A novel assay for determination of diadenosine polyphosphates in human platelets: studies in normotensive subjects and in patients with essential hypertension

Objective Diadenosine polyphosphates (AP(n)As, n = 3-6) are a family of endogenous vasoactive purine dinucleotides which have been isolated from thrombocytes, Diadenosine pentaphosphate (AP(5)A) and diadenosine hexaphosphate (AP(6)A) are more potent than diadenosine tetraphosphate (AP(3)A) and diadenosine triphosphate (AP(3)A) and cause skeletal muscle vasoconstriction in rats. Little is known about their physiological and pathophysiological significance in humans. The aims of the present study were to compare thrombocyte AP(n)A concentrations in patients with essential hypertension (HYP) and in healthy normotensive humans (CON) using a novel quantitative assay and to assess a possible relationship between thrombocyte AP(n)A concentrations and skeletal muscle vascular resistance. Design and methods We describe a novel assay for quantification of AP(n)As in human platelets, involving platelet isolation from human blood, a solid-phase extracting procedure with a derivatized resin, desalting and quantitative determination of the substances with an ion-pair reversed-phase high-performance liquid chromatography (HPLC) system. The structural integrity of the isolated AP(n)As was confirmed by mixed assisted laser desorption ionization -time of flight mass spectrometry (MALDI-TOF) measurements and co-elution with added standards. The detection threshold for all four AP(n)As was 1 pmol/l and the inter-assay coefficients of variation were < 11% (n = 12). After venous blood sampling, mean arterial blood pressure (MAP) and forearm blood flow (FBF, using venous occlusion plethysmography) were measured in HYP and CON. Forearm vascular resistance (FVR) was calculated as MAP/FBF. Results HYP (n = 15) and CON (n = 15) did not significantly differ in platelet AP(3)A and AP(4)A content, but HYP had significantly higher thrombocyte concentrations of AP(5)A (56 +/- 7 Versus 32 +/- 3 ng/<mu>g beta -thromboglobulin, P = 0.003) and AP(6)A (10 +/- 1 versus 6 +/- 1 ng/mug beta -thromboglobulin, P = 0.015) than CON, HYP had significantly elevated FVR (50 +/- 6 versus 33 +/- 2 arbitrary units, P = 0.01) compared to CON. Significant correlations were found between ASA and FVR (p = 0.38, P = 0.04) as well as between AP(6)A and FVR (p = 0.42, P = 0.02). In contrast, there were no significant correlations between AP(n)As and MAP. Conclusions The study shows that thrombocyte concentrations of AP(5)A and AP(6)A are elevated in patients with essential hypertension. Vasoconstriction caused by release of AP(5)A and AP(6)A from thrombocytes may contribute to the increase of vascular resistance in hypertensive patients. (C) 2001 Lippincott Williams & Wilkins.