scholarly journals Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1475-1482 ◽  
Author(s):  
Wei Zhang ◽  
Robert W. Colman
Keyword(s):  
Cyclic Amp ◽  
Platelet Activation ◽  
Selective Inhibition ◽  
Human Platelets ◽  
Intracellular Camp ◽  
Akt Inhibitor ◽  
Camp Content ◽  
Fibrinogen Receptor ◽  
Thrombin Activation ◽  
Camp Hydrolysis

Abstract Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca++ mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y12 inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.

Thrombin Regulates the Camp Level in Human Platelets through Phosphorylation /Activation of Akt.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3538-3538
Author(s):  
Wei Zhang ◽  
Gary Nale ◽  
Robert W. Colman
Keyword(s):  
Kinase Inhibitor ◽  
Specific Inhibitor ◽  
Human Platelets ◽  
Camp Level ◽  
Critical Event ◽  
Intracellular Camp ◽  
Akt Inhibitor ◽  
Dependent Inhibition ◽  
Thrombin Activation ◽  
Phosphoinositide 3 Kinase

Abstract Phosphorylation /activation of Akt is a critical event in platelet activation stimulated by thrombin. We previous demonstrated that the activation of PDE3A was increased concomitantly with the phosphorylation /activation of Akt. In order to demonstrate a link between these two events, in this study, we monitored thrombin-induced cAMP changes in washed platelets. We confirmed that the platelet cAMP level decreased following thrombin stimulation. The thrombin-induced decrease of cAMP was inhibited by an Akt specific inhibitor (1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate). This Akt inhibitor exhibited a concentration-dependent inhibition of thrombin-induced decrease of cAMP. The Akt inhibitor blocked 70 % of thrombin-induced decrease of cAMP. Moreover, a phosphoinositide 3-kinase inhibitor wortmannin also reduced the thrombin effect, consistent with its upstream position relative to Akt. These results suggested that phosphorylation /activation of Akt is required for decrease of cAMP in platelets. In addition, we found that thrombin-induced cAMP level changes were markedly reduced following preincubation of platelets with milrinone, a selective PDE3A inhibitor. We therefore examined PDE3A activity after stimulation of platelets with thrombin with or without the Akt inhibitor or wortmannin. Either the Akt inhibitor or wortmannin inhibited the thrombin-induced activation of PDE3A. Our data indicated that phosphorylation/activation of Akt plays a major role in regulation of cAMP by thrombin. The activation of Akt by thrombin results in activation of PDE3A and a consequent decrease of the intracellular cAMP level, which serves as a positive feedback enhancing the thrombin activation of platelet function. Understanding the mechanisms that are involved in regulation of PDE3A in platelets could provide new targets for therapeutic advances in the treatment of thrombotic disorders.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

Neutrophil P-selectin-glycoprotein-ligand-1 binding to platelet P-selectin enhances metalloproteinase 2 secretion and platelet-neutrophil aggregation

2005 ◽  
Vol 94 (12) ◽  
pp. 1230-1235 ◽  
Author(s):  
Haissam Abou-Saleh ◽  
Jean-François Théorêt ◽  
Daniel Yacoub ◽  
Yahye Merhi
Keyword(s):  
Platelet Activation ◽  
Human Platelets ◽  
Vascular Remodelling ◽  
Adhesive Interaction ◽  
Platelet Binding ◽  
Enhanced Secretion ◽  
Thrombin Activation ◽  
Neutrophil Aggregation ◽  
And Function ◽  
The Impact

SummaryPlatelets and neutrophils constitute a high source of metalloproteinases (MMPs), and their interactions via P-selectin and Pselectin- glycoprotein-ligand-1 (PSGL-1) are involved in thrombosis, vascular remodelling, and restenosis. We investigated the impact of these interactions on platelet MMP-2 secretion and function in platelet and neutrophil aggregation. The secretion of MMP-2 from human platelets was significantly increased threefold after thrombin activation, and enhanced two-fold in the presence of neutrophils. Neutrophil supernatant had no effect on platelet MMP-2 secretion. While no MMP-2 was detected in the supernatant of neutrophils, a high amount of MMP-9 was released by neutrophils, and remained unchanged upon thrombin activation or in the presence of platelets. Platelet P-selectin, which increased significantly after activation, triggered platelet binding to neutrophils that was completely inhibited by P-selectin or PSGL-1 antagonists, and was reduced by 50% with a GPIIb/ IIIa antagonist. P-selectin or PSGL-1 antagonism abolished the enhanced secretion of platelet MMP-2 in the presence of neutrophils and reduced platelet-neutrophil aggregation. Platelet activation and binding to neutrophils enhance the secretion of platelet MMP-2 via an adhesive interaction between P-selectin and PSGL-1, which contribute to increase platelet-neutrophil aggregation.

