(B-46) Attenuating Heme-induced Coagulation with ApoHemoglobin-Haptoglobin

Introduction:: Under conditions of substantial hemolysis, the lysis of red blood cells (RBCs) results in an increased concentration of cell-free hemoglobin (Hb) in the bloodstream. When this cell-free hemoglobin is oxidized into methemoglobin, it is capable of releasing heme. Moreover, labile heme has been found to interact with the coagulation cascade, causing potentially disastrous pro-coagulant consequences in the body. To mediate the effects of excess heme, plasma proteins haptoglobin (Hp) and hemopexin (Hpx) bind to acellular hemoglobin and free heme, respectively. To that end, this work puts forth a novel scavenger protein, apohemoglobin-haptoglobin (apoHb+Hp), to mediate the pro-thrombotic effects of free heme by improving on the body’s innate defense mechanisms against labile heme. ApoHb+Hp mimics the function of hemopexin to collect free heme with greater binding affinity than the naturally occurring hemopexin and uses the clearance mechanism of haptoglobin for effective removal from the plasma. By facilitating the effective clearance of free heme from the plasma, we hypothesize that apoHb+Hp will alleviate pro-coagulatory markers induced by heme.


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 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 be tested.


Results, Conclusions, and Discussions:: Re-injection of the extracted blood volume with lactated ringers, apoHb+Hp, apoHb+Hp and heme HSA, and heme HSA alone corresponded to prothrombin times of 15.33 ±1.42, 18.85 ±1.12, 14.18 ±1.39, 13.80 ±1.99 seconds, respectively. Plasma levels of fibrinogen, thrombin-antithrombin complex, and soluble glycoprotein V all similarly trended towards the control of lactated ringers after 30 minutes of rest when apoHb+Hp was added. No significant differences were found in plasma markers of the intrinsic pathway of the coagulation cascade.


Previous literature has established that heme has significant effects in the extrinsic and common pathways of the coagulation cascade, and these results confirm that heme had the most notable procoagulatory effects in these pathways. When combined with heme HSA, however, apoHb+Hp was able to mediate some of the thrombotic effects induced by the addition of heme HSA to circulation. This is suspected to be the result of apoHb+Hp’s ability to bind to heme with greater affinity and efficiency than naturally occurring defense mechanisms, particularly before heme can bind to fibrinogen and generate fibrin necessary for clotting.



ApoHb+Hp attenuates heme-induced pro-thrombotic states to some extent in the extrinsic and common pathways, but most significantly in connection with fibrinogen. Future studies should test time- and dose-dependency of apoHb+Hp to significantly reduce procoagulatory effects.


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