ISTH

20

17 Poster presented at

ISTH2017 on:

BACKGROUND

OBJECTIVE

RESULTS

CONCLUSION

The PI3K pathway master kinase PDK1 controls platelet activation by regulating

the MAPK pathway via the Raf-1 proto-oncogene, serine/threonine kinase Bhanu Kanth Manne1*, Patrick Münzer2*, Rachit Badolia3, Britta Walker-Allgaier2, Robert Campbell1, Elizabeth Middleton1,

Andrew S Weyrich1, Satya P Kunapuli3, Oliver Borst2, Matthew T. Rondina1,4.

METHODS

RESULTS

Figure 1. GSK3b Inhibition does not rescue 2MeSADP-induced platelet aggregation and thromboxane

generation in human and murine platelets: A)Washed human and murine platelets were left alone or pre-

treated with the PDK1 inhibitor BX795, aspirin, indomethacin, the GSK3b inhibitor SB216763 or DMSO (vehicle

control) for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 5 min at 37˚C.

Platelet aggregation was measured by aggregometry. B) Thromboxane generation was measured in human

(left side) and murine (right side) platelets. Graphs represent mean ± SEM from at least three independent

experiments (*P<0.05).

Figure 2: PDK1 regulates the MAPK signaling pathway through phosphorylation of Raf1: Washed human

platelets were left alone or pre-treated with the PDK1 inhibitor BX795 (1 mM) 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), GSK3b (Ser9), MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204) and cPLA2 (Ser505).

The western blot shown is representative of three independent experiments

Figure 5. Pharmacologic inhibition or genetic ablation of PDK1 delays pulmonary embolism induced

mortality: Survival curve of A) Vehicle (DMSO) or BX795 (10 mg/ml) mice after induction of pulmonary

thromboembolism (n=6/group) (*P<0.05) and B) pdk1fl/fl and pdk1-/- mice after induction of pulmonary

thromboembolism (n=6/group) (*P<0.05). Representative pictures from pdk1fl/fl and pdk1-/- lungs after

thromboembolism are shown.

Figure 4. Genetic ablation of PDK1 reduces 2MeSADP induced platelet aggregation, and abolished

MAPK pathway mediated thromboxane generation: A) Washed platelets from pdk1fl/fl and pdk1-/- mice were

stimulated with 2MeSADP (50 nM) under stirred conditions at 37˚C for 5 min. Platelet aggregation was

measured by aggregometry. B) Thromboxane generation was measured in platelets from pdk1fl/fl and pdk1-/-.

Graphs represent mean ± SEM from at least three different experiments (*P<0.05). C) Washed platelets from

pdk1fl/fl and pdk1-/- mice were stimulated with 2MeSADP (50 nM) under stirred conditions at 37°C for 3

minutes. Platelet proteins were separated by SDS-PAGE and probed against the indicated proteins.

This work was supported by the NHLBI and NIA (HL112311 and HL126547 to M.T.R. and A.S.W.

and AG048022 to M.T.R.). We thank Dr. Oliver Borst, University of Tübingen, Germany for

providing PDK1 knockout mice samples for the experiments.

ACKNOWLEDGEMENT

PDK1 regulates 2MeSADP-induced platelet activation and thromboxane generation via the MAPK

pathway. PDK1 phosphorylates and activates Raf1 in MAPK pathway when platelets are

stimulated with 2MeSADP. Our study provides clear evidence for a novel crosstalk between PI3K

and MAPK pathways, regulating platelet function via PDK1.

Platelets are involved in many processes, ranging from fighting microbial infections and

triggering inflammation to promoting tumor angiogenesis and metastasis.

Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor

4 (PAR4)-induced platelet activation and thrombus formation through glycogen synthase

kinase 3 beta (GSK3β). However, it is unknown whether PDK1 signaling also involves the

ADP receptor and, if so, what the downstream functional consequences for platelet

activation are.

In this study We employed both pharmacological (e.g. with the selective PDK1 inhibitor

BX795) and genetic (platelet-specific deletion of PDK1) approaches to dissect the role of

PDK1 in ADP-induced platelet activation and thrombosis.

We assessed the effect of PDK1 on ADP-induced platelet activation by measuring

aggregation, thromboxane generation and phosphorylation events in the presence of BX

795 (a PDK1 specific inhibitor) and in platelets from PDK1 knockout mice. PDK1 function in

thrombus formation was also assessed using pulmonary embolism model in vivo.

pS6

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Figure 3. PDK1, but not GSK3b or ASK1, regulates 2MeSADP induced secondary aggregation and

thromboxane generation in platelets: Washed human platelets were incubated with BX795 (1 mM),

SB216763 (5 mM), MSC 2032964A (5 mM), LY294002 (25 mM), MK2206 (1 mM) , 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 proteins in MAPK kinase pathway. Respective proteins are used

as loading control for all western blots.

1Department of Internal Medicine, Molecular Medicine Program, University of Utah, Salt Lake City, UT, 84112 USA ,2Department of Cardiology and Cardiovascular Medicine, University of Tübingen, Tübingen, 72076 Germany.,3 Sol Sherry

Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, 19140 USA, 4Department of Internal Medicine, GRECC, George E. Wahlen VAMC, Salt Lake City, UT, 84148

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No relevant conflicts of interest to disclose. (* Both contributed equally to the manuscript)

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1296--PBPatrick Munzer Tuesday, July 11DOI: 10.3252/pso.eu.ISTH2017.2017

Platelet Signaling