This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pawloski, J. R. Articles by Stamler, J. S. Search for Related Content PubMed PubMed Citation Articles by Pawloski, J. R. Articles by Stamler, J. S.

(Circulation. 1998;97:263-267.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Cell-Free and Erythrocytic S-Nitrosohemoglobin Inhibits Human Platelet Aggregation

John R. Pawloski; Rajesh V. Swaminathan; ; Jonathan S. Stamler

From the Howard Hughes Medical Institute (J.S.S.) and Department of Medicine (J.R.P., J.S.S.) and Division of Pulmonary and Cardiovascular Medicine (R.V.S., J.S.S.), Duke University Medical Center, Durham, NC.

Correspondence to Jonathan S. Stamler, MD, Duke University Medical Center, Howard Hughes Medical Institute, Room 321 MSRB, Box 2612, Durham, NC 27710.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Nitric oxide (NO) and related molecules are thought to inhibit human platelet aggregation by raising levels of cGMP.

Methods and Results—Both oxidative stress (reactive oxygen species) and hemoglobin (Hb) seem to oppose NO effects. A major fraction of NO in the blood is bound to thiols of Hb, forming S-nitrosohemoglobin (SNO-Hb), which releases the NO group on deoxygenation in the microcirculation. Here we show that (1) both cell-free and intraerythrocytic SNO-Hb (SNO-RBC) inhibit platelet aggregation, (2) the oxidation state of the hemes in Hb influences the response—SNO-metHb (which is functionally similar to SNO-deoxyHb) has greater platelet inhibitory effects than SNO-oxyHb, and (3) the mechanism of platelet inhibition by SNO-Hb is cGMP independent.

Conclusions—We suggest that the RBC has evolved a means to counteract platelet activation in small vessels and the proaggregatory effects of oxidative stress by forming SNO-Hb.

Key Words: S-nitrosohemoglobin • nitric oxide • platelets

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Inhibition of platelet aggregation by EDRF^{1 2 3} has significant clinical implications, particularly in the arena of vaso-occlusive disorders. Extensive experimental evidence shows that the inhibition of platelet function by EDRF/NO, both in vitro and in vivo, is directly associated with activation of soluble guanylate cyclase, which increases the intraplatelet content of the second messenger cGMP.^{4 5} That said, simulations of NO reactions and diffusion and mechanism of activation suggest that this free radical alone cannot account for all of the EDRF bioactivity.^{6 7} Specifically, NO would be scavenged by hemoglobin in RBCs. Accordingly, it has been suggested that more durable and potent SNOs of glutathione or cysteine may contribute to EDRF activity.⁸ SNOs have both potent vasorelaxant⁹ and platelet inhibitory properties^{10 11} involving activation of soluble guanylate cyclase as well as cGMP-independent actions, including direct modulation of membrane-associated ion channels¹² and enzymes.¹³ This broad range of activity reflects the ability of SNOs to regulate both heme- and thiol-containing proteins.^{13 14}

We have recently shown that hemoglobin in circulating RBCs transports NO as an S-nitroso adduct of Cysß93 and releases it selectively at low oxygen tension (PO₂) or when the heme is oxidized (metHb).¹⁵ This can be understood by appreciating that Hb exists in two alternative structures named R (high O₂ affinity) and T (low O₂ affinity) and that metHb is structurally similar to T-state Hb; thus, NO is transported bound to Cysß93 in R structure (high PO₂) and released to relax blood vessels in T structure (low PO₂).¹⁶ Here we show that (1) SNO-Hb, principally the met form, possesses potent antiaggregatory activity both in cell-free and erythrocytic systems and (2) attenuation of platelet aggregation was not associated with changes in platelet cGMP content as determined by radioimmunoassay. These observations suggest that SNO-Hb can physiologically modulate platelet function without increasing cGMP, ie, in a manner distinct from other SNOs.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolation of PRP and PPP
Venous blood (20 mL) was obtained from healthy volunteers who had not consumed aspirin or other nonsteroidal antiinflammatory drugs for at least 2 weeks and was anticoagulated in 13 mmol/L sodium citrate dihydrate. PRP was prepared by centrifugation of whole blood at 150g for 10 minutes, followed by aspiration of the upper two thirds of the plasma layer. PPP was prepared by centrifuging the remaining blood at 1500g for 15 minutes. An accurate platelet count was obtained with a Coulter Counter (model Z1, Coulter Electronics), and the count was then adjusted to 150 000 platelets per 1 µL of PRP with PPP as the diluent.

