1. by Wei Zhang, and Robert W. Colman
Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A
by Wei Zhang, and Robert W. Colman
Blood
Volume 110(5):
September 1, 2007
©2007 by American Society of Hematology

2. Thrombin and PAR-1 peptide agonist up-regulate cAMP-PDE activity.
Thrombin and PAR-1 peptide agonist up-regulate cAMP-PDE activity. Washed platelets (2 × 108/mL) were treated with (A) thrombin or (B) SFLLRN peptide at 37°C for 3 minutes, and vehicle was added in the sample,which served as control basal line. Activities of cAMP-PDE in the platelet samples were determined by measurement of hydrolysis of cAMP. The changes of hydrolysis of cAMP induced by thrombin or SFLLRN peptide were calculated as that of each value minus the control base line. Data are from 4 individual experiments using different donor platelets. Error bars represent means plus or minus SEM.
Wei Zhang, and Robert W. Colman Blood 2007;110:
©2007 by American Society of Hematology

3. PDE3 selective inhibitors restrict thrombin-induced PDE activity.
PDE3 selective inhibitors restrict thrombin-induced PDE activity. Washed platelets (2 × 108/mL) were incubated with 20 μmol/L EHNA or Bay , or 10 μmol/L milrinone, cilostazol, IBMX, or vehicle at 37°C for 30 minutes. After addition of thrombin (0.5 nmol/L) in each group at 37°C for 3 minutes, the reactions were stopped by addition of 0.5% of Triton X-100. The platelet samples, which were treated with vehicle or the inhibitors but not with thrombin, served as control basal line for each inhibitor. The hydrolysis of cAMP in each sample was determined. The control basal level of hydrolysis of cAMP was subtracted from each test sample. Data are from 4 individual experiments using different donor platelets and are means plus or minus SEM. *Significant compared with control (thrombin alone) (P > .05).
Wei Zhang, and Robert W. Colman Blood 2007;110:
©2007 by American Society of Hematology

4. PP1 reverses thrombin-induced activation of PDE3A.
PP1 reverses thrombin-induced activation of PDE3A. A series of paired samples of washed platelets (2 × 108/mL) were incubated with thrombin at 37°C for 3 minutes in the presence of 20 μmol/L EHNA to limit the PDE2A effect. The reactions were stopped by addition of 0.5% Triton X-100. One of the paired samples was treated with PP1 and the other incubated with vehicle. Dephosphorylation by PP1 was carried out as described in “Materials and methods, Dephosphorylation of PDE3,” and PDE3A activities were immediately measured after the dephosphorylation procedure. Data are from 4 experiments using different donor platelets and are means plus or minus SEM.
Wei Zhang, and Robert W. Colman Blood 2007;110:
©2007 by American Society of Hematology

5. Thrombin stimulates PDE3A by activating Akt.
Thrombin stimulates PDE3A by activating Akt. (A) PDE3A, PI3K/Akt inhibitors reverse thrombin-induced activation of PDE3A. Washed platelets (2 ×108/mL) were first incubated with AktI (2 μmol/L), wortmannin (0.1 μmol/L), milrinone (10 μmol/L), H89 (10 μmol/L), or vehicle as described under “Materials and methods.” Thrombin (0.5 nmol/L) or vehicle was then added at 37°C for 3 minutes. PDE3A activity in the platelet lysate was determined in the presence of 20 μmol/L EHNA to remove the effect of PDE2A. Results are from 4 experiments using different donor platelets. (B) The correlation of thrombin-stimulated Akt and PDE3A activation. Activities of Akt and PDE3A in platelets were measured either after 5 minutes of thrombin stimulation (i) (thrombin concentration-dependent) or with thrombin at 0.5 nmol/L (iii) (time-dependent). The closed squares represent Akt activity and the open squares represent PDE3A activity. The data of (i) and (iii) was from 3 individual experiments. Correlation of Akt kinase and PDE3A activities in dose-response of thrombin (ii) was plotted with Microsoft Excel software (Microsoft, Redmond, WA). Values are means plus or minus SEM.
Wei Zhang, and Robert W. Colman Blood 2007;110:
©2007 by American Society of Hematology

