Can erythrocytes release biologically active NO?
Benz and Fleming Cell Communication and Signaling
Can erythrocytes release biologically active NO?
Peter M. Benz 0 1
Ingrid Fleming 0 1
0 DZHK (German Centre for Cardiovascular Research) partner site Rhine-Main , 60590 Frankfurt am Main , Germany
1 Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University , Frankfurt , Germany
Under physiological conditions, endothelial cells and the endothelial nitric oxide (NO) synthase (eNOS) are the main source of NO in the cardiovascular system. However, several other cell types have also been implicated in the NO-dependent regulation of cell function, including erythrocytes. NO derived from red blood cells has been proposed to regulate erythrocyte membrane fluidity, inhibit platelet activation and induce vasodilation in hypoxic areas, but these proposals are highly controversial. In the current issue of Cell Communication and Signaling, an elegant study by Gambaryan et al., assayed NO production by erythrocytes by monitoring the activation of the platelet intracellular NO receptor, soluble guanylyl cyclase, and its downstream kinase protein kinase G. After systematically testing different combinations of erythrocyte/platelet suspensions, the authors found no evidence for platelet soluble guanylyl cyclase/protein kinase G activation by erythrocytes and conclude that erythrocytes do not release biologically active NO to inhibit platelet activation.
Nitric oxide; NO; Red blood cells; Erythrocytes; eNOS; Platelet inhibition; Hypoxic vasodilation; VASP; Soluble guanylyl cyclase; PKG
It is more than 20 years since nitric oxide (NO) was
recognized as a biological signalling molecule in the
cardiovascular system. NO is generated in endothelial
cells by the constitutive endothelial nitric oxide
synthase (eNOS), which converts L-arginine into NO and
L-citrulline [1, 2]. NO is a short-lived gaseous radical,
which diffuses randomly to other cell types, including
smooth muscle cells, platelets, and immune cells. Most,
if not all, of the biological functions of NO, including
platelet inhibition and smooth muscle relaxation, are
mediated by the soluble guanylate cyclase (sGC), which
converts GTP to cGMP and thereby activates the protein
kinase G (PKG) [3–5] (Fig. 1). The activity of eNOS is
largely dependent on an increase in the intracellular
concentration of calcium [Ca2+]i, released from the
endoplasmic reticulum in response to the activation of
receptordependent ligands such as acetylcholine, bradykinin, or
histamine. eNOS can also be activated, in the absence
of a prolonged increase in [Ca2+]i by stimuli that elicit
the phosphorylation of eNOS on Ser1177. One such
stimulus is the fluid shear stress that acts on the luminal
surface of vascular endothelium [1, 2] (Fig. 1). In addition
to endothelial cells, several other cell types have been
reported to generate and release NO; these include
vascular smooth muscle cells and cardiac muscle cells.
Even though they contain large amounts of the NO
scavenger hemoglobin, red blood cells (RBCs,
erythrocytes) have also been reported to express eNOS  and
generate/release NO [7–9] but these observations are
still highly controversial.
Erythrocytes and platelets represent the major cell
populations in the mammalian blood and clinical observations
indicate a functional interaction between the two cell
types. On the one hand, there is an undisputed role of
erythrocytes in platelet activation as bleeding times are
prolonged in patients with anemia independent of their
platelet count – a condition that can be corrected by
erythrocyte transfusion [10, 11]. Importantly, the
bleeding defects observed are directly associated with
an impaired platelet activation and independent of the
blood coagulation system . Furthermore,
erythrocyte transfusion can increase platelet activation, which
may result in complications in treatment of coronary
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Fig. 1 Role of nitic oxide (NO) in blood vessels and functional interplay between erythrocytes and platelets. NO is generated by the endothelial
NO synthase (eNOS) upon stimulation by fluid shear stress or Ca2+ elevating agonists. The biological actions of NO, including platelet inhibition
and smooth muscle relaxation, are mediated by the soluble guanylate cyclase (sGC), which generates cGMP and the subsequent activation of
protein kinase G (PKG). Erythrocytes play a role in the regulation of platelet activation as ADP and ATP secreted from damaged erythrocytes directly
stimulates platelet purinergic receptors (P2Y12, P2Y1, and P2X1). Secreted hemoglobin (Hb) scavenges endothelial-derived NO and therefore decreases
platelet inhibition. It has been proposed that erythrocytes also play a role in platelet inhibition by generation/release of NO or NO-carriers, such as
SNO. While it is still debatable whether or not erythrocytes can generate NO/SNO, current experimental evidence by Gambaryan et al. concludes that
erythrocytes do not release biologically active NO/SNO
artery diseases . How erythrocytes contribute to
platelet activation is still under debate, but this may involve
activation of the ADP-P2Y12 receptor pathway and/or the
elevated platelet radial movement and interaction with the
endothelium [10, 12].