Syntheses
SNO-cys was prepared by reacting equimolar L-cysteine (in 0.5 eq/L HCl and 0.5 mmol/L EDTA) with NaNO₂ (in distilled water), followed by adjustment of the pH to 7.4 with phosphate buffer. MetHb was prepared by exposing oxyHbA₀ to a 10-fold excess of potassium ferricyanide for 5 minutes; excess ferricyanide was removed by twice desalting the protein over a Sephadex G-25 column. SNO-oxyHb and SNO-metHb were prepared by transnitrosylating oxy or met HbA₀ with a 10-fold excess of SNO-cys for 10 minutes, followed by desalting over a column of Sephadex G-25. The identity and concentration of SNO-oxy/metHb was confirmed by spectroscopic analysis by use of a Perkins-Elmer (Lambda2S) ultraviolet/visual dual-beam spectrophotometer.¹⁷ SNO content was quantified by a modified method of Saville¹⁸ as previously described.¹⁶ The S-nitroso hemoglobin derivatives had 2±0.2 SNO groups per hemoglobin tetramer.

Preparation of Human RBCs Containing S-Nitrosohemoglobin
After removal of PRP and PPP from whole blood as described above, the remaining RBC pellet was resuspended and washed in PBS (pH 7.4) twice, followed by centrifugation at 1000g. The RBC pellet was resuspended in PBS to a hematocrit of 10%. Hemoglobin was converted to the met (Fe³⁺) form by adding PBS saturated with NO gas to the RBC solution at a ratio of 1:4 (vol/vol). After 5 minutes, RBCs were pelleted at 1000g, followed by four washes in PBS.

To prepare SNO-RBCs, the RBC pellet (either oxyRBC or metRBC) was resuspended to a hematocrit of 10% in 2% borate and 0.9% NaCl, pH 9. A twofold excess of SNO-cys to Hb was then added to the RBC solution, which was incubated for 10 minutes. RBCs were pelleted at 1000g and washed four times with PBS. Lack of residual SNO-cys or free SNO-oxy/metHb (from hemolysis) was confirmed by spectroscopic analysis and assay of the supernatant for SNO. To quantify the intracellular concentration of SNO-Hb, a 500-µL aliquot of the final RBC pellet was combined with 1000 µL of distilled H₂O to lyse the RBCs. The lysate was twice desalted over a Sephadex G-25 column, and the concentration of SNO-Hb was determined.

Platelet Aggregation Studies
PRP was incubated with 10^-8 to 10^-4 mol/L native oxyHb, native metHb, SNO-oxyHb, or SNO-metHb for 10 minutes at 37°C in a PAP-4 aggregometer (Biodata). Aggregation was then initiated with 10 µmol/L ADP. Results were quantified by measurement of the rate or extent of change of light transmittance through the sample cuvette and are expressed as a normalized ratio of percent aggregation in the presence of hemoglobin to that obtained in the absence of the hemoprotein.

For SNO-RBC experiments, PRP (containing 150 000 to 300 000 platelets per 1 µL) was incubated at room temperature for 15 minutes with SNO-oxy/met RBCs or native oxy/met RBCs (10^-6 to 10^-4 mol/L) followed by differential centrifugation at 50g for 10 minutes to separate the platelets from RBCs. Control incubations of PPP were treated in the same fashion. The resulting PRP was placed in separate cuvettes, baseline transmittance was calibrated with the PPP retrieved from control incubates to normalize platelet counts, and platelet aggregation was initiated by the addition of 10 µmol/L ADP.

Platelet cGMP Determination
ADP (10 µmol/L) was added to 450 uL of PRP containing 150 000 platelets per 1 µL and native oxyHb, SNO-oxyHb, or SNO-metHb. After 1 minute, 300 µL of 10% (wt/vol) trichloroacetic acid was added to the platelet mixture to precipitate the proteins. Intraplatelet cGMP was assessed by radioimmunoassay as previously described.¹⁹

Materials
Purified human hemoglobin A₀ was kindly supplied by Apex Bioscience. All other chemicals were obtained from Sigma Chemical Co and were of the highest grade.

