PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway

Abstract

Essentials Phosphoinositide 3-kinase and MAPK pathways crosstalk via PDK1. PDK1 is required for adenosine diphosphate-induced platelet activation and thromboxane generation. PDK1 regulates RAF proto-oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway. Genetic ablation of PDK1 protects against platelet-dependent thrombosis in vivo.

Summary: Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3β. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1^-/- mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.

Keywords: phosphoinositide dependent kinase 1; platelet activation; purinergic receptor P2Y12; thrombosis; thromboxane A2.

Figure 1. GSK3β Inhibition does not rescue 2MeSADP-induced platelet aggregation and thromboxane generation in human and murine platelets

A) Washed human and murine (e.g. mouse) platelets were pre-treated with the DMSO (vehicle control), BX-795 (1 μM), aspirin (10 μM) or indomethacin (10 μM) at 37°C for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. B) Washed human and murine platelets were pre-treated with DMSO (vehicle control), the PDK1 inhibitor BX-795 (1 μM) or the GSK3β inhibitor SB-216763 (5 μM) or both for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 3.5 min at 37°C. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. C) Thromboxane generation was measured as described in the experimental section. Graphs represent mean ± SD from at least three independent experiments.

Figure 2. PDK1 regulates the MAPK signaling pathway through phosphorylation of Raf1

(A) Washed human platelets were left alone or pre-treated with the PDK1 inhibitor BX-795 (1 μM) for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 5 minutes. Western blots were then probed for phospho Akt (Thr308), Akt (Ser473), GSK3β (Ser9), MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204) and cPLA2 (Ser505). The western blot shown is representative of three independent experiments. (B) Washed human platelets were pre-treated with BX-795 (1 μM) for 5 min followed by stimulation 2MeSADP (50 nM) under stirred conditions for 5 minutes. Platelets were then lysed in NP40 lysis buffer and immunoprecipitated for MEK1/2. A MEK1/2 kinase activity assay was performed on the platelet lysates to measure phosphorylation of the MEK1/2 substrate myelin basic protein (pMBP). The Western blot shown is representative of three experiments. C) Washed human platelets were left alone or pre-treated with the PDK1 inhibitor BX-795 (1 μM) for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 5 minutes. Western blots were then probed for phospho Raf1 (Ser338), and total beta-actin. The western blot shown is representative of three independent experiments. D) Washed human platelets were pre-treated with the DMSO (vehicle control), BX-795 (1 μM, 37°C 5 min) followed by stimulation with 2MeSADP (50 nM) (Black and Blue), arachidonic acid (10 μM) (Red), or both (Purple) while stirring Platelet aggregation was measured by aggregometry. Tracings representative of n≥3 independent experiments.

Figure 3. PDK1, but not ASK1, regulates 2MeSADP induced secondary aggregation and thromboxane generation in platelets

(A) Washed human platelets were pre-treated with the ASK1 inhibitor DMSO (vehicle control), MSC 2032964A (5 μM), BX-795 (1 μM), or both at 37°C for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions. Platelet aggregation was measured by aggregometry. The shown tracings are representative of at least three independent experiments. (B) Washed human platelets were pre-treated with DMSO (vehicle control), MSC 2032964A (5 μM), BX-795 (1 μM), or both for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 3.5 min at 37°C. Thromboxane generation was measured as described in the experimental section. Graphs represent mean ± SEM from at least 3 different experiments (*P<0.05). (C) Washed human platelets were incubated BX-795 (1 μM), SB-216763 (5 μM), MSC 2032964A (5 μM), LY294002 (25 μM), MK2206 (1 μM), or DMSO (vehicle control) followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 5 minutes. Platelet proteins were separated using SDS-PAGE and probed for phospho Raf1 (Ser338), MEK1/2 (Ser212/221), ERK1/2 (Thr202/Tyr204), and cPLA2 (S505). Respective proteins are used as loading control for all western blots. The results shown are representative of data from at least three independent experiments.

