Novel Regulation of Glycoprotein VI Signaling in platelets

Transcription

1 Novel Regulation of Glycoprotein VI Signaling in platelets Satya P. Kunapuli, Ph.D. Department of Physiology, Sol Sherry Thrombosis Research Center Temple University School of Medicine, Philadelphia, PA 19140, USA

2 Platelets Adhesion Receptors and Hemostasis vwf = Von Willebrand factor

3 Phosphoinositide 3-Kinase (PI3K) PI3K An important signaling molecule that is activated upon platelet activation. leads to generation of Phosphatidylinositol (3,4,5)- trisphosphate (PIP 3 ) in the plasma membrane. Recruits PH domain containing proteins to the membrane. BTK and more. FcRg- chain Syk P P Collagen GPVI SFK PI3K PIP 2 PIP 3 P BTK GPVI = Glycoprotein VI PIP 2 = Phosphatidylinositol (4,5)-bisphosphate BTK = Bruton s tyrosine kinase P P P P Our laboratory sought out to screen for proteins that interacts with PIP 3 in human platelets.

4 Analysis of proteins interacting with PIP 3 in human platelets GelCode Blue kda Buffer Platelet lysate Mass spectrometry analysis In Dr. Salim Merali s laboratory Control beads PIP 3 beads Control beads PIP 3 beads Source: Human platelet lysate

5 Results from mass spectrometry 29 proteins were selectively found to interact with PIP 3 in platelets. Summary of PH domain containing proteins. Accession Protein MW [kda] Q14644 Ras GTPase-activating protein Q06187 Tyrosine-protein kinase BTK 76.2 Q99418 Cytohesin Q9UN19 Dual adapter for phosphotyrosine and 3-phosphotyrosine and phosphoinositide Q92556 Engulfment and cell motility protein

6 Engulfment and cell Motility (ELMO) ELMO scaffold with no intrinsic catalytic activity. There are three isoforms of ELMO as follows: ELMO1 (83.8 kda) ELMO2 (82.6 kda) ELMO3 (81.5 kda) Function: ELMO1 and ELMO2 are known to regulate actin cytoskeletal reorganization via Rac1 and are involved in phagocytosis, cell spreading, and cell motility. ELMO proteins contain PH domain at the c-terminus.

7 ELMO1 interacts with PIP 3 in human platelets kda 250 IP: PIP 3 WB: ELMO ELMO1 50 MW MW = Molecular weight markers WCL = whole cell lysate Source: human platelets ELMO1 is present in human platelets. ELMO1 interacts with PIP 3 in human platelets.

8 ELMO1 in platelets ELMO1 is expressed in platelets however its function is unknown. (Goggs, R et al, JBC 2013) Active RhoG, a small GTPase, is known to interact with ELMO1 in platelets. However, the functional implication of this interaction is not understood. (Goggs, R et al, JBC 2013)

9 We obtained ELMO1 deficient mice (ELMO1 -/- ) from Dr. Kodi S. Ravichandran. ELMO1 -/- mice are viable and do not have any gross abnormalities.

10 ELMO1 is expressed in human and murine platelets MW = Molecular weight marker

11 Hematologic parameters are normal in ELMO1 -/- mice WT ELMO1 -/- White blood cells (x 10 9 cells/l) 3.39 ± ± 0.39 Neutrophils (x 10 9 cells/l) 0.31 ± ± 0.12 Lymphocytes (x 10 9 cells/l) 2.87 ± ± 0.26 Red blood cells (x 10 9 cells/l) 7.89 ± ± 0.11 Platelets (x 10 9 cells/l) ± ± Mean platelet volume (fl) 5.77 ± ± 0.03 The data is represented as mean ± SE. N = 5.

12 ELMO1 -/- platelets respond normally to GPCR agonist A. B. D. C. AYPGKF = PAR4 agonist The data is represented as mean ± SE. N = 5.

13 GPVI agonist potentiates ELMO1 -/- platelet functional responses CRP = collagen-related peptide.

14 GPVI agonist potentiates ELMO1 -/- platelet functional responses * * * * The data is represented as mean ± SE. N = 5. (*) = p<0.05. CRP = collagen-related peptide.

15 GPVI agonist potentiates ELMO1 -/- platelet functional responses

16 GPVI and αiibβ 3 expression are unaffected in ELMO1 -/- platelets A. GPVI B. Integrin αiib Sample Name WT Isotype control ELMO1 -/- Isotype control WT ELMO1 -/-

17 Enhanced thromboxane generation in ELMO1 -/- platelets with GPVI agonists The data is represented as mean ± SE. N = 5. (*) = p<0.05

18 ELMO1-/- platelets exhibit enhanced thrombus formation Flow over collagen WT ELMO1-/- Flow 50 μg/ml collagen

19 Evaluation of ELMO1 -/- mice using tail bleeding assay p<0.05 Mean ± SE Count WT ± ELMO1 -/ ± Age: 5 weeks There was faster bleeding times with ELMO1 -/- mice compared to the Wildtype (WT).

20 Analysis of ELMO1 -/- mice using pulmonary thromboembolism model Collagen: 400 μg/kg Epinephrine: 60 μg/kg P<0.01 The ELMO1 -/- mice exhibit shorter survival rate compared to the wildtype (WT).

21 Analysis of thrombus formation with ELMO1 -/- mice FeCl 3 injury model * The ELMO1 -/- mice showed shorter time to occlusion compared to wildtype (WT). * = P<0.01. Count for WT = 12 and count for ELMO1 -/- = 14

22 Conclusions ELMO1 interacts with PIP 3 in platelets. ELMO1 is a negative regulator of GPVI-mediated platelet activation.

23 Evaluation of signaling molecules downstream of GPVI CRP = collagen related peptide PLCγ2 = PhospholipaseCγ2 ELMO1 is upstream of Syk in GPVI pathway.

24 Ras homology Growth-related (RhoG) Is a small monomeric GTP-binding protein. In platelets: RhoG positively regulates GPVI-mediated platelet activation and thrombus formation. (Goggs, R et al, JBC 2013 & Kim, S et al, JBC 2013) It was found to be upstream of SYK and PLCγ2 (Kim, S et al, JBC 2013). Active RhoG was also found to interact with ELMO1 (Goggs, R et al, JBC 2013).

25 Agonist-induced platelet aggregation and dense granule secretion in RhoGdeficient platelets. Soochong Kim et al. J. Biol. Chem. 2013;288:

26 Enhanced RhoG activity in ELMO1 -/- platelets

27 Current model Collagen FcRg- chain GPVI RhoG ELMO1 GTP? P P Syk PI3K SFK ELMO1 negatively regulates GPVI mediated platelet function upon binding to RhoG. Granule secretion TxA2 aiibb3- platelet aggregation Thrombus formation

28 Acknowledgements Laboratory: Carol Dangelmaier Dr. Soochung Kim Dr. John Kostyak Bhanukanth Manne Dheeraj Bhavanasi Rachit Badolia Vaishali Inamdar Collaborators: Dr. Kodi Ravichandran Dr. Joseph Aslan Dr. Salim Merali Dr. Lawrence Goldfinger

29 Thank you