Statistical Analysis
All values are expressed as the mean±SEM. Differences in platelet aggregation and platelet cGMP levels in the presence or absence of the various hemoglobins or RBCs were evaluated with a one-way ANOVA, with posttest assessment done with the Bonferroni method. Statistical significance was assumed for P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of S-Nitrosylated Oxy (Fe²⁺) or Met (Fe³⁺) Hemoglobin on Platelet Aggregation
SNO-metHb caused a dose-dependent (10^-8 to 10^-4 mol/L) inhibition of ADP-evoked platelet aggregation with an IC₅₀ of 5.2±0.8 µmol/L (Fig 1a, open circles). In contrast, native metHb (Fig 1a, closed circles) had little effect on platelet aggregation. SNO-oxyHb (10^-8 to 10^-4 mol/L) had appreciably less effect on ADP-evoked platelet aggregation (Fig 1b, closed circles) than SNO-metHb.

View larger version (12K): [in this window] [in a new window]   Figure 1. Effects of SNO-Hb on platelet aggregation. a, Effect of cell-free SNO-metHb () and native metHb () on platelet aggregation. Baseline light transmittance was established with 10-6 to 10-4 mol/L SNO-metHb or native met-Hb in PPP. Data represent the extent of platelet aggregation normalized to control aggregation in the absence of hemoglobin and are presented as mean±SEM (n=4), * P<.05. b, Effect of cell-free SNO-oxyHb () and native oxyHb () on platelet aggregation. Platelets were exposed to SNO-oxyHb or native oxy-Hb for 15 minutes before the addition of ADP to induce aggregation. Baseline light transmittance was established by use of 10-6 to 10-4 mol/L SNO-oxyHb or native oxy-Hb in PPP. Data represent the extent of platelet aggregation normalized to control aggregation in the absence of hemoglobin. Data presented as mean±SEM (n=4), * P<.05.

Effect of RBCs Containing SNO-RBCs on Platelet Aggregation
Platelets were coincubated with SNO-oxyRBCs, SNO-metRBCs, native oxyRBCs, or native metRBCs for 15 minutes, followed by retrieval of the platelets by differential centrifugation. The coincubation of PRP with 10^-6 to 10^-4 mol/L SNO-metRBCs (Fig 2a, hatched bars) produced a dose-dependent attenuation of ADP-induced platelet aggregation that was significantly (P<.05) different from native metRBCs (Fig 2a, solid bars). SNO-oxyRBCs [final (SNO-oxyHb) of 10^-6 to 10^-4 mol/L] had no effect on ADP-induced platelet aggregation (Fig 2b, gray bars) compared with controls. Fig 3 is a representative platelet aggregation tracing illustrating the dose-dependent manner by which SNO-metRBCs inhibited platelet aggregation.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of SNO-RBCs on platelet aggregation. a, Effect of SNO-metRBC (solid bars) and native met-RBCs (open bars) on platelet aggregation. Baseline light transmittance was established by use of PPP coincubated with 10-6 to 10-4 mol/L met SNO-RBCs or native met-Hb followed by centrifugation at 50g (see "Methods"). Data are presented as mean±SEM (n=4), *P<.05. b, Effects of SNO-oxyRBCs (solid bars) and native oxy-RBCs (open bars) on platelet aggregation. Baseline light transmittance was established by use of PPP coincubated with 10-6 to 10-4 mol/L oxy SNO-RBCs or native oxy-Hb followed by centrifugation at 50g. Data are presented as mean±SEM (n=4). *P<.05.

View larger version (23K): [in this window] [in a new window]   Figure 3. Representative tracing illustrating dose-dependent inhibition of platelet aggregation by SNO-metRBCs. PRP was incubated with SNO-metRBCs (10-6 to 10-4 mol/L) for 15 minutes followed by separation by differential centrifugation as described in "Methods." Platelet aggregation was initiated by addition of 10 µmol/L ADP to a cuvette containing PRP and allowed to proceed for 5 to 7 minutes or until aggregation stabilized. Results are expressed as change in light transmittance per unit time (minutes).

