Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans

Abstract

Extracellular ATP and ADP trigger vasodilatatory and prothrombotic signalling events in the vasculature. Here, we tested the hypothesis that nucleotide turnover is activated in the bloodstream of exercising humans thus contributing to the enhanced platelet reactivity and haemostasis. Right atrial, arterial and venous blood samples were collected from endurance-trained athletes at rest, during submaximal and maximal cycle ergometer exercise, and after early recovery. ATP-specific bioluminescent assay, together with high-performance liquid chromatographic analysis, revealed that plasma ATP and ADP concentrations increased up to 2.5-fold during maximal exercise. Subsequent flow cytometric analysis showed that plasma from exercising subjects significantly up-regulated the surface expression of P-selectin in human platelets and these prothrombotic effects were diminished after scavenging plasma nucleotides with exogenous apyrase. Next, using thin layer chromatographic assays with [γ-32 P]ATP and 3 H/ 14 C-labelled nucleotides, we showed that two soluble nucleotide-inactivating enzymes, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase, constitutively circulate in human bloodstream. Strikingly, serum nucleotide pyrophosphatase and hydrolase activities rose during maximal exercise by 20-25 and 80-100%, respectively, and then declined after 30 min recovery. Likewise, soluble nucleotidases were transiently up-regulated in the venous blood of sedentary subjects during exhaustive exercise. Human serum also contains 5 -nucleotidase, adenylate kinase and nucleoside diphosphate (NDP) kinase; however, these activities remain unchanged during exercise. In conclusion, intravascular ADP significantly augments platelet activity during strenuous exercise and these prothrombotic responses are counteracted by concurrent release of soluble nucleotide-inactivating enzymes. These findings provide a novel insight into the mechanisms underlying the enhanced risk of occlusive thrombus formation under exercising conditions.

Cellular function and molecular structure of ecto-nucleotidases

“…Minor amounts of soluble NTPDase activity were reported to constitutively circulate in human bloodstream [152]. But the truly soluble nature of the enzyme(s) has not been verified by phase partitioning and the molecular identity has not been determined.…”

“…Soluble forms have also been described for NPP1 [365,417], NPP3 [422], NPP6 [388], and NPP7 [423]. E-NPPs are found in human serum [152,424,425]. The endogenous mechanisms for shedding the membrane-bound forms need to be further characterized.…”

The touching story of purinergic signaling in epithelial and endothelial cells

“…The matching of oxygen supply with demand requires a mechanism that increases blood flow in response to decreased tissue oxygen levels. Several reports suggest that the RBC acts as a sensor for hypoxia and different mechanisms have been suggested by which the deoxygenated RBC stimulates vasodilatation [98][99][100][101]. RBCs contain millimolar amounts of ATP and possess the membrane-bound glycolytic enzymes necessary for its production [93,102,103].…”

“…RBCs contain millimolar amounts of ATP and possess the membrane-bound glycolytic enzymes necessary for its production [93,102,103]. ATP is released in response to reductions in oxygen tension and pH [99,100]. It has been shown in vitro that vessels dilate in response to low O 2 levels only when blood vessels are perfused with RBCs [104].…”

“…It has been shown in vitro that vessels dilate in response to low O 2 levels only when blood vessels are perfused with RBCs [104]. ATP is released in working human skeletal muscle circulation depending on the number of unoccupied hemoglobin O 2 -binding sites [99,100]. The released ATP then binds to P2Y receptors on the endothelium and stimulates vasodilatation.…”