Skip to main content
SpringerLink
Log in

The Relative Role of Soluble Guanylyl Cylase Dependent and Independent Pathways in Nitric Oxide Inhibition of Platelet Aggregation Under Flow

  • Published:
Cellular and Molecular Bioengineering Aims and scope Submit manuscript

Abstract

Endothelial cell derived nitric oxide (NO) inhibits the activation and aggregation of platelets. NO inhibition occurs through the intracellular receptor soluble guanylyl cyclase (sGC)-dependent pathways, but there is also evidence of sGC-independent pathways at high NO concentrations. In this study, we integrated a NO-releasing polymer into a microfluidic vascular injury model to measure the relative roles of sGC-dependent and sGC-independent pathways as a function of NO flux and shear rate. Whole blood was perfused at 200–1000 s−1 over collagen with NO wall fluxes of 0.4 and 6.8 × 10−10 mol cm−2 min−1, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to inhibit sGC. A sGC-independent pathway dominated inhibition of platelet aggregation at high NO flux, while the sGC-dependent pathway dominated at low NO flux independent of shear rate. Experiments performed with inhibitors of thrombin or an antagonist of the ADP receptor P2Y12 showed that platelet aggregation was primarily driven by ADP, but that the sGC-independent pathway dominated in both cases at high NO flux. These data suggest that a sGC-independent pathway may play an important role under conditions where NO flux is elevated such as inducible nitric oxide mediated NO production at the site of a vascular injury.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Antl, M., et al. IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation. Blood 109:552–559, 2007.

    Article  Google Scholar 

  2. Ballou, D. P., Y. Zhao, P. E. Brandish, and M. A. Marletta. Revisiting the kinetics of nitric oxide (NO) binding to soluble guanylate cyclase: the simple NO-binding model is incorrect. Proc. Natl. Acad. Sci. USA 99:12097–12101, 2002.

    Article  Google Scholar 

  3. Batchelor, M. M., et al. More lipophilic dialkyldiamine-based diazeniumdiolates: synthesis, characterization, and application in preparing thromboresistant nitric oxide release polymeric coatings. J. Med. Chem. 46:5153–5161, 2003.

    Article  Google Scholar 

  4. Bolte, S., and F. P. Cordelières. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224:213–232, 2006.

    Article  MathSciNet  Google Scholar 

  5. Chatterjee, M. S., J. E. Purvis, L. F. Brass, and S. L. Diamond. Pairwise agonist scanning predicts cellular signaling responses to combinatorial stimuli. Nat. Biotechnol. 28:727–732, 2010.

    Article  Google Scholar 

  6. Condorelli, P., and S. C. George. In vivo control of soluble guanylate cyclase activation by nitric oxide: a kinetic analysis. Biophys. J. 80:2110–2119, 2001.

    Article  Google Scholar 

  7. Dangel, O., E. Mergia, K. Karlisch, D. Groneberg, D. Koesling, and A. Friebe. Nitric oxide-sensitive guanylyl cyclase is the only nitric oxide receptor mediating platelet inhibition. J. Thromb. Haemost. 8:1343–1352, 2010.

    Article  Google Scholar 

  8. Doni, M. G., L. Cavallini, and A. Alexandre. Ca2+ influx in platelets: activation by thrombin and by the depletion of the stores. Effect of cyclic nucleotides. Biochem. J. 303:599–605, 1994.

    Google Scholar 

  9. Folie, B. J., and L. V. McIntire. Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow. Biophys. J. 56:1121–1141, 2005.

    Article  Google Scholar 

  10. Garthwaite, J., E. Southam, C. L. Boulton, E. B. Nielsen, K. Schmidt, and B. Mayer. Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol. Pharmacol. 48:184–188, 1995.

    Google Scholar 

  11. Hansen, R. R., A. A. Tipnis, T. C. White-Adams, J. A. Di Paola, and K. B. Neeves. Characterization of collagen thin films for von Willebrand factor binding and platelet adhesion. Langmuir 27:13648–13658, 2011.

    Article  Google Scholar 

  12. Hansen, R. R., A. R. Wufsus, S. T. Barton, A. A. Onasoga, R. M. Johnson-Paben, and K. B. Neeves. High content evaluation of shear dependent platelet function in a Microfluidic flow assay. Ann. Biomed. Eng. 41:250–263, 2012.