6. Effects of different signaling pathway inhibitors on thrombin-induced decrease of the platelet cAMP.
Effects of different signaling pathway inhibitors on thrombin-induced decrease of the platelet cAMP. (A) Washed platelets (2 ×106/mL) were incubated with 2 μmol/L AktI, 0.1 μmol/L wortmannin, 10 μmol/L milrinone or IBMX, 10 μmol/L GF , 10 μmol/L H89, or vehicle at conditions as described under “Materials and methods.” The samples were stimulated by thrombin (0.5 nmol/L) at 37°C for 3 minutes after incubation with 10 μmol/L forskolin for 3 minutes. The results are expressed as the percentage of baseline cAMP level in each condition without thrombin. (B–D) The changes of cAMP contents medicated by agonist thrombin (0.5 nmol/L at 37°C for 3 minutes), PMA (1 μmol/L at 37°C for 10 minutes), and TPO (100 ng/mL 37°C for 10 minutes) in the absence or in the presence of a variety of pharmacological agents were monitored, respectively. The results are normalized as the percentage of baseline cAMP level in each condition without agonist. cAMP levels were determined by Biotrak Enzyme Immunoassay kit. Data are from 4 independent experiments using platelets from different donors and are means plus or minus SEM. ∗, significant by ANOVA for wortmannin, AktI, milrinone, and IBMX compared with thrombin alone (P > .05).
Wei Zhang, and Robert W. Colman Blood 2007;110:
©2007 by American Society of Hematology

7. Akt phosphorylates PDE3A during thrombin activation of platelets.
Akt phosphorylates PDE3A during thrombin activation of platelets. (A) Washed platelets were stimulated with thrombin at 37°C for 3 minutes. The samples were separated in 4%-15% SDS-PAGE gels and transferred into PVDF membranes. The membranes were probed by anti-Akt mAb, antiphospho-Akt (Ser473) mAb, or anti-phospho-Akt substrate mAb, respectively. (B) The purified PDE3A protein were immunodetected with anti-phospho-Akt substrate mAb. Lanes 1 and 2 represent the purified platelet PDE3A proteins from the vehicle control platelets and the thrombin-treated platelets, respectively (thrombin at 0.25 nmol/L). The data are representative of the 3 similar experiments. (C) Identification of thrombin-induced PDE3A phosphorylation with immunoprecipitated method. Washed platelets incubated with AktI (2 μmol/L) or vehicle were treated by thrombin (0.25 nmol/L at 37°C for 3 minutes) or vehicle. Platelet lysates were immunoprecipitated by the anti-Akt phosphorylated-substrate monoclonal antibody and immunoblotted by PDE3A antibody.
Wei Zhang, and Robert W. Colman Blood 2007;110:
©2007 by American Society of Hematology

8. Regulation of intracellular cAMP in platelets by thrombin.
Regulation of intracellular cAMP in platelets by thrombin. Thrombin restricts intracellular cAMP content by suppression of synthesis from ATP and acceleration of hydrolysis of cAMP to 5′AMP by PDE3A. After activation of PAR-1, thrombin inhibits adenylate cyclase either through PAR1-coupled Gi protein directly, through Gq protein directly, or through Gq protein-induced of ADP from dense granules via the PLCβ pathway. In the latter, secreted ADP binds to the outside surface P2Y12 receptor, which is coupled to Gi2 to inhibit adenylate cyclase. Furthermore, thrombin via PAR-1 links to Gq, stimulates PI3K and PDK1, which in turn phosphorylates Akt, which in turn phosphorylates PDE3A. Elevation of PDE3A lowers intracellular cAMP content.
Wei Zhang, and Robert W. Colman Blood 2007;110:
©2007 by American Society of Hematology