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(Circulation. 1996;93:208-209.)
© 1996 American Heart Association, Inc.

Articles

Cell-Free Hemoglobin as an Oxygen Carrier Removes Nitric Oxide, Resulting in Defective Thromboregulation

Aaron J. Marcus, MD; M. Johan Broekman, PhD

From the Divisions of Hematology and Medical Oncology, Departments of Medicine and Pathology, Department of Veterans Affairs Medical Center and Cornell University Medical College, New York, NY.

Correspondence to Aaron J. Marcus, MD and M. Johan Broekman, PhD, Thrombosis Research Laboratory, Room 13028W, Cornell University Medical College and Department of Veterans Affairs Medical Center, 423 E 23rd St, New York, NY 10010-5050. E-mail mjbroek@med.cornell.edu.

Key Words: Editorials • hemoglobin • endothelium-derived factors • thrombosis

	Introduction

Top Introduction Thromboregulation References

 
Development of a clinically safe and effective substitute for erythrocytes that is capable of efficient oxygen delivery in vivo has progressed to the stage where cross-linked hemoglobin preparations are now undergoing clinical trials.¹ Such preparations withstand storage for prolonged periods of time, can be administered without the need for cross-matching, and are free of contamination by infectious agents.

Utilization of cell-free hemoglobin as an erythrocyte substitute was initially hampered by nephrotoxicity and an affinity for oxygen that prevented efficient oxygen delivery to tissues.² These disadvantages were overcome when Bunn and Jandl³ chemically cross-linked the hemoglobin molecule to produce stable hemoglobin oligomers that do not pass through the glomerular filtrate. In addition, Benesch and Benesch⁴ developed reagents that modified the 2,3-diphosphoglycerate binding site of hemoglobin, thereby reducing its oxygen affinity.

Administration of cell-free hemoglobin solutions results in systemic vasoconstriction in research animals.⁵ This is thought to be a consequence of the high avidity of hemoglobin for nitric oxide (NO, endothelium-derived relaxing factor [EDRF]), which it binds and inactivates. The NO-hemoglobin interaction results in rapid formation of nitrite/nitrate and methemoglobin. This blocks vasodilation induced by NO via activation of vascular smooth muscle cell guanylate cyclase.^{5 6 7}

Removal of NO by hemoglobin will also reduce activity of platelet guanylate cyclase. This increases platelet reactivity, resulting in platelet deposition on prothrombotic surfaces such as injured vessel wall. This phenomenon was indeed demonstrated by the experiments of Olsen et al⁸ as reported in this issue of Circulation. Using a rat microsurgical carotid endarterectomy model, the authors showed that infusion of a cross-linked hemoglobin preparation (Hb) led to significant enhancement of platelet deposition on the injured blood vessel surface. This was due to the NO-scavenging property of Hb as demonstrated by the following observations: (1) Increased platelet deposition resulted from infusion of Hb as well as infusion of an inhibitor of NO synthase, N^G-monomethyl-L-arginine (NMMA), and (2) increased platelet deposition after Hb or NMMA administration was reversed by infusion of L-arginine, the precursor of NO. Thus, the data obtained in this model system document and emphasize the importance of NO as an endogenous thromboregulator.

Oral administration of aspirin failed to prevent the increase in platelet deposition induced by Hb infusion, although a small, beneficial effect cannot be excluded (Fig 2 in reference 8). Thus, the proaggregatory effect of cross-linked hemoglobin appears to occur via EDRF removal alone. Earlier, Broekman et al⁹ had shown that EDRF/NO could block platelet reactivity in an aspirin-insensitive manner.

	Thromboregulation

Top Introduction Thromboregulation References

 
There is experimental evidence for at least three independent mechanisms in endothelial cells that act concurrently to downregulate platelet reactivity and defend against accumulation of an occlusive platelet-rich thrombus (the Figure). Loss of platelet reactivity in the presence of endothelial cells occurs via one or more of the following mechanisms (Table): (1) Formation of eicosanoids such as prostacyclin, either endogenously or via transcellular metabolism of released precursors from activated platelets¹⁰ ; (2) formation of EDRF/NO⁹ ; and (3) metabolism of prothrombotic, platelet-released ADP by endothelial cell ecto-ADPase.^{11 12 13}

View larger version (57K): [in this window] [in a new window]   Figure 1. Diagram shows that when circulating platelets become activated, endothelial cells respond to limit or reverse the consequences of platelet adhesion, aggregation, and recruitment. We define this process as endothelial thromboregulation. In vitro, platelets become unresponsive to all agonists in the presence of endothelial cell suspensions. This is due to at least three separate thromboregulatory systems: (1) formation of eicosanoids from arachidonic acid; (2) generation of endothelium-dependent relaxing factor/nitric oxide (EDRF/NO) from arginine; and (3) ecto-nucleotidase(s) with both ADPase and ATPase activities.13 14 Activation of endothelial cells by agonists such as thrombin results in formation of prostacyclin via cyclooxygenation of arachidonic acid. Prostacyclin reacts with a specific receptor on the platelet surface and initiates a G protein–linked signal transduction pathway, resulting in formation of cAMP. cAMP is a strong inhibitor of platelet function via antagonism of calcium-mediated platelet responses. EDRF/NO is an aspirin-insensitive fluid-phase autacoid produced by vascular endothelium and a variety of other cells and stimulates the soluble guanylyl cyclase in target cells. The resulting elevation in cGMP blocks responsiveness of activated platelets. In endothelial cells, NO is produced constitutively from L-arginine by a specific isoform of NO synthase.15 The third endothelial thromboregulatory system involves ecto-nucleotidase(s) on the cell surface. These ecto-nucleotidase(s) are aspirin insensitive and metabolize released platelet ADP to AMP and adenosine, thereby limiting platelet recruitment.11 EC indicates endothelial cell; PLT, platelet; PLT*, activated platelet; SMC, smooth muscle cell; RBC, erythrocyte; and PMN, neutrophil.