Platelet cGMP Levels After Exposure to Native OxyHb, SNO-oxyHb, or SNO-metHb
Because low-molecular-weight SNOs inhibit platelet aggregation by activation of soluble guanylate cyclase and increases in the second messenger cGMP, the effects of cell-free SNO-oxy/metHb on platelet cGMP levels were also assessed. Interestingly, neither SNO-metHb (Fig 4, open bars) nor SNO-oxyHb (Fig 4, hatched bars) had any effect on platelet cGMP content compared with cGMP levels in the presence of native oxyHb (Fig 4, solid bars).

View larger version (30K): [in this window] [in a new window]   Figure 4. Intraplatelet cGMP concentrations following incubation with native hemoglobin, oxy (Fe2+) SNO-Hb, or met (Fe3+) SNO-Hb (10-6 to 10-4 mol/L). Exposure of platelets to any of the above hemoglobins had no effect on intraplatelet cGMP levels. Data are presented as mean±SEM (n=4). *P<.05.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
A substantial body of evidence has accumulated highlighting the proaggregatory properties of the RBC. Erythrocytes markedly enhance the platelet release reaction, eicosanoid biosynthesis, and platelet recruitment,^{20 21} effects that can be attenuated in human volunteers by daily ingestion of aspirin.²² RBCs are also rich sources of ADP, the spontaneous and/or evoked release of which has powerful platelet-activating effects in vitro.^{23 24} Likewise, RBCs contain hemoglobin, which readily scavenges EDRF and NO. Thus, not surprisingly, RBCs have been shown to reverse EDRF- and NO-induced inhibition of intravascular platelet aggregation.^{25 26}

How, then, does the platelet contend with the proaggregatory properties of RBCs? The answer may reside, in part, within the platelet. Specifically, both constitutive endothelial and inducible isoforms of NO synthase in the platelet cytosol^{27 28} release NO-related molecules in quantities similar to those liberated by endothelial cells. Thus, the platelet may act in an autocrine or paracrine fashion to inhibit its own activation. The answer may also lie in the vascular endothelium. Indeed, Sprague et al²⁹ recently found that human RBCs release ATP in response to mechanical deformation; this ATP evokes the release of NO, thereby lowering pulmonary vascular resistance. Endothelial activation in this manner should attenuate platelet aggregation. However, paradoxically, the RBC can mitigate NO-related activity by sequestering it. To counterbalance NO scavenging by the hemes in Hb, RBCs also carry NO attached to HbCysß93.¹⁶ SNO is released from SNO-Hb in the peripheral vasculature under low oxygen tension and on heme oxidation, thereby modulating arterial blood pressure and blood flow to tissues.^{15 16} In the present study, we expand the biologic properties of SNO-Hb to include inhibition of human platelet aggregation.

The incongruity in SNO-metHb and SNO-oxyHb activities derives partly from their structural differences. Human Hb exists in two conformational states named R (relaxed, high O₂ affinity) and T (tense, low O₂ affinity).³⁰ Hb transitions from R to T after liberation of the second O₂ molecule, mainly in the flow-regulating resistance blood vessels.³⁰ The allosteric transition affects NO group release from the thiol [ie, thiol affinity for NO is high in the R-(oxy) structure and low in the T-(deoxy) structure]. The structural and functional behavior of T-state Hb closely resembles that of metHb. That is, SNO-metHb, like T-state SNO-deoxyHb, dilates blood vessels, whereas SNO-oxyHb constricts them.¹⁶ Here we applied these structure-function relations to the platelet by showing the increased potency of SNO-metHb over SNO-oxyHb.

Interestingly, 10% of circulating Hb is normally oxidized to the met form in a 24-hour period, and metHb levels increase during oxidative stresses. NO group release from cysß93 on heme iron oxidation may serve to inhibit platelet activation engendered by oxidative stress. Thus, this protective function of RBCs may constitute a novel antioxidative mechanism. Equally important, SNO-metHb provides insight into the antiplatelet potential of SNO-oxyHb on assuming the deoxy (T) configuration in peripheral resistance microvessels and capillaries.

