Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation.
BACKGROUND
Platelets (PLTs) contain purinergic receptors for ATP (P2X1) and ADP (P2Y1 and P2Y12) that rapidly desensitize upon stimulation with these nucleotides. In vivo, this is antagonized by ectonucleotidases on the surface of endothelial cells and white blood cells (WBCs). The receptor desensitization of ATP- and ADP-induced responses of PLTs stored in plasma without WBCs was investigated.


METHODS
ATP- and ADP-induced PLT shape change (shear-induced) aggregation and Ca2+ signaling were measured in the presence or absence of plasma. Degradation of nucleotides in plasma was quantified by high-performance liquid chromatography.


RESULTS
Washed PLTs became refractory for ATP and ADP in shape change, aggregation, and Ca2+ responses during a 90-minute incubation at 37 degrees C. The PLT responses mediated by P2X1, P2Y1, and P2Y12 receptors gradually reduced or disappeared. When plasma was present, however, the PLTs persistently showed high responses to ATP and ADP. Heat treatment of plasma abolished this effect. Also under conditions of flow and high shear, PLTs in plasma kept high P2X1 activity, mediating aggregate formation. In isolated plasma, not containing WBCs, nucleotides were degraded in the order of ADP/UDP>ATP/UTP. Degradation of ATP was partly inhibited by blocking the ecto-NTPDase CD39, whereas degradation of both ATP and ADP was inhibited by blocking ectopyrophosphatase/phosphodiesterase activity. Part of the nucleotide-degrading activities appeared to be membrane-bound.


CONCLUSIONS
Ectonucleotidases in plasma preserve the functionality of P2X1 and P2Y receptors. Upon PLT storage, these plasma activities are essential to ensure adequate (shear-dependent) formation of aggregates and thrombi.
Cauwenberghs
Sandra
S
Department of Biochemistry (CARIM), the Sanquin Blood Bank South-East, the Department of Health Risk Analysis and Toxicology (NUTRIM), Maastricht University, Maastricht, the Netherlands.
Feijge
Marion A H
MA
Hageman
Geja
G
Hoylaerts
Marc
M
Akkerman
Jan-Willem N
JW
Curvers
Joyce
J
Heemskerk
Johan W M
JW
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000502:physiology*; D001793:Blood Preservation; D020013:Calcium Signaling; D006801:Humans; D010949:Plasma / Q000201:enzymology; D015539:Platelet Activation; D011755:Pyrophosphatases / Q000097:blood / Q000502:physiology*; D011983:Receptors, Purinergic / Q000378:metabolism*; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D013927:Thrombosis / Q000209:etiology*
D011983:Receptors, Purinergic; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; C092064:ectonucleotide pyrophosphohydrolase