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 end ogenous vasoactive purine dinucleotides which have been isolated from throm bocytes, Diadenosine pentaphosphate (AP(5)A) and diadenosine hexaphosphate (AP(6)A) are more potent than diadenosine tetraphosphate (AP(3)A) and diade nosine triphosphate (AP(3)A) and cause skeletal muscle vasoconstriction in rats. Little is known about their physiological and pathophysiological sign ificance in humans. The aims of the present study were to compare thrombocy te AP(n)A concentrations in patients with essential hypertension (HYP) and in healthy normotensive humans (CON) using a novel quantitative assay and t o 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 quantita tive determination of the substances with an ion-pair reversed-phase high-p erformance liquid chromatography (HPLC) system. The structural integrity of the isolated AP(n)As was confirmed by mixed assisted laser desorption ioni zation -time of flight mass spectrometry (MALDI-TOF) measurements and co-el ution with added standards. The detection threshold for all four AP(n)As wa s 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. R esults HYP (n = 15) and CON (n = 15) did not significantly differ in platel et 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 -thrombogl obulin, P = 0.003) and AP(6)A (10 +/- 1 versus 6 +/- 1 ng/mug beta -thrombo globulin, P = 0.015) than CON, HYP had significantly elevated FVR (50 +/- 6 versus 33 +/- 2 arbitrary units, P = 0.01) compared to CON. Significant co rrelations were found between ASA and FVR (p = 0.38, P = 0.04) as well as b etween AP(6)A and FVR (p = 0.42, P = 0.02). In contrast, there were no sign ificant correlations between AP(n)As and MAP. Conclusions The study shows that thrombocyte concentrations of AP(5)A and A P(6)A are elevated in patients with essential hypertension. Vasoconstrictio n caused by release of AP(5)A and AP(6)A from thrombocytes may contribute t o the increase of vascular resistance in hypertensive patients. (C) 2001 Li ppincott Williams & Wilkins.