SNO-Hb did not affect platelet cGMP content. Although the lack of an increase in platelet cGMP associated with the activity of SNOs is unprecedented, the notion that cGMP does not fully account for the biologic properties of NO/SNO is not new. Indeed, SNO can activate calcium-dependent potassium channels in vascular smooth muscle,³¹ reduce cytosolic Ca²⁺ in guanylate cyclase–deficient fibroblasts,³² and modulate Ca²⁺ influx in PC12–64 cells,³³ all in a cGMP-independent manner. Significantly, SNOs can inhibit ATP-dependent Ca²⁺ uptake into platelet membrane vesicles in the absence of guanylate cyclase or cGMP.³⁴ The precise cellular mechanism of SNO-Hb–evoked inhibition of platelet aggregation, however, remains to be determined.

In summary, we demonstrate the capacity of SNO-Hb to modulate platelet function independently of cGMP and suggest regulation of this pathway by cellular redox state. These data expand the understanding of NO regulation in general and of SNO-Hb physiology in particular. The potential also exists for exploitation of this system for therapeutic purposes.

	Selected Abbreviations and Acronyms

 

Cysß93 = cysteine in the ß chain of hemoglobin (93 position) EDRF = endothelium-derived relaxing factor NO = nitric oxide PPP = platelet-poor plasma PRP = platelet-rich plasma RBC = red blood cell SNO = S-nitrosothiol SNO-cys = S-nitrosocysteine SNO-metHb = S-nitroso methemoglobin SNO-oxyHb = S-nitroso oxyhemoglobin SNO-RBC = intraerythrocytic S-nitrosylated oxy/met hemoglobin

	Acknowledgments

 
We wish to thank the members of the Stamler laboratory for their repeated contributions of whole blood for these studies.

Received June 26, 1997; revision received September 29, 1997; accepted September 30, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Azuma H, Ishikawa, M, Sekizaki S. Endothelium-dependent inhibition of platelet aggregation. Br J Pharmacol. 1986;88:411–415.[Medline] [Order article via Infotrieve]

2. Furlong B, Henderson AH, Lewis MJ, Smith JA. Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol. 1987;90:687–692.[Medline] [Order article via Infotrieve]

3. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet. 1987;2:1057–1058.[Medline] [Order article via Infotrieve]

4. Mellion TB, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3',5'-cyclic monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood. 1981;57:946–955.[Free Full Text]

5. Rovin JD, Stamler JS, Loscalzo J, Folts JD. Sodium nitroprusside, an endothelium-derived relaxing factor congener, increases platelet cyclic GMP levels and inhibits epinephrine-exacerbated in vivo platelet thrombus formation in stenosed canine coronary arteries. J Cardiovasc Pharmacol. 1993;22:626–631.[Medline] [Order article via Infotrieve]

6. Lancaster Jr. JR. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc Natl Acad Sci U S A. 1994;91:8137–8141.[Abstract/Free Full Text]

7. Makings LR, Tsien RY. Caged nitric oxide. Stable organic molecules from which nitric oxide can be photoreleased. J Biol Chem. 1994;269:6282–6285.[Abstract/Free Full Text]

8. Stamler JS. A radical vascular connection. Nature. 1996;380:108–111.[Medline] [Order article via Infotrieve]

9. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, Singel DJ, Loscalzo J. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A. 1992;89:444–448.[Abstract/Free Full Text]

10. Radomski MW, Rees DD, Dutra A, Moncada S. S-nitrosoglutathione inhibits platelet activation in vitro and in vivo. Br J Pharmacol. 1992;107:745–749.[Medline] [Order article via Infotrieve]

11. Simon DI, Stamler JS, Jaraki O, Keaney JF, Osborne JA, Francis SA, Singel DJ, Loscalzo J. Antiplatelet properties of protein S-nitrosothiols derived from nitric oxide and endothelium-derived relaxing factor. Arterioscler Thromb. 1993;13:791–799.[Abstract/Free Full Text]

12. Meszaros LG, Minarovic I, Zahradnikova A. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett. 1996;380:49–52.[Medline] [Order article via Infotrieve]

13. Stamler JS. Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell.. 1994;78:931–936.[Medline] [Order article via Infotrieve]

14. Hausladen A, Privalle CT, Keng T, DeAngelo J, Stamler JS. Nitrosative stress: activation of the transcription factor oxyR. Cell. 1996;86:719–729.[Medline] [Order article via Infotrieve]