Several recent studies have indicated a role for
erythrocytes in platelet inhibition by virtue of their ability to
release NO [7–9]. Three contradictory mechanisms have
been proposed by which erythrocytes may provide NO.
The first suggestion was that the nitrosation of a conserved
cysteine within the hemoglobin (Hb) β-chain (β93 cysteine)
by NO results in the formation of S-nitrosohemoglobin
(SNO-Hb), which could function as an NO carrier .
The second proposal was that deoxyhemoglobin
(deoxyHb) may act as a nitrite reductase, and catalyze the
formation of NO (and methemoglobin) from inorganic nitrite
[15–17]. The third hypothesis is that NO derived from
eNOS protein expressed in erythrocytes may directly
inhibit platelet aggregation . All of these hypotheses have
been the subject of intense debate and all have been
challenged by other authors. For example, the generation of
a knockin mouse model that replaced hemoglobin β
cysteine93 with alanine helped to conclude that SNO-Hb
is not essential for the coupling of erythrocyte
deoxygenation with increased NO bioactivity in vivo .
Moreover, detailed electron paramagnetic resonance (EPR)
spectroscopy of nitrite–methemoglobin complexes
questioned the proposed role of deoxyhemoglobin in
generating NO by the reduction of nitrite . Finally, other
authors failed to detect a functional eNOS in erythrocytes
General doubt about the release of NO or NO-carriers
from erythrocytes comes from the fact that Hb is an avid
scavenger of NO. Notably, physiological NO
concentrations range between 100 pM (or below) up to ~ 5 nM, at
least six orders of magnitude below the Hb concentration
in erythrocytes . Thus while the erythrocyte membrane
and the cell free zone established in resistance-sized
arteries can reduce the apparent rate at which
endotheliumderived NO is consumed by Hb [22, 23], the situation is
dramatically different inside red blood cells. As outlined
above, most of the biological actions of NO, including
platelet inhibition and smooth muscle relaxation, are
mediated by the sGC/cGMP/PKG pathway (Fig. 1). An
important limitation of all of the publications, which
claimed platelet inhibition by erythrocyte-derived NO,
is that direct activation of the sGC/cGMP/PKG
pathway in platelets was not investigated. Together with the
conflicting experimental results described above, the
ongoing debate about generation and release of NO (or NO
carriers) from erythrocytes has precluded a consensus on
the physiological role of erythrocytes in platelet inhibition.
In the current issue of Cell Communication and
Signaling, Gambaryan et al.  set out to solve the discrepancy
regarding erythrocyte-mediated platelet inhibition by
asking a very simple and logical question. If erythrocytes
indeed release physiological relevant levels of NO or SNO
to inhibit platelet activation, then the sGC/cGMP/PKG
pathway in platelets should be activated, irrespective of
the mechanism by which NO/SNO are generated in red
blood cells. The authors monitored the effects of NO in
platelets by assessing the activity of purified sGC in the
presence of erythrocytes as well as the NO/sGC/cGMP/
PKG-dependent phosphorylation of vasodilator-stimulated
phosphoprotein (VASP). The latter is a highly sensitive
and reliable assay, used in numerous studies to assess
NO-dependent effects and which is widely used in clinical
diagnosis of platelet reactivity [25–29]. Other assays that
aim to assess the role of NO in platelets, e.g. by the
ELISA-base measurement of cGMP, seem to be prone to
artefacts, especially in the presence of nitrite or
erythrocytes, which both interfere with these assays .
Gambaryan et al. systematically tested all possible
combinations of erythrocyte/platelet suspensions over time,
with varying erythrocyte concentrations, as well as
studying erythrocytes containing Hb in different states
(oxy-Hb, deoxy-Hb, NO-Hb), with or without nitrite,
and platelets isolated under normal or deoxygenated
conditions . They found no evidence of platelet and
purified sGC activation by erythrocytes. Instead, the
authors discovered a strong scavenging effect of NO by
erythrocytes - even if NO was added exogenously. Consequently,
the authors came to the conclusion that erythrocytes, under
all of the conditions tested, cannot inhibit platelet activation
by the release of physiological active NO, but in strong
contrast act as potent NO scavengers.
