1. Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) and NTPDase2 have implications for thromboregulation
by Jean Sévigny, Christian Sundberg, Norbert Braun, Olaf Guckelberger, Eva Csizmadia, Imrana Qawi, Masato Imai, Herbert Zimmermann, and Simon C. Robson
Blood
Volume 99(8):
April 15, 2002
©2002 by American Society of Hematology

2. Biochemical activity of murine tissues and recombinant NTPDase1 and NTPDase2.NTPDase activities of protein preparations were determined by measurement of phosphate release from the respective substrates.
Biochemical activity of murine tissues and recombinant NTPDase1 and NTPDase2.NTPDase activities of protein preparations were determined by measurement of phosphate release from the respective substrates. Data are expressed as the mean ± SD for specific ATPase (□) or ADPase (■) activities. (A) NTPDase activities ofcd39 +/+ (n = 5) andcd39 −/− (n = 5) mouse lung and heart preparations were measured. (B) NTPDase activity of protein extracts from NTPDase1- or NTPDase2-transfected cells were compared with control cells—untransfected (COS) or those transfected with pcDNA3 empty vector (n = 3 different transfections).
Jean Sévigny et al. Blood 2002;99:
©2002 by American Society of Hematology

3. Opposing effects of NTPDase1 and NTPDase2 on platelet aggregation
Opposing effects of NTPDase1 and NTPDase2 on platelet aggregation.PRP prepared from human donors was tested for platelet activation in the presence of exogenous nucleotides (ATP and/or ADP) and protein extracts from COS cells transfected with pcDNA3 encodin...
Opposing effects of NTPDase1 and NTPDase2 on platelet aggregation.PRP prepared from human donors was tested for platelet activation in the presence of exogenous nucleotides (ATP and/or ADP) and protein extracts from COS cells transfected with pcDNA3 encoding NTPDase1 or NTPDase2. Light transmittance (%) was recorded over a period of 8 (B-D) or 10 minutes (A). Protein extracts from COS cells transfected with vector DNA were added as controls at the required protein level. Representative aggregation profiles from 3 to 6 experiments are shown. (A) An arrow indicates the addition of 20 μM ATP to PRP followed 3 minutes later by the addition of 20 μg NTPDase1 or NTPDase2 protein preparation (second arrow). (B) PRP was preincubated for 25 seconds with 20 μg NTPDase1 (0.3 U ATPase) or NTPDase2 (0.1 U ATPase) protein extracts before the addition of 40 μM ATP. (C) PRP was preincubated for 25 seconds with 0.06 units of ATPase activity of NTPDase1 (4 μg) or NTPDase2 (12 μg) prior to the addition of a mixture of nucleotides (4 μM ATP plus 1 μM ADP). (D) PRP was preincubated for 25 seconds with 0.06 units of ATPase activity with either NTPDase1 (4 μg = 0.043 U ADPase) or NTPDase2 (12 μg) prior to the addition of 5 μM ADP.
Jean Sévigny et al. Blood 2002;99:
©2002 by American Society of Hematology

4. Specificity of antisera for murine NTPDase1 and NTPDase2
Specificity of antisera for murine NTPDase1 and NTPDase2.Protein samples (5 μg) from COS cells, transiently transfected with NTPDase1 or NTPDase2 cDNA constructs or from cells transfected with empty vector DNA (as a negative control), were fractionated on 1...
Specificity of antisera for murine NTPDase1 and NTPDase2.Protein samples (5 μg) from COS cells, transiently transfected with NTPDase1 or NTPDase2 cDNA constructs or from cells transfected with empty vector DNA (as a negative control), were fractionated on 10% acrylamide SDS–polyacrylamide gel electrophoresis under nonreducing conditions. Separated proteins were then transferred to an Immobilon-P membrane and incubated with pAbs directed against NTPDase1 or NTPDase2. Each antiserum detected protein bands only in the appropriately transfected cells.
Jean Sévigny et al. Blood 2002;99:
©2002 by American Society of Hematology

5. Immunohistologic localization of NTPDases in cardiac vasculature
Immunohistologic localization of NTPDases in cardiac vasculature.Serial sections of a mouse heart were stained by immunohistochemistry with various antibodies detecting (A) NTPDase1, (B) NTPDase2, (C) α-actin (a marker of smooth muscle), (D) CD31 (a marker ...
Immunohistologic localization of NTPDases in cardiac vasculature.Serial sections of a mouse heart were stained by immunohistochemistry with various antibodies detecting (A) NTPDase1, (B) NTPDase2, (C) α-actin (a marker of smooth muscle), (D) CD31 (a marker of endothelium), (E) NG2 (a marker of pericytes and smooth muscle cells), and (F) laminin (a marker of basement membranes). Positive reactions were seen as rust color (A-F), and sections were counterstained with hematoxylin (blue). In panels G-I, immunofluorescence and confocal analysis were performed: (G) CD31 in blue and NTPDase1 in green (colocalization as aquamarine); (H) as for panel G with addition of NTPDase2 in red; (I) NTPDase1 in green, smooth muscle α-actin in blue, and NTPDase2 in red (no colocalization). In medium-sized vessels, CD31 and NTPDase1 were found to colocalize while NTPDase2 did not colocalize with these 2 markers or the smooth muscle α-actin–expressing cells. Original magnifications, ×200.
Jean Sévigny et al. Blood 2002;99:
©2002 by American Society of Hematology

6. Immunohistologic localization of NTPDases in the cardiac microvasculature.Immunohistochemistry of mouse heart sections (A-G) are shown in the same order as in Figure 4.
Immunohistologic localization of NTPDases in the cardiac microvasculature.Immunohistochemistry of mouse heart sections (A-G) are shown in the same order as in Figure 4. Antibody specificity: (A) NTPDase1, (B) NTPDase2, (C) α-actin (a marker of smooth muscle), (D) CD31 (a marker of endothelium), (E) NG2 (a marker of pericytes and smooth muscle cells), (F) laminin (a marker of basement membranes), and (G) a higher magnification of NTPDase2. Sections were counterstained with hematoxylin. In panels H-M, immunofluorescence and confocal analysis were performed: (H) NTPDase1 in green and CD31 in blue (colocalization is aquamarine); (I) NTPDase1 in green and NTPDase2 in red; (J) CD31 in blue and NTPDase2 in red (no colocalization); (K) pericyte marker NG2 in green; (L) NTPDase2 in red; (M) panels K and L combined (colocalization is yellow). In the microvasculature, NTPDase1 colocalized with an endothelial cell marker (CD31) while NTPDase2 colocalized with a pericyte marker (NG2). Original magnifications A-F and H-M, ×200; G, ×400.
Jean Sévigny et al. Blood 2002;99:
©2002 by American Society of Hematology