15. Stamler JS, Jia L, Eu JP, McMahon TJ, Demchenko IT, Bonaventura J, Gernert K, Piantadosi CA. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science. 1997;276:2034–2037.[Abstract/Free Full Text]

16. Jia L, Bonaventura C, Bonaventura J, Stamler JS. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature. 1996;380:221–226.[Medline] [Order article via Infotrieve]

17. Kharitonov VG, Bonaventura J, Sharma VS. Interactions of nitric oxide with heme proteins using UV-Vis spectroscopy. In: Feelisch M, Stamler JS. Eds. Methods in Nitric Oxide Research. West Sussex, England: John Wiley & Sons Ltd; 1996:39–47.

18. Saville B. A scheme for the colorimetric determination of microgram amounts of thiols. Analyst. 1958;83:670–672.

19. Stamler JS, Mendelsohn ME, Amarante P, Smick D, Andon N, Davies PF, Cooke JP, Loscalzo J. N-acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor. Circ Res. 1989;65:789–795.[Abstract/Free Full Text]

20. Santos MT, Valles J, Marcus AJ, Safier LB, Broekman MJ, Islam N, Ullman HL, Eiroa AM, Aznar J. Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. J Clin Invest. 1991;87:571–580.

21. Valles J, Santos MT, Marcus AJ, Martinez-Sales V, Portoles M, Broekman MJ, Safier LB. Erythrocytes metabolically enhance platelet responsiveness via increased thromboxane production, ADP release, and recruitment. Blood. 1991;78:154–162.[Abstract/Free Full Text]

22. Santos MT, Valles J, Aznar J, Marcus AJ, Broekman MJ, Safier LB. Prothrombotic effects of erythrocytes on platelet activity. Reduction by aspirin. Circulation. 1997;95:63–68.[Abstract/Free Full Text]

23. Goldsmith HL, Bell DN, Braovac S, Steinberg A, McIntosh F. Physical and chemical effects of red cells in the shear-induced aggregation of human platelets. Biophys J. 1995;69:1584–1595.[Medline] [Order article via Infotrieve]

24. Saniabadi AR, Lowe GD, Barbenel JC, Forbes CD. Effect of dipyridamole on spontaneous platelet aggregation in whole blood decreases with the time after venipuncture: evidence for the role of ADP. Thromb Haemost. 1987;58:744–748.[Medline] [Order article via Infotrieve]

25. Ohno M, Kishimoto T, Jidoi J, Tada M. Assessment of production of endothelium-derived relaxing factor (EDRF) by cultured human vascular endothelial cells based on its anti-aggregatory effect on human platelets. Hum Cell. 1994;7:68–71.[Medline] [Order article via Infotrieve]

26. Houston DS, Robinson P, Gerrard JM. Inhibition of intravascular platelet aggregation by endothelium-derived relaxing factor: reversal by red blood cells. Blood. 1990;76:953–958.[Abstract/Free Full Text]

27. Mehta JL, Chen LY, Kone BC, Mehta P, Turner P. Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. J Lab Clin Med. 1995;125:370–377.[Medline] [Order article via Infotrieve]

28. Sase K, Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci. 1995;57:2049–2055.[Medline] [Order article via Infotrieve]

29. Sprague RS, Ellsworth ML, Stephenson AH, Lonigro AJ. ATP: the red blood cell link to NO and local control of the pulmonary circulation. Am J Physiol. 1996;271:H2717–H2722.[Abstract/Free Full Text]

30. Perutz MF. Molecular anatomy, physiology and pathology of hemoglobin. In: Stammatayanopoulos G, ed. Molecular Basis of Blood Diseases. Philadelphia, Pa; WB Saunders; 1987:127–178.

31. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature. 1994;368:850–853.[Medline] [Order article via Infotrieve]

32. Garg UC, Hassid A. Nitric oxide decreases cytosolic free calcium in Balb/c 3T3 fibroblasts by a cyclic GMP-independent mechanism. J Biol Chem. 1991;266:9–12.[Abstract/Free Full Text]

33. Clementi E, Vecchio I, Corasaniti MT, Nistico G. Nitric oxide modulates agonist-evoked Ca²⁺ release and influx responses in PC12–64 cells. Eur J Pharmacol. 1995;289:113–115.[Medline] [Order article via Infotrieve]

34. Pernollet MG, Lantoine F, Devynck MA. Nitric oxide inhibits ATP-dependent Ca²⁺ uptake into platelet membrane vesicles. Biochem Biophys Res Commun. 1996;222:780–785.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				E. Gkaliagkousi, J. Ritter, and A. Ferro Platelet-Derived Nitric Oxide Signaling and Regulation Circ. Res., September 28, 2007; 101(7): 654 - 662. [Abstract] [Full Text] [PDF]

				P. Sonveaux, I. I. Lobysheva, O. Feron, and T. J. McMahon Transport and Peripheral Bioactivities of Nitrogen Oxides Carried by Red Blood Cell Hemoglobin: Role in Oxygen Delivery Physiology, April 1, 2007; 22(2): 97 - 112. [Abstract] [Full Text] [PDF]

				J.-P. Bin, A. Doctor, J. Lindner, E. M. Hendersen, D. E. Le, H. Leong-Poi, N. G. Fisher, J. Christiansen, and S. Kaul Effects of Nitroglycerin on Erythrocyte Rheology and Oxygen Unloading: Novel Role of S-Nitrosohemoglobin in Relieving Myocardial Ischemia Circulation, May 30, 2006; 113(21): 2502 - 2508. [Abstract] [Full Text] [PDF]

				T. J. McMahon and A. Doctor Extrapulmonary effects of inhaled nitric oxide: role of reversible s-nitrosylation of erythrocytic hemoglobin. Proceedings of the ATS, January 1, 2006; 3(2): 153 - 160. [Abstract] [Full Text] [PDF]

				B. A. Klarl, P. A. Lang, D. S. Kempe, O. M. Niemoeller, A. Akel, M. Sobiesiak, K. Eisele, M. Podolski, S. M. Huber, T. Wieder, et al. Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion Am J Physiol Cell Physiol, January 1, 2006; 290(1): C244 - C253. [Abstract] [Full Text] [PDF]

				P. Sonveaux, A. M. Kaz, S. A. Snyder, R. A. Richardson, L. I. Cardenas-Navia, R. D. Braun, J. R. Pawloski, G. M. Tozer, J. Bonaventura, T. J. McMahon, et al. Oxygen Regulation of Tumor Perfusion by S-Nitrosohemoglobin Reveals a Pressor Activity of Nitric Oxide Circ. Res., May 27, 2005; 96(10): 1119 - 1126. [Abstract] [Full Text] [PDF]

				C. N. Morrell, K. Matsushita, K. Chiles, R. B. Scharpf, M. Yamakuchi, R. J. A. Mason, W. Bergmeier, J. L. Mankowski, W. M. Baldwin III, N. Faraday, et al. Regulation of platelet granule exocytosis by S-nitrosylation PNAS, March 8, 2005; 102(10): 3782 - 3787. [Abstract] [Full Text] [PDF]

				J. R. Pawloski, D. T. Hess, and J. S. Stamler Impaired vasodilation by red blood cells in sickle cell disease PNAS, February 15, 2005; 102(7): 2531 - 2536. [Abstract] [Full Text] [PDF]

				M. Beghetti, C. Sparling, P. N. Cox, D. Stephens, and I. Adatia Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H637 - H642. [Abstract] [Full Text] [PDF]

				T. J. McMahon, J. R. Pawloski, D. T. Hess, C. A. Piantadosi, B. P. Luchsinger, D. J. Singel, J. S. Stamler, J. H. Crawford, C. R. White, and R. P. Patel S-nitrosohemoglobin is distinguished from other nitrosovasodilators by unique oxygen-dependent responses that support an allosteric mechanism of action Blood, July 1, 2003; 102(1): 410 - 411. [Full Text] [PDF]

				J. H. Crawford, C. R. White, and R. P. Patel Vasoactivity of S-nitrosohemoglobin: role of oxygen, heme, and NO oxidation states Blood, June 1, 2003; 101(11): 4408 - 4415. [Abstract] [Full Text] [PDF]