Figure 4. Role of p38 Kinase in 2MeSADP induced platelet activation

(A) Washed human platelets were incubated BX-795 (1 μM), SB-203580 (5 μM), or DMSO (vehicle control) followed by stimulation with 2MeSADP (2.5 nM) under stirred conditions for 5 minutes. Platelet proteins were separated using SDS-PAGE and probed for phosphorylation of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204), and cPLA2 (S505). (B) Washed human platelets were incubated with SB-203580 (5 μM) followed by stimulation with 2MeSADP at various concentrations under stirred conditions for 5 minutes. Platelet proteins were separated using SDS-PAGE and probed for phosphorylation of p38 (Thr180/Tyr182). Actin is loading control. Representative of n≥3 independent experiments. (C, D) Proposed models for ADP-induced activation of cPLA2 and p38.

Figure 5. Genetic ablation of PDK1 reduces 2MeSADP induced platelet aggregation, and abolished MAPK pathway mediated thromboxane generation

(A) Washed platelets from pdk1^fl/fl and pdk1^−/− mice were stimulated with 2MeSADP (50 nM) under stirred conditions at 37°C for 5 min. Aggregation was measured using light transmission aggregometry. Light transmission and area under the curve (mean ± SEM) are calculated from six independent experiments. (B) Washed platelets from pdk1^fl/fl and pdk1^−/− mice were stimulated with 2MeSADP (50 nM) under stirred conditions for 5 min at 37°C. Thromboxane generation was measured using ELISA. Graphs represent mean ± SD from at least three different experiments (P=0.0014). (C) Washed platelets from pdk1^fl/fl and pdk1^−/− mice were stimulated with 2MeSADP (50 nM) under stirred conditions for 3 minutes. Platelet proteins were separated by SDS-PAGE, immunoblotted and probed for phospho Raf1 (Ser338), MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204), cPLA2 (Ser505) and Akt (Thr308). The Western blot shown is a representative of three independent experiments.

Figure 6. Pharmacologic inhibition or genetic ablation of PDK1 delays pulmonary thromboembolism induced mortality

(A) Wildtype mice were treated with BX-795 (10 μg/ml) or vehicle (DMSO) by intraperitionial injection once a day for 0, 3, or 5 days. Phosphorylation of Akt (T308) in isolated platelets stimulated with 2MeSADP ex vivo was measured to determine the duration of BX-795 treatment necessary to completely block phosphorylation of AKT. (B) Wildtype mice were treated with different concentrations of BX-795 (or vehicle) by intraperitoneal injection once a day for 5 days at various concentrations. Phosphorylation of Akt (T308) in isolated platelets stimulated with 2MeSADP ex vivo was measured. For Panels A and B, actin is a loading control and the immunoblot is representative of n=3 independent experiments. (C, D) Wildtype mice were treated with BX795 (10 μg/ml) or vehicle for 5 days. Pulmonary thromboembolism was induced by injecting (C) 2MeSADP (20mg/kg) plus epinephrine (30mg/kg) or (D) collagen (0.4 mg/kg) plus epinephrine (30mg/kg). N=7 mice/group for Panels C and D. Representative histological sections from lungs of mice injected with ADP are shown above. (E) pdk1^fl/fl or pdk1^−/− mice after induction of pulmonary thromboembolism (n=6/group). Representative histological sections from pdk1^fl/fl or pdk1^−/−lungs are shown to the right.

Figure 7. Proposed model illustrating PDK1 regulation of the MAPK signaling pathway in platelets stimulated with 2MeSADP

We propose that upon activation of P2Y12 in platelets, PDK1 becomes activated and then phosphorylates Raf1. Phosphorylated Raf1, in turn, regulates activation of MEK1/2, ERK1/2 and cPLA2 proteins, which are necessary for 2MeSADP-induced thromboxane generation and platelet aggregation.