Both ADP and thrombin regulate arteriolar thrombus stabilization and embolization, but are not involved in initial hemostasis as induced by micropuncture.
OBJECTIVE
Thrombosis and embolization are main causes of morbidity and mortality. Up to now, the relative importance of mediators involved is only partly known. It was the aim of this study to investigate the involvement of ADP and thrombin in subsequent phases of arteriolar hemostasis and thromboembolism in vivo.


METHODS
Rabbit mesenteric arterioles were punctured, which induced bleeding, hemostasis, and subsequent thromboembolism. This reaction as well as the activation state of platelets involved ([Ca(2+)](i)), was monitored in real time by intravital (fluorescence) microscopy.


RESULTS
Neither inhibition of thrombin formation or thrombin activity nor blockade of platelet ADP receptors P2Y(1) and P2Y(12) influenced the initial hemostatic reaction: in all experiments initial bleeding was stopped by a primary thrombus within 2-3 s. On the other hand, both thrombin inhibition and P2Y(1) blockade increased rebleeding frequency, which indicates reduced thrombus stability in the long term. Finally, inhibition of either thrombin or ADP (via both receptors) reduced aggregate formation during the embolization phase by at least 90%. While most participating platelets exhibited a transient increase in [Ca(2+)](i) during embolization, an increased percentage of platelets showed no calcium response at all during P2Y(1) blockade, which was accompanied by reduced platelet-platelet interaction strength.


CONCLUSIONS
Whereas thrombin and ADP are not involved in the initial hemostatic reaction, both substances appear to be essential to prevent rebleedings in the long term. During subsequent embolization, ADP (via both receptors) and small amounts of thrombin are involved in platelet activation.
van Gestel
Miriam A
MA
Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
Reitsma
Sietze
S
Slaaf
Dick W
DW
Heijnen
Viviane V Th
VV
Feijge
Marion A H
MA
Lindhout
Theo
T
van Zandvoort
Marc A M J
MA
Elg
Margareta
M
Reneman
Robert S
RS
Heemskerk
Johan W M
JW
Egbrink
Mirjam G A Oude
MG
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D006470:Hemorrhage / Q000378:metabolism*; D006487:Hemostasis / Q000187:drug effects; D006490:Hemostatics / Q000494:pharmacology*; D008565:Membrane Proteins / Q000819:agonists; D008638:Mesenteric Arteries / Q000293:injuries / Q000503:physiopathology; D011677:Punctures; D058910:Purinergic P2 Receptor Agonists; D011817:Rabbits; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D013917:Thrombin / Q000494:pharmacology*; D013923:Thromboembolism / Q000378:metabolism*
D006490:Hemostatics; D008565:Membrane Proteins; C546384:P2ry1 protein, rat; C553058:P2ry12 protein, rat; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D000244:Adenosine Diphosphate; D013917:Thrombin