				D. A. Andrews, L. Yang, and P. S. Low Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells Blood, October 16, 2002; 100(9): 3392 - 3399. [Abstract] [Full Text] [PDF]

				Y.-C. Tyan, J.-D. Liao, Y.-T. Wu, and R. Klauser Anticoagulant Activity of Immobilized Heparin on the Polypropylene Nonwoven Fabric Surface Depending upon the pH of Processing Environment J Biomater Appl, October 1, 2002; 17(2): 153 - 178. [Abstract] [PDF]

				U. Schmidt, R. O. Han, T. G. DiSalvo, J. L. Guerrero, H. K. Gold, W. M. Zapol, K. D. Bloch, and M. J. Semigran Cessation of platelet-mediated cyclic canine coronary occlusion after thrombolysis by combining nitric oxide inhalation with phosphodiesterase-5 inhibition J. Am. Coll. Cardiol., June 1, 2001; 37(7): 1981 - 1988. [Abstract] [Full Text] [PDF]

				J. Bonaventura and V. P. Lance Nitric Oxide, Invertebrates and Hemoglobin Integr. Comp. Biol., April 1, 2001; 41(2): 346 - 359. [Abstract] [Full Text] [PDF]

				A.C. Mendes Ribeiro, T.M.C. Brunini, J.C. Ellory, and G.E. Mann Abnormalities in L-arginine transport and nitric oxide biosynthesis in chronic renal and heart failure Cardiovasc Res, March 1, 2001; 49(4): 697 - 712. [Abstract] [Full Text] [PDF]

				N. P. Andrews, M. Husain, N. Dakak, and A. A. Quyyumi Platelet inhibitory effect of nitric oxide in the human coronary circulation: impact of endothelial dysfunction J. Am. Coll. Cardiol., February 1, 2001; 37(2): 510 - 516. [Abstract] [Full Text] [PDF]

				N. Ikebe, T. Akaike, Y. Miyamoto, K. Hayashida, J. Yoshitake, M. Ogawa, and H. Maeda Protective Effect of S-Nitrosylated alpha 1-Protease Inhibitor on Hepatic Ischemia-Reperfusion Injury J. Pharmacol. Exp. Ther., December 1, 2000; 295(3): 904 - 911. [Abstract] [Full Text]

				M. Wolzt, R. J. MacAllister, D. Davis, M. Feelisch, S. Moncada, P. Vallance, and A. J. Hobbs Biochemical Characterization of S-Nitrosohemoglobin. MECHANISMS UNDERLYING SYNTHESIS, NO RELEASE, AND BIOLOGICAL ACTIVITY J. Biol. Chem., October 8, 1999; 274(41): 28983 - 28990. [Abstract] [Full Text] [PDF]

				R. E. Glover, E. D. Ivy, E. P. Orringer, H. Maeda, and R. P. Mason Detection of Nitrosyl Hemoglobin in Venous Blood in the Treatment of Sickle Cell Anemia with Hydroxyurea Mol. Pharmacol., June 1, 1999; 55(6): 1006 - 1010. [Abstract] [Full Text]

				R. P. Patel, N. Hogg, N. Y. Spencer, B. Kalyanaraman, S. Matalon, and V. M. Darley-Usmar Biochemical Characterization of Human S-Nitrosohemoglobin. EFFECTS ON OXYGEN BINDING AND TRANSNITROSATION J. Biol. Chem., May 28, 1999; 274(22): 15487 - 15492. [Abstract] [Full Text] [PDF]

				T. O. Nossuli, R. Hayward, D. Jensen, R. Scalia, and A. M. Lefer Mechanisms of cardioprotection by peroxynitrite in myocardial ischemia and reperfusion injury Am J Physiol Heart Circ Physiol, August 1, 1998; 275(2): H509 - H519. [Abstract] [Full Text] [PDF]

				T. J. McMahon, A. Exton Stone, J. Bonaventura, D. J. Singel, and J. Solomon Stamler Functional Coupling of Oxygen Binding and Vasoactivity in S-Nitrosohemoglobin J. Biol. Chem., May 26, 2000; 275(22): 16738 - 16745. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pawloski, J. R. Articles by Stamler, J. S. Search for Related Content PubMed PubMed Citation Articles by Pawloski, J. R. Articles by Stamler, J. S.