Plasma Hemoglobin and Heme Trigger Weibel Palade Body Exocytosis and Vaso-Occlusion in Transgenic Sickle Mice
Authors: Belcher John D , Nguyen Julia , Chen Chunsheng , Smith Ann , Alayash Abdu I , Nair Sethu L , Somani Arif , Hebbel Robert P , Vercellotti Gregory M 
Published: October 2019

Abstract 896

Sickle cell disease (SCD) is a devastating hemolytic disease characterized by recurring episodes of painful vaso-occlusive crises and endothelial dysfunction. We hypothesize that hemoglobin (Hb) or hemin (ferri-protoporphyrin IX), released from HbS derived from hemolyzed sickle red blood cells, is fundamental to vaso-occlusion and vasculopathy in SCD. Utilizing intravital microscopy and dorsal skin fold chambers implanted on mice we previously demonstrated transient stasis in subcutaneous venules in response to hypoxia in transgenic sickle, but not normal mice. In the current studies we used NY1DD sickle mice; Townes-AA, -AS, and -SS mice; and C57 normal mice to test whether Hb or heme induce vaso-occlusion in the absence of a hypoxic stimulus. Four groups of 3–6 mice were given a bolus infusion (0.012 ml/g i.v.) of the following: 1) saline; 2) stroma-free HbA (32 umols heme/kg); 3) hemin (32 umols/kg); or 4) water (to induce intravascular hemolysis). In NY1DD mice, Hb, hemin or water induced a range of 36–46% stasis at 1 and 4 hours post-infusion compared to only 4–7% stasis at 1 and 4 hours in saline controls (Fig 1A, p<0.001 for all groups vs saline). In contrast, Hb, hemin or saline induced only 0–6% stasis in C57 mice (Fig 1B, p<0.001 for Hb or hemin in C57 vs NY1DD). Similarly, Townes-AA, -AS and –SS mice had a range of 5–15%, 27–37% and 40–50% stasis, respectively, in response to HbA (p<0.05 for all Townes pairs).

The response to Hb or hemin was dose dependent with as little as 0.32 umols heme/kg, equivalent to ∼5 uM heme, a level found in SCD patients. These data support that heme, derived from Hb, promotes vascular stasis in sickle mice. The plasma of SCD patients is deficient in the two primary Hb/heme scavenging proteins haptoglobin (Hp) and hemopexin (Hpx). Therefore we infused equimolar Hp-Hb or Hpx-hemin into NY1DD mice and inhibited Hb- and heme-induced stasis at 1 hour by 79 and 88%, respectively (p<0.001).

To examine whether nitrogen derivatives contribute to the mechanism of Hb- or heme-induced stasis, we measured plasma nitrite and nitrate (NOx) in NY1DD sickle mice 4 hours after infusion of Hb or hemin. NOx levels decreased in mice infused with Hb, but not in mice infused with hemin suggesting that NO consumption does not play a role in heme-induced stasis.

Previous studies have demonstrated a role for P-selectin in vaso-occlusion in sickle mice. We tested the ability of heme to trigger Weibel Palade body (WPB) exocytosis in cultured primary human umbilical vein endothelial cells (HUVEC). Cells were treated with 10 uM hemin for 2, 5, 10, 15, 30 or 60 minutes and then fixed and stained (without permeabilization) for surface expression of P-selectin and von Willebrand factor (vWF). Cells treated with 100 uM histamine and vehicle served as positive and negative controls, respectively. There was rapid expression of P-selectin as well as vWF strings on the surface of HUVEC within 5 minutes of hemin addition. This is the first report that heme is an agonist for WPB exocytosis. Recently, heme has been shown to be an extracellular inflammatory signaling molecule with strict binding specificity for toll-like receptor-4 (TLR4). A specific small molecule inhibitor of TLR4 (TAK-242) completely prevented heme-induced P-selectin expression in vitro. In vivo the pulmonary veins and arteries of sickle mice injected with hemin expressed surface P-selectin within 15 minutes. Supporting this novel mechanism, blocking antibodies to P-selectin or the drug TAK-242 inhibited heme-induced stasis and thus provide a potential therapy for vaso-occlusion.

These data strongly support that heme, released from hemolyzed sickle red blood cells, is fundamental to vaso-occlusion and vasculopathy in SCD.

We speculate that removal of Hb and heme with Hp and Hpx, or as we've previously shown, detoxifying heme with heme oxygenase-1, would decrease the oxidative stress, inflammation and vaso-occlusion in SCD that cause endothelial cell dysfunction. Novel therapies focusing on the consequences of endothelial cell/heme interactions such as TLR4 or P-selectin antagonists, in addition to Hp and Hpx modulators should be considered in SCD.

Disclosures:

Belcher: Sangart, Inc: Research Funding. Nguyen:Sangart Inc: Research Funding. Chen:Sangart, Inc: Research Funding. Vercellotti:Sangart, Inc: Research Funding.