Sickle Cell Disease

Sickle cell disease (SCD) effects millions children and adults globally and about 100,000 in the United States. The average life expectancy of a SCD patient at birth is 42-47 years in United States, compared to about 79 years for the general U.S. population. SCD profoundly effects the quality of life of patients with the disease. The therapeutic options for SCD include hydration, blood transfusions, hydroxyurea, L-glutamine, crizanlizumab and voxelotor. SCD patients often develop sickle crisis and vasoocclusive disease. In its most devastating form, arterial thrombosis leads to cerebral infarction and stroke as early as in childhood (Shet et al, Haematologica, 2020)  SCD remains one of the most common causes of stroke in children (Usmani and Machado, Clin Hemorheol Microcirc, 2018). Thrombotic vasculopathy in SCD is accompanied by significant organ dysfunction, morbidity from diminished quality of life, and mortality. Despite recent therapeutic advances, SCD patients remain at a high risk of developing vascular complications due to the poorly understood prothrombotic and thromboinflammatory state.

Thromboinflammatory processes in SCD are characterized by interactions between platelets, leukocytes and activated endothelial cells leading to vascular occlusion and inflammatory processes (Carden and Little, Haematologica, 2019). Most notably, endothelial inflammation leads to surface expression of adhesion molecules including P-selectin and release of prothrombotic granule contents (von Willebrand factor and FVIII), both effects enhancing leukocyte/platelet adhesion (Shet et al, Haematologica, 2020). Intravascular release of tissue factor (TF) also contributes the polarization toward a prothrombotic state (Shet et al, Blood, 2003). Release of cell-free heme activates converging inflammatory pathways, such as TLR signaling, formation of neutrophil extracellular traps (NETs) and priming of the inflammasome (NLRP3) (Shet et al, Haematologica, 2020). 



(Sung et al, nature communications, 2019)

Under physiological conditions, free heme is scavenged by the plasma protein hemopexin, and is subsequently catabolized by heme oxygenase-1 into carbon monoxide, biliverdin and ferrous iron (Fe2+). However, acute or/and chronic hemolysis exhausts the scavenging system leading to an increase in free heme in the blood. Upon release, reduced heme is rapidly and spontaneously oxidized in the blood into ferric (Fe3+) form, hemin, with increased levels observed in hemolytic diseases such as SCD. Interestingly, hemin induces rapid aggregation of human washed platelets which is abolished by pre-incubation of hemin with a recombinant dimeric form of C-type-lectin-like receptor 2 (CLEC2). Platelet CLEC2 may play a role in sickle cell mediated platelet activation (Bourne et al, Haematologica, 2020). 

CLEC2 is a potential therapeutic target in SCD. CLEC2 signaling on platelets lead to release of platelet exosomes and microvesicles that stimulate the CELC5A and TLR2 receptors on neutrophils, respectively (Sung et al, nature communications, 2019). Subsequent neutrophil activation leads to activation of the inflammasome, and release of neutrophil extracellular traps and pro-inflammatory cytokines, which is consistent with SCD pathology. Interestingly, upon activation, CLEC2 receptor undergoes tyrosine phosphorylation mediated by thromboxane A2 (TxA2). This leads to downstream phosphorylation of spleen tyrosine kinase (Syk) and phospholipase γ2 (PLCγ2), against potentiated by TxA2. This cooperation between CLEC2 and TxA2 signaling is critical for platelet activation (Badolia et al, JBC, 2017) 

Thromboxane A2 (TxA2), a key mediator of thrombosis, is released by platelets, endothelial cells, macrophages, and neutrophils. TxA2 binds to the thromboxane-prostanoid (TP) receptor on platelets, thereby stimulating activation and aggregation of platelets. The eicosanoid axis has been examined in SCD patients since SCD is a prothrombotic disease associated with microvascular thrombosis. TxB2 and 2,3-dinor-TxB2, a terminal metabolite of TxB2 and a marker of in vivo platelet activation, were significantly elevated in the urine and plasma of steady state sickle cell patients compared to healthy HbAA controls (Kurantsin-Mills et al, Br J Haematol, 1994). Therefore, TxA2 is likely to play a major role in sickle cell-associated thrombotic and cardiovascular complications and potentially CLEC2 signaling.

