Phosphatidyl Serine Exposure in the Membrane of RBCs after

inhibition of PKC and ScramblaseMaster ThesisBy 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 membraneThe 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, “Phosphatidylserine-related 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