    Article  Google Scholar 

  13. Heemskerk, J., K. S. Sakariassen, J. J. Zwaginga, L. F. Brass, S. P. Jackson, and R. W. Farndale. Collagen surfaces to measure thrombus formation under flow: possibilities for standardization. J. Thromb. Haemost. 9:856–858, 2011.

    Article  Google Scholar 

  14. Homer, K. L., and J. C. Wanstall. Cyclic GMP-independent relaxation of rat pulmonary artery by spermine NONOate, a diazeniumdiolate nitric oxide donor. Br. J. Pharmacol. 131:673–682, 2000.

    Article  Google Scholar 

  15. Homer, K. L., and J. C. Wanstall. Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate. Br. J. Pharmacol. 137:1071–1081, 2002.

    Article  Google Scholar 

  16. Kibbe, M. Inducible nitric oxide synthase and vascular injury. Cardiovasc. Res. 43:650–657, 1999.

    Article  Google Scholar 

  17. Lies, B., D. Groneberg, S. Gambaryan, and A. Friebe. Lack of effect of ODQ does not exclude cGMP signalling via NO-sensitive guanylyl cyclase. Br. J. Pharmacol. 170:317–327, 2013.

    Article  Google Scholar 

  18. Marcondes, S., et al. Cyclic GMP-independent mechanisms contribute to the inhibition of platelet adhesion by nitric oxide donor: a role for α-actinin nitration. Proc. Natl. Acad. Sci. USA 103:3434–3439, 2006.

    Article  Google Scholar 

  19. Marjanovic, J. A., Z. Li, A. Stojanovic, and X. Du. Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation. J. Biol. Chem. 280:37430–37438, 2005.

    Article  Google Scholar 

  20. Morrell, C. N. Regulation of platelet granule exocytosis by S-nitrosylation. Proc. Natl. Acad. Sci. USA 102:3782–3787, 2005.

    Article  Google Scholar 

  21. Naseem, K. M., and W. Roberts. Nitric oxide at a glance. Platelets 22:148–152, 2011.

    Article  Google Scholar 

  22. Neeves, K. B., O. J. T. McCarty, A. J. Reininger, M. Sugimoto, M. R. King, and The Biorheology Subcommittee of the SSC of the ISTH. Flow-dependent thrombin and fibrin generation in vitro: opportunities for standardization: communication from SSC of the ISTH. J. Thromb. Haemost. 12:418–420, 2014.

    Article  Google Scholar 

  23. Onasoga-Jarvis, A. A., T. J. Puls, S. K. O’Brien, L. Kuang, H. J. Liang, and K. B. Neeves. Thrombin generation and fibrin formation under flow on biomimetic tissue factor rich surfaces. J. Thromb. Haemost. 12:373–382, 2014.

    Article  Google Scholar 

  24. Plata, A. M., S. J. Sherwin, and R. Krams. Endothelial nitric oxide production and transport in flow chambers: the importance of convection. Ann. Biomed. Eng. 38:2805–2816, 2010.

    Article  Google Scholar 

  25. Pugh, N., A. M. C. Simpson, P. A. Smethurst, P. G. de Groot, N. Raynal, and R. W. Farndale. Synergism between platelet collagen receptors defined using receptor-specific collagen-mimetic peptide substrata in flowing blood. Blood 115:5069–5079, 2010.

    Article  Google Scholar 

  26. Roberts, W., R. Riba, S. Homer-Vanniasinkam, R. W. Farndale, and K. M. Naseem. Nitric oxide specifically inhibits integrin-mediated platelet adhesion and spreading on collagen. J. Thromb. Haemost. 6:2175–2185, 2008.

    Article  Google Scholar 

  27. Roest, M., A. Reininger, J. J. Zwaginga, M. R. King, and J. W. M. Heemskerk. Flow chamber-based assays to measure thrombus formation in vitro: requirements for standardization. J. Thromb. Haemost. 9:2322–2324, 2011.

    Article  Google Scholar 

  28. Ruggeri, Z. M., and G. L. Mendolicchio. Adhesion mechanisms in platelet function. Circ. Res. 100:1673–1685, 2007.

    Article  Google Scholar 

  29. Schrammel, A., S. Behrends, K. Schmidt, D. Koesling, and B. Mayer. Characterization of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase. Mol. Pharmacol. 50:1–5, 1996.

