Identification and partial characterization of an adenosine(5′)tetraphospho(5′)adenosine hydrolase on intact bovine aortic endothelial cells

Abstract

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.

NPP4 is a procoagulant enzyme on the surface of vascular endothelium

“…Our identification of NPP4 as a slow hydrolyzing Ap3A enzyme comes many years after the presence of such an entity in the circulatory system was first hypothesized by Luthje and Olgilvie. 9 The researchers subsequently detected an enzyme with Ap3A and Ap4A hydrolytic activity on the surface of bovine vascular endothelium 11 contemporaneously with a second group who discovered an Ap3A hydrolase on porcine vascular endothelium. 10 Our search of the NCBI-GEO database also revealed NPP4 gene expression in a variety of human vascular endothelial cells.…”

“…10,11,30,31 ADP released during platelet degranulation stimulates further platelet aggregation and release, and thus the process is autocatalytic. To prevent unregulated thrombus formation, ADP is rapidly degraded-1M ADP is degraded in whole blood within 3.2 minutes, and may be even faster in vivo because of ectoenzymes present on cell surfaces.…”

“…10,11 We therefore stained by immunofluorescence a highly vascular human organ (brain, which accepts ϳ 30% of cardiac output blood flow) for NPP4 (Figure 1). Immunofluorescence of NPP4 in brain reveals that NPP4 is enriched on blood vessel surfaces and localizes to vascular walls, as highlighted in numerous branched vessels in the brain parenchyma (Figure 1).…”

“…9 The mechanism by which Ap3A promotes aggregation suggests that Ap3A is stored as a metabolically inactive or chemically masked molecule, which on release into the extracellular space is converted into a hemodynamically active form by an enzyme that liberates ADP from the dinucleotide. An enzyme capable of hydrolyzing Ap3A into AMP and ADP was partially characterized on the surface of intact porcine 10 and bovine 11 vascular endothelial cells more than 20 years ago. Before this, Luthje and Olgilvie linked weak Ap3A hydrolase activity in PRP to an extracellular glycoprotein, neither stored within nor released by platelets, with optimal enzymatic activity around pH 8.5 to 9.0, and a divalent cation dependence.…”

Di(1,N6‐ethenoadenosine)5′, 5‴‐P1,P4‐tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

“…K= values for Ap4A cleaving enzymes from bovine adrenal chromaffin and aortic endothelial cells lie in the low micromolar range [8, 22,24]. The results reported here demonstrate that e-(Ap4A) is biologically active at similar concentrations as is Ap4A, and open up the possibility to assay Ap4A cleaving enzymes by direct fluorescence measurements as a complementary or even alternative approach to the use of radiometric or lurninometric techniques.…”