A predominant role for cAMP-dependent protein kinase in the cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein and platelet inhibition in humans.

The vasodilator-stimulated phosphoprotein (VASP) plays an important role in cGMP-induced platelet inhibition. Since VASP is an in vitro substrate for cGMP-dependent protein kinase (PKG), it has been presumed that VASP phosphorylation induced by cGMP is mediated by PKG. Here we show that, in human platelets, phosphorylation of VASP at Ser239 induced by either cGMP analogs or nitric oxide (NO) donor glyco-SNAP1 is inhibited by PKA inhibitors KT5720, PKI, Rp-Br-cAMPS, and H89, but not by PKG inhibitors KT5823 or Rp-pCPT-cGMPS. Unlike human platelets, cGMP analog-induced phosphorylation of VASP in mouse platelets is inhibited by both PKG and PKA inhibitors. Ineffectiveness of PKG inhibitors in inhibiting VASP phosphorylation in human platelets is not due to an inability to inhibit PKG, as these PKG inhibitors but not PKA inhibitors inhibit a different cGMP-induced intracellular signaling event: phosphorylation of extracellular signal-responsive kinase. Furthermore, PKA inhibitors reverse cGMP-induced inhibition of thrombin-induced platelet aggregation, whereas PKG inhibitors further enhance the inhibitory effect of cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of VASP and platelet inhibition in human platelets.

[1]  F. Hofmann,et al.  A Stimulatory Role for cGMP-Dependent Protein Kinase in Platelet Activation , 2003, Cell.

[2]  R. Haslam,et al.  Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen‐induced platelet aggregation by nitroprusside * , 2002, British journal of haematology.

[3]  Xiaoping Du,et al.  A Mitogen-activated Protein Kinase-dependent Signaling Pathway in the Activation of Platelet Integrin αIIbβ3 * , 2001, The Journal of Biological Chemistry.

[4]  R. Bodnar,et al.  Regulation of von Willebrand Factor Binding to the Platelet Glycoprotein Ib-IX by a Membrane Skeleton-dependent Inside-out Signal* , 2001, The Journal of Biological Chemistry.

[5]  U. Walter,et al.  Actin-based motility: stop and go with Ena/VASP proteins. , 2001, Trends in biochemical sciences.

[6]  FranzHofmann,et al.  Mechanisms of NO/cGMP-Dependent Vasorelaxation , 2000 .

[7]  U. Walter,et al.  KT5823 Inhibits cGMP-dependent Protein Kinase Activity in Vitro but Not in Intact Human Platelets and Rat Mesangial Cells* , 2000, Journal of Biological Chemistry.

[8]  T. Lincoln,et al.  Activation of Mitogen-activated Protein Kinase Pathways by Cyclic GMP and Cyclic GMP-dependent Protein Kinase in Contractile Vascular Smooth Muscle Cells* , 1999, The Journal of Biological Chemistry.

[9]  R. Haslam,et al.  Cyclic Nucleotides and Phosphodiesterases in Platelets , 1999, Thrombosis and Haemostasis.

[10]  K. Knobeloch,et al.  Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Klatt,et al.  Increased Adhesion and Aggregation of Platelets Lacking Cyclic Guanosine 3′,5′-Monophosphate Kinase I , 1999, The Journal of experimental medicine.

[12]  A. Aszódi,et al.  The vasodilator‐stimulated phosphoprotein (VASP) is involved in cGMP‐ and cAMP‐mediated inhibition of agonist‐induced platelet aggregation, but is dispensable for smooth muscle function , 1999, The EMBO journal.

[13]  H. Granger,et al.  Protein Kinase G Mediates Vascular Endothelial Growth Factor-induced Raf-1 Activation and Proliferation in Human Endothelial Cells* , 1998, The Journal of Biological Chemistry.

[14]  U. Walter,et al.  Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody* , 1998, The Journal of Biological Chemistry.

[15]  T. Lincoln,et al.  Mechanism of platelet inhibition by nitric oxide: in vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Wehland,et al.  Mena, a Relative of VASP and Drosophila Enabled, Is Implicated in the Control of Microfilament Dynamics , 1996, Cell.

[17]  M. Trueba,et al.  Thrombin-stimulated phospholipase C activity is inhibited without visible delay by a rapid increase in the cyclic GMP levels induced by sodium nitroprusside. , 1996, Molecular pharmacology.

[18]  U. Walter,et al.  Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. , 1994, European journal of biochemistry.

[19]  M. Krieger,et al.  cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. , 1994, The Journal of biological chemistry.

[20]  U. Walter,et al.  Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia. , 1993, The Journal of biological chemistry.

[21]  J. Beavo,et al.  Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP. , 1992, Biochemical pharmacology.

[22]  Xiaoping Du,et al.  Ligands “activate” integrin α IIb β 3 (platelet GPIIb-IIIa) , 1991, Cell.

[23]  R. Haslam,et al.  Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP. , 1990, Molecular pharmacology.

[24]  M. Hagiwara,et al.  Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. , 1990, The Journal of biological chemistry.

[25]  U. Walter,et al.  Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators. , 1990, The Journal of biological chemistry.

[26]  U. Walter,et al.  Purification of a vasodilator-regulated phosphoprotein from human platelets. , 1989, European journal of biochemistry.

[27]  U. Walter,et al.  Vasodilator-stimulated protein phosphorylation in platelets is mediated by cAMP- and cGMP-dependent protein kinases. , 1987, European journal of biochemistry.

[28]  R. Colman,et al.  Purification and characterization of a human platelet cyclic nucleotide phosphodiesterase. , 1984, Biochemistry.

[29]  M. Haddox,et al.  Cyclic GMP metabolism and involvement in biological regulation. , 1977, Annual review of biochemistry.

[30]  E. Beachey,et al.  Interaction of a chick skin collagen fragment (alpha1-CB5) with human platelets. Biochemical studies during the aggregation and release reaction. , 1975, The Journal of biological chemistry.

[31]  R. Haslam,et al.  Cytochalasin B, the blood platelet release reaction and cyclic GMP , 1975, Nature.