At least one additional physiological function has been
attributed to erythrocyte-derived NO i.e., the phenomenon
of hypoxic vasodilation. According to a hypothesis put
forward several years ago , erythrocytes have an essential
role in matching blood flow to local metabolic demand
that can be explained by NO release from Hb at low
oxygen saturation of blood Hb to elicit vasodilatation
[31, 32]. To-date it is unclear what form this NO has to
take to avoid efficient scavenging by oxygen bound to
the heme of oxyhemoglobin or with the unoccupied
heme of deoxyhemoglobin , but NO release form
RBCs has been proposed to occur via the reduction of
nitrite, the SNO-hemoglobin pathway, as well as
endogenous erythrocyte eNOS activity . Even though no
contribution to hypoxic vasodilatation was specifically
studied, Gambaryan et al.  found no evidence for
NO/SNO release form RBCs in erythrocyte/platelet
suspensions using the sensitive platelet sGC/cGMP/
PKG pathway as NO sensor. Given that activation of
the same pathway in smooth muscle cells would be
mandatory for NO-dependent hypoxic vasodilation, it
seems very unlikely that any RBC-derived NO/SNO
could diffuse to the much more distant smooth muscle
In summary, it is still debatable whether or not erythrocytes
possess the functional machinery to generate NO/SNO.
However, irrespective of this question, the study by
Gambaryan et al.  seems to resolve some of the
current controversy by concluding that erythrocytes do
not release biologically active NO.
cGMP: Cyclic guanosine monophosphate; eNOS: Endothelial NO synthase;
EPR: Electron paramagnetic resonance; NO: Nitric oxide; PKG: Protein
kinase G; RBCs: Red blood cells; sGC: Soluble guanylyl cyclase;
SNO-Hb: S-nitrosohemoglobin; VASP: Vasodilator-stimulated phosphoprotein
1. Fleming I. Molecular mechanisms underlying the activation of eNOS . Pflugers Arch . 2010 ; 459 : 793 - 806 .
2. Siragusa M , Fleming I. The eNOS signalosome and its link to endothelial dysfunction . Pflugers Arch . 2016 ; 468 : 1125 - 37 .
3. Koesling D , Friebe A. Soluble guanylyl cyclase: structure and regulation . Rev Physiol Biochem Pharmacol . 1999 ; 135 : 41 - 65 .
4. Martin E , Berka V , Tsai AL , Murad F. Soluble guanylyl cyclase: the nitric oxide receptor . Methods Enzymol . 2005 ; 396 : 478 - 92 .
5. Tsai AL , Berka V , Sharina I , Martin E. Dynamic ligand exchange in soluble guanylyl cyclase (sGC): implications for sGC regulation and desensitization . J Biol Chem . 2011 ; 286 : 43182 - 92 .
6. Cortese-Krott MM , Rodriguez-Mateos A , Sansone R , Kuhnle GG , Thasian-Sivarajah S , Krenz T , Horn P , Krisp C , Wolters D , Heiss C , et al. Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease . Blood . 2012 ; 120 : 4229 - 37 .
7. Srihirun S , Sriwantana T , Unchern S , Kittikool D , Noulsri E , Pattanapanyasat K , Fucharoen S , Piknova B , Schechter AN , Sibmooh N. Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation . PLoS One . 2012 ; 7 : e30380 .
8. Corti P , Xue J , Tejero J , Wajih N , Sun M , Stolz DB , Tsang M , Kim-Shapiro DB , Gladwin MT . Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells . Proc Natl Acad Sci U S A . 2016 ; 113 : 8538 - 43 .
9. Akrawinthawong K , Park JW , Piknova B , Sibmooh N , Fucharoen S , Schechter AN. A flow cytometric analysis of the inhibition of platelet reactivity due to nitrite reduction by deoxygenated erythrocytes . PLoS One . 2014 ; 9 : e92435 .
10. Anand A , Feffer SE . Hematocrit and bleeding time: an update . South Med J . 1994 ; 87 : 299 - 301 .
11. Boneu B , Fernandez F. The role of the hematocrit in bleeding . Transfus Med Rev . 1987 ; 1 : 182 - 5 .
12. Silvain J , Abtan J , Kerneis M , Martin R , Finzi J , Vignalou JB , Barthelemy O , O'Connor SA , Luyt CE , Brechot N , et al. Impact of red blood cell transfusion on platelet aggregation and inflammatory response in anemic coronary and noncoronary patients: the TRANSFUSION-2 study (impact of transfusion of red blood cell on platelet activation and aggregation studied with flow cytometry use and light transmission aggregometry) . J Am Coll Cardiol . 2014 ; 63 : 1289 - 96 .
13. Kumbhani DJ , Bhatt DL . Platelet activation: yet another strike against routine TRANSFUSION . Eur Heart J . 2010 ; 31 : 2712 - 4 .
14. Jia L , Bonaventura C , Bonaventura J , Stamler JS. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control . Nature . 1996 ; 380 : 221 - 6 .
15. Tejero J , Basu S , Helms C , Hogg N , King SB , Kim-Shapiro DB , Gladwin MT . Low NO concentration dependence of reductive nitrosylation reaction of hemoglobin . J Biol Chem . 2012 ; 287 : 18262 - 74 .
