD activity may be implicated in the pathophysiology of SCD vasculopathy in endothelial cells, transgenic sickle mice, and in a prospective cohort of patients with sickle cell anemia.
In vitro: We exposed endothelial cells (EA.hy926, ATTC® CRL-2922TM; Manassas, VA) to incremental doses of cell-free hemoglobin. With higher doses of cell-free hemoglobin we observed reduced surface endothelial cell THBD activity at 6 hours of incubation, assessed by cleavage of chromogenic substrate for activated protein C in the presence of thrombin (Figure 1A). In conjunction with the reduced endothelial THBD activity, there were increased concentrations of cleaved THBD in the supernatant by ELISA (R&D Systems, Minneapolis, MN) (Figure 1B).
Transgenic sickle mice: At 6 months of age, transgenic sickle mice (Townes model, Jackson Laboratory; Bar Harbor, Maine) had higher plasma concentrations of cleaved non-functional THBD versus hemoglobin AA mice (Figure 2A). Furthermore, staining of the glomerular microvasculature demonstrated decreased endothelial-bound THBD (Figure 2B).
Patients with sickle cell anemia: We evaluated whether plasma concentrations of cleaved non-functional THBD are predictive of acute multiorgan failure syndrome (PMID 8109600) in a cohort of 103 SCD patients recruited into a longitudinal kidney cohort study. Clinical and laboratory variables and plasma samples were obtained at the time of enrolment and the patients were monitored prospectively for acute multiorgan failure syndrome. Hemoglobinuria was defined by urine dipstick positive for blood with &lt; 2 red blood cells/high power field. The median age of this cohort was 35 years (interquartile range, 28 - 44 years), 46% were female, 87% had hemoglobin SS genotype, and 47% were on hydroxyurea at the time of enrolment. In cross-sectional analysis, plasma THBD concentrations were greater in patients with hemoglobinuria, a marker of intravascular hemolysis-derived cell-free hemoglobin in circulation exceeding scavenging capability and filtering through the glomerulus, versus without hemoglobinuria (6.1 ± 0.6 µg/mL vs. 3.6 ± 0.4 µg/mL, P = 0.004) (Figure 3A). With a median follow up of 5.5 years (interquartile range, 1.4 - 5.9 years), 18 (17%) SCD patients had a multiorgan failure event. SCD patients with a multiorgan failure event were older (43 vs. 34 years, P = 0.01) but without significant differences in sex, SCD genotype, or hydroxyurea therapy. After adjusting for age, baseline THBD concentrations predicted a greater risk for multi-organ failure syndrome (log-transformed OR 4.0, 95% CI: 1.2 - 13.3; P = 0.01) (Figure 3B).
In conclusion, circulating non-functional THBD, a protein that normally functions to reduce vasculopathy when bound to the endothelium, is increased after endothelial cell exposure to incremental doses of hemoglobin in vitro, in hemoglobin SS vs. AA mice, and in SCD patients with versus without hemoglobinuria. Furthermore, circulating THBD is a biomarker that predicted the risk for multi-organ failure syndrome on longitudinal follow up in SCD patients. Future studies investigating the role of THBD in SCD vasculopathy may help improve our understanding for the catastrophic multiorgan failure syndrome and therapies to augment endothelial cell THBD function may guide future intervention practices.
Figure 1

Disclosures
Gordeuk: Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding. Saraf:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.