Effects of PGI2 and Its More Stable Forms on the Aggregation and on the Camp Content of Human Platelets

1979 ◽  
Author(s):  
E. Nemesánszky ◽  
I. Blaskó ◽  
I. Stadler ◽  
G. Sas ◽  
L. A. Pálos
Keyword(s):  
Ethyl Ester ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Intracellular Camp ◽  
Camp Content ◽  
Prolonged Duration ◽  
Potent Inhibitory Effect ◽  
Stable Forms ◽  
Derivatives Of ◽  
Intracellular Camp Content

The authors investigated the anti-aggregating properties of some relatively stable derivatives of PGI2 concurrently determining the intracellular cAMP content of platelets. The ethyl-ester derivative of PGI2 and the complexes of PGI2-ethyl ester with β-cyclodextrine proved to be more stable than PGI2-sodium salt. Their stimulating effect on adenyl-cyclase correlated with the potent inhibitory effect on platelet aggregation as well. The enhancing effect of these compounds for the intracellular cAMP content have a prolonged duration time -lasting 300 minutes- compared with the only a few minutes’ effect of PGE1.These stable forms of PGI2 might be clinically useful as highly effective antiaggregating compounds.

The Roles of Akt1 and Akt2 in GPIb-IX-Mediated Platelet Activation Signaling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3635-3635
Author(s):  
Hong Yin ◽  
Aleksandra Stojanovic ◽  
Nissim Hay ◽  
Xiaoping Du
Keyword(s):  
Shear Stress ◽  
Signaling Pathway ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Integrin Activation ◽  
Akt Inhibitor ◽  
Platelet Spreading ◽  
Inside Out

Abstract The platelet von Willebrand factor (VWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. To understand the downstream signaling pathway of GPIb-IX signaling, we investigated the roles of PI3K effector kinases, Akt1 and Akt2, in VWF/GPIb-IX-induced platelet activation. VWF/GPIb-IX-induced platelet aggregation was impaired in Akt1- or Akt2-knockout mouse platelets and in human and mouse platelets treated with an Akt inhibitor, SH-6. GPIb-IX-mediated platelet stable adhesion to VWF under shear stress was also inhibited in mouse platelets deficient in Akt1 or Akt2, and in human platelets treated with SH-6. Interestingly, while deficiency of Akt1 or Akt2 caused nearly complete inhibition of stable platelet adhesion to VWF under shear stress, stable platelet adhesion was only partially reduced in platelets treated with both P2Y1 and P2Y12 ADP receptor antagonists, A3P5P and 2MeSAMP or thromboxane A2 pathway inhibitor, aspirin or Syk inhibitor, piceatannol. Therefore, Akt1 and Akt2 are important in early GPIb-IX signaling independent of Syk, ADP or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of either Akt1 or Akt2 diminished platelet spreading on VWF, but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both VWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1 or Akt2 deficiency diminished VWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by low concentration of exogenous cGMP, indicating that Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway. In conclusion, both Akt1 and Akt2 mediate VWF/GPIb-IX-induced signaling pathway leading to platelet activation and the consequent stable platelet adhesion, spreading and aggregation.

scholarly journals LAT Is Required for Tyrosine Phosphorylation of Phospholipase Cγ2 and Platelet Activation by the Collagen Receptor GPVI

1999 ◽  
Vol 19 (12) ◽  
pp. 8326-8334 ◽  
Author(s):  
Jean-Max Pasquet ◽  
Barbara Gross ◽  
Lynn Quek ◽  
Naoki Asazuma ◽  
Weiguo Zhang ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Human Platelets ◽  
Major Pathway ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Protein Receptor ◽  
Thrombin Activation ◽  
Granule Secretion ◽  
A Minor ◽  
Collagen Receptor

ABSTRACT In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cγ2 (PLCγ2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcγRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCγ2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCγ2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin αIIbβ3 in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCγ2, leading to downstream responses such as α-granule secretion and activation of integrin αIIbβ3. The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCγ2. We propose a model in which LAT and SLP-76 are required for PLCγ2 phosphorylation but are regulated through independent pathways downstream of Syk.

scholarly journals Glycoprotein IIIa is phosphorylated in intact human platelets

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2363-2368 ◽  
Author(s):  
LV Parise ◽  
AB Criss ◽  
L Nannizzi ◽  
MR Wardell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Transmembrane Protein ◽  
Specific Protein ◽  
Human Platelets ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Kinase C ◽  
Glycoprotein Iiia

Abstract The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP IIb-IIIa during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP IIIa, but not GP IIb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP IIIa phosphorylation increased four-fold in platelets activated with thrombin or phorbol 12-myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP IIIa during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP IIb-IIIa function.