Nitric Oxide

Cell-free plasma hemoglobin resulting from intravascular hemolysis consumes nitric oxide very rapidly (Liu et al, JBC, 1998), dramatically limiting NO bioavailability in patients with sickle cell disease (Reiter et al, Nat Med, 2002). Following arterial injury, NO has been shown to serve many vasoprotective roles, including inhibition of platelet aggregation and adherence to the site of injury, inhibition of leukocyte adherence, inhibition of vascular smooth muscle cell proliferation and migration, and stimulation of endothelial cell growth (Kibbe et al, Cardiovascular Research, 1999). TxA2 directly inhibits nitric oxide synthase. Nitrite accumulation was enhanced by TP receptor antagonists, seratrodast or ramatroban, in a model of IL-1β stimulated rat aortic smooth muscle cells (Shiokoshi et al, Journal of Hypertension, 2002). Therefore, TxA2 may play a role in NO deficiency in sickle cell disease which would be alleviated by TPr blockade.

Other Thromboinflammatory Processes in SCD

Leukocyte adhesion (ICAM-1 and VCAM-1)

RBC-endothelial cell interactions induces production of oxygen radicals by NF-kappaB, which in turn, upregulates the production of endothelial adhesion molecules such as E-selectin, VCAM-1 and ICAM-1 Sultana et a, Blood, 1998). Expression of leukocyte adhesion molecules including VCAM-1 and ICAM-1 are mediated part by TPr stimulation with TxA2 (Ishizuka et al, Clinical and Experimental Immunology, 1998). In human vascular endothelial cells stimulated with a TPr agonist, protein and mRNA levels of VCAM-1, ICAM-1 or ELAM-1 were marked upregulated. Conversely, pretreatment with a TPr antagonist diminished the expression of leukocyte adhesion molecules. It was concluded that ICAM-1 or ELAM-1 expression of HUVEC stimulated via TXA2 receptors is augmented by induction of NF-kappaB and AP-1 binding activity through the PKC system, and that VCAM-1 expression is augmented by induction of NF-kappaB binding activity.


Elevated levels of plasma soluble P-selectin reflect platelet activation in SCD (Voskaridou et al, Blood, 2019) P-selectin is a well-known therapeutic target in SCD and is inhibited by crizanlizumab, a humanized monoclonal antibody targeting P-selectin (Ataga et al, NEJM, 2016) TxA2 plays a role in platelet expression of P-selectin. It was demonstrated that the percentage of P-selectin-positive platelets in TP receptor knockout mice on day 1 was significantly reduced compared with that in wild type mice (Matsui et al, Cancer Science, 2012) P-selectin inhibition with crizanlizumab in patients with SCD led to significantly lower rates of sickle cell-related pain crises and was associated with a lower incidence of adverse events (Ataga et al, NEJM, 2016).

Tissue factor

Tissue factor (TF) expression has long been investigated as markers of coagulation in patients with SCD. TxA2 has been shown to mediate tissue factor (TF) expression on both endothelial cells and monocytes (Bode and Mackman, Vascular Pharmacology, 2014) TPr antagonism induced TF expression in endothelial cells, while a TPr antagonist reduced endothelial expression of TF after TNF-α induction.  Similarly, LPS induced TF expression on human monocytes was abrogated by a TP receptor antagonist (Eligini et al, Cardiovascular Research, 2006). 

Therefore TxA2 regulates many of the thromboinflammatory processes that contribute to the vascular complications of SCD. 


Conran and De Paula, Haematologica, 2020

Ramatroban (Elpidazo)

Ramatroban, is a surmountable and potent antagonist of two prostanoid receptors; first, the thromboxane A2 prostanoid receptor (TPr) and second, the D-prostanoid receptor 2 (DPr2, formerly known as CRTH2) for prostaglandin D2 (PGD2). Ramatroban has been used in Japan for the past 20 years as treatment for allergic rhinitis. Ramatroban is 100 times more potent than aspirin in inhibiting platelet aggregation and P-selectin expression (Ishizuka et al, Cardiovascular Drug Reviews, 2004 ; Kariyazono et al, Blood Coagul Fibrinolysis, 2004). In addition to its anti-platelet action, ramatroban also improves vascular responsiveness; while inhibiting endothelial surface expression of ICAM-1 and VCAM-1; inhibiting MCP-1 expression in response to TNF-α or platelet activating factor; and inhibiting macrophage infiltration (Ishizuka et al, Cardiovascular Drug Reviews, 2004). In a rat model of endotoxic shock, ramatroban prevented hypotension and reduced mortality by 45%; effects attributable to marked reduction in myeloperoxidase levels in lungs, ileum and heart and > 90% reduction in plasma TNF-α levels (Altavilla et al, Pharmacol Res, 1994) Therefore, ramatroban has the potential to target hemolysis induced platelet activation, neutrophil extracellular trap formation, leukocyte adhesion and many other thromboinflammatory pathways that contribute to vaso-occlusive disease and thrombosis in SCD.