Phosphatidyl Serine Exposure in
the Membrane of RBCs after
inhibition of PKC and
Scramblase
Master Thesis
By Judy Mutua
Outline
•
•
•
•
Introduction
Method
Results
Conclusion
Introduction
• Red Blood Cells:
• Transport oxygen
• 120 days life span
• Produced in Bone Marrow
• Role in blood clot
formation.
qmul.ac.uk
Introduction
• Structure of RBC membrane
The human RBC membrane consists of: Lipids, Proteins
& Carbohydrates.
• The main lipids are cholesterol and phospholipids
Outer monolayer: (PC); (SM)
Inner monolayer: (PE); (PI); (PS)
Introduction
RBC Membrane
Modifiziert nach Zwaal et al., 2005
Introduction
Introduction
Introduction
Introduction
Calphostatin c
R5421
Method
• Wash 500 µl blood with physiological solution
3X
• Incubate in 1 µM X-Rhod-1 (30 min)
• Incubate with inhibitor
• Add Annexin V-FITC
• Measure in FACS
• flourescent microscope
Results
Nguyen et al. 2011
Results
PS-Exposure of RBCs after 30 min activation with LPA, A23187 or PMA (Nguyen et al., 2011)
Results
Conclusion
• Activation of RBCs by LPA ,A23187 or PMA
leads to increase in the intracellular calcium.
• Increasing of intracellular calcium activates
the calcium pump, this leads to decrease in
intracellular calcium with time.
• Cells with increased intracellular calcium
content and showing PS exposure don’t
always correlate together. Further
experiments will solve this discrepancy!
References
• L. Wagner, D.B. Nguyen, A. Jung, P. Steffen, C. Wagner, L. Kaestner, T.
Mueller, I. Bernhardt, „Phosphatidylserine Exposure and Aggregation of
Red Blood Cells“ (2011) Red Cell Club, Philadelphia
• B. N. Yamaja Setty, S. Kulkarni, M. J. Stuart, “Role of erythrocyte
phosphatidylserine in sickle red cell-endothelial adhesion” (2002) blood
2002 99: 1564-1571
• D. B. Nguyen, „Phosphatidylserine exposure in red blood cells: A
suggestion for the active role of red blood cells in blood clot formation”
(2010) Dissetation Saarbrücken
• C. Le Closse. J. Dachary-Prigent, M. R. Boisseau, “Phosphatidylserinerelated adhesion of human erythrocytes to vascular endothelium“ (1999)
British Journal of Haematology, 1999, 107, 300±302
• J. Connor, C. C. Pak, A. J. Schroit, “Exposure of Phosphatidylserine in the
Outer Leafle of Human Red Blood Cells” (1993) THE JOLIRNAOFL
BIOUXICACLHEMISTRY