    Google Scholar 

  30. Sylman, J. L., S. M. Lantvit, M. C. VeDepo, M. M. Reynolds, and K. B. Neeves. Transport limitations of nitric oxide inhibition of platelet aggregation under flow. Ann. Biomed. Eng. 274:2193–2205, 2013.

    Article  Google Scholar 

  31. Trepakova, E. S., R. A. Cohen, and V. M. Bolotina. Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase dependent refilling of Ca2+ stores. Circ. Res. 84:201–209, 1999.

    Article  Google Scholar 

  32. Tsai, M., et al. In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology. J. Clin. Investig. 122:408–418, 2012.

    Article  Google Scholar 

  33. van Gestel, M. A. In vivo blockade of platelet ADP receptor P2Y12 reduces embolus and thrombus formation but not thrombus stability. Arterioscler. Thromb. Vasc. Biol. 23:518–523, 2003.

    Article  Google Scholar 

  34. Van Kruchten, R., J. M. Cosemans, and J. W. Heemskerk. Measurement of whole blood thrombus formation using parallel-plate flow chambers—a practical guide. Platelets 23:229–242, 2012.

    Article  Google Scholar 

  35. Vaughn, M. W., L. Kuo, and J. C. Liao. Estimation of nitric oxide production and reaction rates in tissue by use of a mathematical model. Am. J. Physiol. Heart Circ. Physiol. 274:H2163–H2176, 1998.

    Google Scholar 

  36. Wanstall, J. C., K. L. Homer, and S. A. Doggrell. Evidence for, and importance of, cGMP-independent mechanisms with NO and NO donors on blood vessels and platelets. Curr. Vasc. Pharmacol. 3:41–53, 2005.

    Article  Google Scholar 

  37. Zhang, G., et al. Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation. Blood 118:3670–3679, 2011.

    Article  Google Scholar 

Download references

Acknowledgments

This material is based upon work supported by the National Science Foundation under Grant No. CBET-1351672, the American Heart Association (10SDG2610066, K.B.N.) and Boettcher Foundation Webb-Waring Biomedical Research Awards (K.B.N., M.M.R.).

Conflict of interest

JL.S., S.M.L, M.M.R. and K.B.N declare that they have no conflicts of interest.

Ethical Standards

All human subjects research was carried out in accordance with the Declaration of Helsinki and under the University of Colorado, Boulder Institutional Review Board approval. No animal studies were carried out by the authors for this article.

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Colorado School of Mines, 1500 Illinois Street, Golden, CO, 80401, USA

    J. L. Sylman & Keith B. Neeves

  2. Department of Chemistry, Colorado State University, Fort Collins, CO, USA

    S. M. Lantvit & M. M. Reynolds

  3. School of Biomedical Engineering, Colorado State University, Fort Collins, CO, USA

    M. M. Reynolds

  4. Department of Pediatrics, University of Colorado Denver, Aurora, CO, USA

    Keith B. Neeves

Authors
  1. J. L. Sylman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. M. Lantvit
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. M. Reynolds
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Keith B. Neeves
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Keith B. Neeves.

Additional information

Associate Editor Michael R. King oversaw the review of this article.

This paper is part of the 2014 Young Innovators Issue.

Keith B. Neeves is an assistant professor in Chemical and Biological Engineering at the Colorado School of Mines. He obtained his B.S. in chemical engineering from the University of Colorado, Boulder and a Ph.D. in chemical and biomolecular engineering at Cornell University. He was an NIH NRSA postdoctoral fellow at the University of Pennsylvania. His laboratory focuses on transport phenomena in biological tissues. His research has been recognized with a NSF CAREER award, a Scientist Development Grant from the American Heart Association, and Early Career Investigator Awards from the Boettcher Foundation Waring-Webb Biomedical Research Foundation and the Bayer Hemophilia Awards Program.

figure a

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 542 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sylman, J.L., Lantvit, S.M., Reynolds, M.M. et al. The Relative Role of Soluble Guanylyl Cylase Dependent and Independent Pathways in Nitric Oxide Inhibition of Platelet Aggregation Under Flow. Cel. Mol. Bioeng. 7, 421–431 (2014). https://doi.org/10.1007/s12195-014-0331-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12195-014-0331-1

Keywords

Search

Navigation