The Effects of Apohemoglobin-Haptoglobin on Heme-Induced Coagulation
Authors: Martinez Jacinda , Ricardo Mia , Munoz Carlos J , Lucas Daniela , Muller Cynthia R , Gopal Srila , Cabrales Pedro 
Published: December 2023

Introduction: In conditions with significant hemolysis, like sickle cell and thalassemia, red blood cells break down, leading to higher levels of cell-free hemoglobin (Hb) in the bloodstream. When this cell-free Hb is oxidized into methemoglobin, it releases heme, a crucial iron-protoporphyrin complex essential for oxygen transport. However, excessive free heme can become problematic, since plasma concentrations of the body protective proteins, haptoglobin and hemopexin, to scavenge and clear hemoglobin and heme is very limited. Thus, these protective mechanisms can get overwhelmed by high levels of free Hb or heme in the blood. Additionally, heme can interact with the coagulation cascade, potentially causing harmful pro-coagulant effects in the body. To address this, we introduce a new synthetic scavenger protein called apohemoglobin-haptoglobin (apoHb+Hp). It mimics hemopexin's function and binds to free heme with even greater affinity. Moreover, it utilizes haptoglobin's clearance mechanism for efficient removal from the plasma. Our aim is for apoHb+Hp to effectively clear free heme from the plasma, thereby alleviating pro-coagulatory markers induced by heme. This novel approach may enhance the body's natural defense against heme, leading to potential therapeutic benefits.

Materials and Methods: This study used male Sprague-Dawley rats to investigate the effect of apoHb+Hp on coagulation markers during conditions of excess heme. To do so, 20% of the blood volume, estimated as 7% of body mass, was removed from the animal over a 10 minute period. After 10 minutes of rest, the removed blood volume was re-infused to the animal as either 20% lactated ringers, 20% apoHb+Hp, 10% heme bound to human serum albumin (HSA) and 10% apoHb+Hp, or 20% heme HSA. After a 30-minute rest period, 70 µm and 5 mL blood samples were drawn from the tail vein and arterial catheter, respectively, to evaluate measures of the coagulation cascade including prothrombin time, active partial thromboplastin time, fibrinogen, soluble glycoprotein V, Thrombin/ antithrombin complex (TAT), thromboelastography (TEG).

Results: Re-injection of Heme HSA resulted in activation of the coagulation system as evidenced by an increase in TAT and soluble glycoprotein V. This effect was reversed with co administration of ApoHb-Hp. Heme HSA caused activation of the extrinsic pathway, as measured by TEG (EXTEM) and a reduction in PT, this effect was ameliorated upon treatment with apoHb-Hp. No notable changes were seen in the intrinsic pathway factors as evidenced by the PTT or TEG/ INTEM.

Discussion: Previous literature has established that heme has significant effects in the extrinsic and common pathways of the coagulation cascade, and our study is consistent with these results. When combined with heme HSA, however, apoHb+Hp was able to improve some of the thrombotic effects induced by the addition of heme HSA to circulation.

Conclusions: Excess free heme can contribute to a hypercoagulable state. As expected, our findings showed significantly notable pro-coagulatory state in the presence of excessive heme in the blood. ApoHb+Hp attenuates heme-induced prothrombotic states to some extent in the extrinsic and common pathways. ApoHb-Hp may be a potential therapeutic strategy in hemolytic anemias and further studies to evaluate its role in pathologic states associated with hemolysis is warranted.