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.