title: Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis.
abstract: Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets.
  
  
  
  To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASP(S239)) by flow cytometry and Western blot. ANOVA followed by Bonferroni's test and Student's t-test were used as appropriate.
  
  
  
  We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite.
  
  
  
  All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.
authors: Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Grombuehlstraße 12, D-97080, Wuerzburg, Germany. gambaryan@klin-biochem.uni-wuerzburg.de.|Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, St, Petersburg, 194223, Russia. gambaryan@klin-biochem.uni-wuerzburg.de.|Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany. gambaryan@klin-biochem.uni-wuerzburg.de.
authors: Stepan
authors: S
authors: Gambaryan
authors: Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Hariharan
authors: H
authors: Subramanian
authors: Institute of Physiology, University of Wuerzburg, Wuerzburg, Germany.
authors: Linda
authors: L
authors: Kehrer
authors: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, St, Petersburg, 194223, Russia.
authors: Igor
authors: I
authors: Mindukshev
authors: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, St, Petersburg, 194223, Russia.
authors: Julia
authors: J
authors: Sudnitsyna
authors: Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany.
authors: Cora
authors: C
authors: Reiss
authors: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, St, Petersburg, 194223, Russia.
authors: Natalia
authors: N
authors: Rukoyatkina
authors: Institute of Physiology, University of Wuerzburg, Wuerzburg, Germany.
authors: Andreas
authors: A
authors: Friebe
authors: Department of Internal Medicine, Division of Cardiology, University of Texas Houston Medical School, Houston, USA.
authors: Iraida
authors: I
authors: Sharina
authors: Department of Internal Medicine, Division of Cardiology, University of Texas Houston Medical School, Houston, USA.
authors: Emil
authors: E
authors: Martin
authors: Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany.|German Centre for Cardiovascular Research (DZHK) RheinMain, Mainz, Germany.
authors: Ulrich
authors: U
authors: Walter
mesh_terms: D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*
chemical_list: D009569:Nitric Oxide; D006162:Guanylate Cyclase
keywords: Erythrocytes; Hemoglobin; Nitric oxide; Platelets; Soluble guanylate cyclase