Molecular mechanisms of von Willebrand-factor (VWF)-stimulated, NO-independent acitivation of soluble guanylyl cyclase (sGC) in human platelets
Final Report Abstract
The fine-tuned interplay of stimulatory and inhibitory pathways affecting thrombus formation and platelet function is physiologically essential and impaired in many diseases. The main focus of our research for many years addresses the mechanisms of cyclic nucleotide-mediated platelet inhibition. Recently, we focused on feed-back inhibitory mechanisms mediated by cGMP/PKG and cAMP/PKA pathways. For cAMP/PKA system we established a new feedback inhibitory signalling pathway in thrombin and collagen stimulated platelets mediated by dissociation of PKAc from NFkB-lkB complex. In this project, we studied the NOS-independent regulation of the platelet inhibitory pathway sGC/ cGMP/ PKG by vWF and other platelet agonists. We tested several mechanisms of sGC activation mediated by posttranslational modifications (translocation of sGC to the plasma membrane, interaction with Hsp and CCT proteins, phosphorylation of other sGC tyrosines and/or Ser/Thr) and did not find any changes in platelets stimulated with VWF compared to control platelets. We then generated a platelet specific sGC KO mouse model (PS-GCKO), tested several other compounds (hirudin, refludan, argotraban) which also NOS-independently activate platelet sGC and stimulate VASP phosphorylation. We also established and achieved other preliminary data by in vitro sGC activation experiments. We show that low molecular weight (≤ 3 kDa) platelet filtrate can activate purified sGC. Taking into account that feed-back inhibitory mechanisms play very important role in platelet function and thrombus formation we will continue these projects in the future. We will focus on the nature of platelet specific endogenous low molecular weight (LMM; ≤ 3 kDa ) sGC activator which could represent a) some unknown natural sGC activator, and/or b) NO produced / released from NOS-independent sources. Using several different approaches and newly developed tools we will continued to elucidate the potentially very important mechanism of NOS-independent platelet sGC activation by platelet agonists such as vWF and others.
Publications
- cGMP and cGMP-Dependent Protein Kinase in Platelets and Blood Cells. Handb Exp Pharmacol. 2009.191: 533-48
Walter U, Gambaryan S
- (2010). cGMP and PKG signaling in platelets. In: RA. Bradshaw and EA. Dennis eds. Handbook of Cell Signaling 2nd edition. Oxford: Academic Press, pp. 1563 -1568
Gambaryan S., Walter U.
- (2010). Thrombin and collagen induce a feedback inhibitory signaling pathway in platelets involving dissociation of the catalytic subunit of PKA from an NF-kB-IkB complex. J. Biol. Chem. 285:18352 - 18363
Gambaryan S, Kobsar A, Rukoyatkina N, Herterich S, Geiger J, Smolenski A, Lohmann SM, Walter U
- (2011). Differentiation of cGMP-dependent and -independent nitric oxide effects on platelet apoptosis and reactive oxygen species production using platelets lacking soluble guanylyl cyclase. Thromb. Haemost. 106: 922 - 933
Rukoyatkina N, Walter U, Friebe A, Gambaryan S
- (2012). Does the NO/sGC/cGMP/PKG pathway play a sfimulatory role in platelets? Blood. 119: 535 - 536
Gambaryan S, Friebe A, Walter U
- (2012). DT-2 is a specific PKG I inhibitor only in vitro, but not in living cells. Br J Pharmacol.
Gambaryan S, Butt E, Kobsar A, Geiger J, Rukoyatkina N, Parnova R, Nikolaev VO, Walter U
(See online at https://doi.org/10.1111/j.1476-5381.2012.02044.x) - (2012). The thrombin inhibitors, hirudin und Refludan activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets. Thromb Haemost. 107: 521 - 529
Kobsar A, Koessler J, Kehrer L, Gambaryan S, Walter U
(See online at https://doi.org/10.1160/TH11-07-0461)