View this table: [in this window] [in a new window]   Table 1. Human Endothelial Cell Thromboregulation

In the study by Olsen and colleagues,⁸ prevention of cyclooxygenase-catalyzed eicosanoid formation by aspirin treatment did not protect against platelet deposition after infusion of Hb. Although not examined by Olsen et al, endothelial cell ecto-ADPase can completely inhibit platelet reactivity in vitro, even if cyclooxygenase-catalyzed eicosanoid formation and EDRF/NO production are blocked. The data of Olsen et al demonstrate conclusively that infusion of Hb results in destruction of EDRF/NO, thereby promoting platelet deposition at the site of experimental injury. Thus, infusion of Hb results in a breach in one of the components of the thromboregulatory system.

Results of these experiments demonstrate that a previously unappreciated property of hemoglobin, destruction of EDRF/NO, can lead to a phenomenon with important clinical implications, ie, platelet deposition at sites of vascular injury, possibly leading to an aspirin-insensitive thrombotic diathesis.

	Acknowledgments

 
This work was supported in part by grants from the National Institutes of Health (HL-18828 SCOR, HL-47073, and HL-46403) and the Department of Veterans Affairs.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top Introduction Thromboregulation References

 

1. Shoemaker SA, Gerber MJ, Evans GL, Archer-Paik LE, Scoggin CH. Initial clinical experience with a rationally designed, genetically engineered recombinant human hemoglobin. Artif Cells Blood Substit Immobil Biotechnol. 1994;22:457-465. [Medline] [Order article via Infotrieve]
   
2. Lieberthal W. Stroma-free hemoglobin: a potential blood substitute. J Lab Clin Med. 1995;126:231-232. Editorial. [Medline] [Order article via Infotrieve]
   
3. Bunn HF, Jandl JH. The renal handling of hemoglobin, II: catabolism. J Exp Med. 1969;129:925-934. [Abstract]
   
4. Benesch R, Benesch RE. The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochem Biophys Res Commun. 1967;26:162-167. [Medline] [Order article via Infotrieve]
   
5. Matheson-Urbaitis B, Lu YS, Fronticelli C, Bucci E. Renal and systemic-hemodynamic response to isovolumic exchange transfusion with hemoglobin cross-linked with bis-(3,5-dibromosalicyl) fumarate or albumin. J Lab Clin Med. 1995;126:250-260. [Medline] [Order article via Infotrieve]
   
6. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987;84:9265-9269. [Abstract/Free Full Text]
   
7. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993;329:2002-2012. [Free Full Text]
   
8. Olsen SO, Tang DB, Jackson MR, Gomez ER, Ayala B, Alving BM. Enhancement of platelet deposition by cross-linked hemoglobin in a rat carotid endarterectomy model. Circulation. 1996;93:327-332. [Abstract/Free Full Text]
   
9. Broekman MJ, Eiroa AM, Marcus AJ. Inhibition of human platelet reactivity by endothelium-derived relaxing factor from human umbilical vein endothelial cells in suspension: blockade of aggregation and secretion by an aspirin-insensitive mechanism. Blood. 1991;78:1033-1040. [Abstract/Free Full Text]
   
10. Marcus AJ, Weksler BB, Jaffe EA, Broekman MJ. Synthesis of prostacyclin from platelet-derived endoperoxides by cultured human endothelial cells. J Clin Invest. 1980;66:979-986.
    
11. Marcus AJ, Safier LB, Hajjar KA, Ullman HL, Islam N, Broekman MJ, Eiroa AM. Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase: thromboregulation by endothelial cells. J Clin Invest. 1991;88:1690-1696.
    
12. Marcus AJ, Safier LB, Broekman MJ, Islam N, Fliessbach JH, Hajjar KA, Kaminski WE, Jendraschak E, Silverstein RL, von Schacky C. Thrombosis and inflammation as multicellular processes: significance of cell-cell interactions. Thromb Haemost. 1995;74:213-217. [Medline] [Order article via Infotrieve]
    
13. Marcus AJ, Safier LB. Thromboregulation: multicellular modulation of platelet reactivity in hemostasis and thrombosis. FASEB J. 1993;7:516-522. [Abstract]
    
14. Yagi K, Shinbo M, Hashizume M, Shimba LS, Kurimura S, Miura Y. ATP diphosphohydrolase is responsible for ecto-ATPase and ecto-ADPase activities in bovine aorta endothelial and smooth muscle cells. Biochem Biophys Res Commun. 1991;180:1200-1206. [Medline] [Order article via Infotrieve]
    
15. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994;298:249-258.
    

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