Protease-Activated Receptors and Platelet Function

1999 ◽  
Vol 82 (08) ◽  
pp. 353-356 ◽  
Author(s):  
Shaun Coughlin
Keyword(s):  
Platelet Activation ◽  
Human Platelets ◽  
The Body ◽  
Amino Terminus ◽  
Protease Activated Receptors ◽  
Amino Terminal ◽  
Thrombin Activation ◽  
High Concentrations ◽  
Blocking Antibodies

IntroductionPlatelet activation is critical for normal hemostasis, and platelet-dependent arterial thrombosis underlies most myocardial infarctions. Thrombin is the most potent activator of platelets.1,2 For this reason, understanding the process by which thrombin activates platelets is necessary for understanding hemostasis and thrombosis and may yield novel anti-platelet therapies. This chapter focuses on our recent studies of the receptors that mediate activation of human platelets by thrombin.3,4 Thrombin signaling is mediated, at least in part, by a family of G protein-coupled protease-activated receptors (PARs), for which PAR1 is the prototype.5,6 PAR1 is activated when thrombin binds to and cleaves its amino terminal exodomain to unmask a new receptor amino terminus.5 This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling.5,7,8 The synthetic peptide SFLLRN, which mimics the first six amino acids of the new amino terminus unmasked by receptor cleavage, functions as a PAR1 agonist and activates the receptor independent of thrombin and proteolysis.5,9,10 Such peptides have been used as pharmacological probes of PAR function in various cell types.Our understanding of the role of PARs in platelet activation is evolving rapidly. PAR1 mRNA and protein were detected in human platelets,5,11-13 SFLLRN-activated human platelets,5,9,10 and PAR1-blocking antibodies inhibited human platelet activation by low, but not high, concentrations of thrombin.11,12 These data suggested a role for PAR1 in activation of human platelets by thrombin but left open the possibility that other receptors contribute.Curiously, PAR1 appeared to play no role in mouse platelets.14-16 PAR1-activating peptides did not activate rodent platelets, and platelets from PAR1-deficient mice responded like wild-type platelets to thrombin.16 The latter observation prompted a search for additional thrombin receptors and led to the identification of PAR3.17 PAR3 is activated by thrombin and is expressed in mouse platelets. PAR3 blocking antibodies inhibited mouse platelet activation by low, but not high, concentrations of thrombin,18 and knockout of PAR3 abolished mouse platelet responses to low, but not high, concentrations of thrombin.3 These results established that PAR3 is necessary for normal thrombin signaling in mouse platelets but also pointed to the existence of another mouse platelet thrombin receptor. Such a receptor, PAR4, was recently identified.3,19 PAR4 appears to function in both mouse and human platelets.3 The role of PAR3 in human platelets, if any, remains to be determined, and whether still unidentified receptors contribute to thrombin activation of platelets is unknown. Nonetheless, available data suggest a testable, working model in which PAR3 and PAR4 mediate thrombin activation of mouse platelets and PAR1 and PAR4 mediate activation of human platelets.To determine the roles of PAR1, PAR3, and PAR4 in activation of human platelets by thrombin, we examined PAR mRNA and protein expression in platelets and probed PAR function using specific peptide agonists. We also examined the effect of receptor desensitization, receptor blocking antibodies, and a PAR1 antagonist, used alone and in combination, on platelet activation.4

scholarly journals Glycoprotein IIIa is phosphorylated in intact human platelets

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2363-2368
Author(s):  
LV Parise ◽  
AB Criss ◽  
L Nannizzi ◽  
MR Wardell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Transmembrane Protein ◽  
Specific Protein ◽  
Human Platelets ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Kinase C ◽  
Glycoprotein Iiia

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP IIb-IIIa during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP IIIa, but not GP IIb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP IIIa phosphorylation increased four-fold in platelets activated with thrombin or phorbol 12-myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP IIIa during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP IIb-IIIa function.

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