1. CELL MEMBRANE MICROPARTICLES IN BLOOD AND BLOOD PRODUCTS: POTENTIALLY PATHOGENIC AGENTS AND BIOMARKERS JAN SIMAK, Ph.D. Laboratory of Cellular Hematology, DH/OBRR, CBER FDA

2. Phospholipid vesicles of 0.05 –1.5  m in size shed from the plasma membrane of eukaryotic cells Phospholipid vesicles of 0.05 –1.5  m in size shed from the plasma membrane of eukaryotic cells Contain membrane phospholipids and express antigens characteristic of their cell of origin Contain membrane phospholipids and express antigens characteristic of their cell of origin Derived from PLTs, RBCs,WBCs, ECs, and some other cell types, circulate in blood Derived from PLTs, RBCs,WBCs, ECs, and some other cell types, circulate in blood Specific MP phenotypes are elevated in blood in different pathologies Specific MP phenotypes are elevated in blood in different pathologies Cell Membrane Microparticles (MPs):

3. Potentially Pathogenic Activities of MPs for review: Simak J & Gelderman MP, Transf Med Rev 2006 Intercellular transfer of membrane proteins and lipids Intercellular transfer of membrane proteins and lipids Prothrombotic effects Prothrombotic effects Proinflammatory effects Proinflammatory effects Impairment of endothelial function Impairment of endothelial function Modulation of vascular tone Modulation of vascular tone Induction of angiogenesis and enhancement of cancer cells metastasis Induction of angiogenesis and enhancement of cancer cells metastasis Biologically active molecules in plasma: Soluble or MP-associated ? Biologically active molecules in plasma: Soluble or MP-associated ?

4. FDA/CBER Mission Relevance MPs as biomarkers: QC in vitro testing of blood products and transfusion devices QC in vitro testing of blood products and transfusion devices In vivo testing of adverse effects of biologics, devices and drugs In vivo testing of adverse effects of biologics, devices and drugs Diagnosis of vascular, hematologic, and other diseases Diagnosis of vascular, hematologic, and other diseases Potentially pathogenic activities of MPs in blood products: Transfusion adverse events Transfusion adverse events

5. MP Research Program Selection of MP phenotypes Flow cytometric analysis of MP surface antigens Development of MP assays and investigation of pathogenic or diagnostic importance of selected MP phenotypes In Vitro Studies Clinical Studies Analysis of MPs in cellular blood products Testing of biologics, drugs and devices

6. CD105 (EC) CD41(PLT) CD45 (WBC) CD41 (PLT) CD45 (WBC) IgG IC Flow Cytometric Analysis of MPs in Plasma Simak J et al., British J Haematol, 2004

7. MP Analysis in Vascular Diseases Circulating MPs in PNH and SCD (study with NHLBI) Hemolytic diseases with vascular injury: to mimic transfusion adverse events MPs derived from PLTs, RBCs, WBCs, and ECs found elevated in SCD Endothelial MPs found elevated in both SCD and PNH Identified specific endothelial MP phenotypes with potential diagnostic importance for severity and character of vascular injury Simak J et al., British J Haematol, 2004 Simak J et al., British J Haematol, 2004

8. MP Analysis in Vascular Diseases II Circulating MPs in acute ischemic stroke (study with NINDS) Quantitative evaluation of vascular injury: Ischemic lesion volume measured by DWI-MRI correlated with brain lesion volume Specific endothelial MP phenotype correlated with brain lesion volume Different endothelial MP phenotype in admission samples correlated with clinical outcome Different endothelial MP phenotype in admission samples correlated with clinical outcome Simak J et al., J Thromb Haemost, 2006 (in press) Simak J et al., J Thromb Haemost, 2006 (in press)

9. MP Analysis in Apheresis Platelets (APs) Gelderman MP et al., ASH 2005 MPs derived from PLTs, WBCs, RBCs, and ECs, were elevated in APs compared to normal plasma MPs derived from PLTs, WBCs, RBCs, and ECs, were elevated in APs compared to normal plasma TF+MPs and MP-associated TF activity (FX activation) were elevated in APs compared to normal plasma TF+MPs and MP-associated TF activity (FX activation) were elevated in APs compared to normal plasma AP storage 5 vs 7 days: No MP increase with exception of WBC MPs including TF+ WBC MPs (CD142+CD45+). AP storage 5 vs 7 days: No MP increase with exception of WBC MPs including TF+ WBC MPs (CD142+CD45+). Effects of Washed MPs from APs on HUVEC Culture Increase expression of ICAM-1 and TF on cells Increase expression of ICAM-1 and TF on cells G1 arrest and cell apoptosis G1 arrest and cell apoptosis Extracellular calcium influx (in PC12 cells) Extracellular calcium influx (in PC12 cells)

10. MONOCYTE TF APHERESIS STORAGE TF ACTIVATION PSGL-1 PLT P-SEL PLT TF CD41 PLT RESTINGACTIVATION Possible origin of TF + MP in APs Modifed in vivo mechanism proposed in B. Furie lab (Throm. Haemost. 2004) Future Plans Investigation of TF + MPs in APs

11. Future Plans, cont. Study of MPs in APs and red blood cell transfusion products Study of MPs in APs and red blood cell transfusion products Association of different MP phenotypes with in vitro proinflammatory and proapoptotic effects of MPs Association of different MP phenotypes with in vitro proinflammatory and proapoptotic effects of MPs Effects of donor characteristics, collection devices, leukofiltration, pathogen reduction treatments, and storage, on MP content in cellular blood products Effects of donor characteristics, collection devices, leukofiltration, pathogen reduction treatments, and storage, on MP content in cellular blood products Design of clinical study protocols: Association of different MP phenotypes in cellular blood products with transfusion adverse events. Design of clinical study protocols: Association of different MP phenotypes in cellular blood products with transfusion adverse events. FDA Collaborative project: To study the interaction of FDA Collaborative project: To study the interaction of nanomaterials with plasma membrane of blood and nanomaterials with plasma membrane of blood and endothelial cells endothelial cells

12. ACKNOWLEDGEMENTSCBER Monique P. Gelderman, PhD Karel Holada, PhD Jaroslav Novak, PhD Jan Zivny, MD, PhD Laura B. Carter, MT Jaroslav G. Vostal, MD, PhD NHLBI Neal S. Young, MD, PhD Antonio M. Risitano, MD, PhD NINDS Alison E. Baird, FRACP, PhD Hua Yu, MD Violet Wright, RN, DNSc NIH CC Department of Transfusion Medicine USUHS Olga Simakova, MS