16. Basu S , Grubina R , Huang J , Conradie J , Huang Z , Jeffers A , Jiang A , He X , Azarov I , Seibert R , et al. Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin . Nat Chem Biol . 2007 ; 3 : 785 - 94 .
17. Cosby K , Partovi KS , Crawford JH , Patel RP , Reiter CD , Martyr S , Yang BK , Waclawiw MA , Zalos G , Xu X , et al. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation . Nat Med . 2003 ; 9 : 1498 - 505 .
18. Isbell TS , Sun CW , Wu LC , Teng X , Vitturi DA , Branch BG , Kevil CG , Peng N , Wyss JM , Ambalavanan N , et al. SNO-hemoglobin is not essential for red blood cell-dependent hypoxic vasodilation . Nat Med . 2008 ; 14 : 773 - 7 .
19. Schwab DE , Stamler JS , Singel DJ . Nitrite-methemoglobin inadequate for hypoxic vasodilation . Nat Chem Biol . 2009 ; 5 :366. author reply 367.
20. Bohmer A , Beckmann B , Sandmann J , Tsikas D. Doubts concerning functional endothelial nitric oxide synthase in human erythrocytes . Blood . 2012 ; 119 : 1322 - 3 .
21. Hall CN , Garthwaite J. What is the real physiological NO concentration in vivo? Nitric Oxide . 2009 ; 21 : 92 - 103 .
22. Azarov I , Huang KT , Basu S , Gladwin MT , Hogg N , Kim-Shapiro DB . Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation . J Biol Chem . 2005 ; 280 : 39024 - 32 .
23. Azarov I , Liu C , Reynolds H , Tsekouras Z , Lee JS , Gladwin MT , Kim-Shapiro DB . Mechanisms of slower nitric oxide uptake by red blood cells and other hemoglobin-containing vesicles . J Biol Chem . 2011 ; 286 : 33567 - 79 .
24. Gambaryan S , Subramanian H , Kehrer L , Mindukshev I , Sudnitsyna J , Reiss C , Rukoyatkina N , Friebe A , Sharina I , Martin E , Walter U. Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis . Cell Commun Signal . 2016 ; 14 : 16 .
25. Munzel T , Feil R , Mulsch A , Lohmann SM , Hofmann F , Walter U. Physiology and pathophysiology of vascular signaling controlled by guanosine 3',5'-cyclic monophosphate-dependent protein kinase [corrected] . Circulation . 2003 ; 108 : 2172 - 83 .
26. Bonello L , Camoin-Jau L , Arques S , Boyer C , Panagides D , Wittenberg O , Simeoni MC , Barragan P , Dignat-George F , Paganelli F. Adjusted clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation index decrease rate of major adverse cardiovascular events in patients with clopidogrel resistance: a multicenter randomized prospective study . J Am Coll Cardiol . 2008 ; 51 : 1404 - 11 .
27. Bonello L , Paganelli F , Arpin-Bornet M , Auquier P , Sampol J , Dignat-George F , Barragan P , Camoin-Jau L. Vasodilator-stimulated phosphoprotein phosphorylation analysis prior to percutaneous coronary intervention for exclusion of postprocedural major adverse cardiovascular events . J Thromb Haemost . 2007 ; 5 : 1630 - 6 .
28. Schwarz UR , Geiger J , Walter U , Eigenthaler M. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets-definition and detection of ticlopidine/clopidogrel effects . Thromb Haemost . 1999 ; 82 : 1145 - 52 .
29. Walter U , Gambaryan S. cGMP and cGMP-dependent protein kinase in platelets and blood cells . Handb Exp Pharmacol . 2009 ; 191 : 533 - 48 .
30. Gambaryan S , Tsikas D. A review and discussion of platelet nitric oxide and nitric oxide synthase: do blood platelets produce nitric oxide from L-arginine or nitrite? Amino Acids . 2015 ; 47 : 1779 - 93 .
31. Singel DJ , Stamler JS . Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin . Annu Rev Physiol . 2005 ; 67 : 99 - 145 .
32. Diesen DL , Hess DT , Stamler JS . Hypoxic vasodilation by red blood cells: evidence for an s-nitrosothiol-based signal . Circ Res . 2008 ; 103 : 545 - 53 .
33. Robinson JM , Lancaster Jr JR . Hemoglobin-mediated, hypoxia-induced vasodilation via nitric oxide: mechanism(s) and physiologic versus pathophysiologic relevance . Am J Respir Cell Mol Biol . 2005 ; 32 : 257 - 61 .
34. Kulandavelu S , Balkan W , Hare JM . Regulation of oxygen delivery to the body via hypoxic vasodilation . Proc Natl Acad Sci U S A . 2015 ; 112 : 6254 - 5 .