Raised levels of CD39 in leucocytosis result in marked inhibition of ADP-induced platelet aggregation via rapid ADP hydrolysis

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Abstract
We observed previously that the extent of ADP-induced platelet aggregation in blood from patients with leucocytosis is markedly reduced. We obtained evidence that this is via enhanced ADP metabolism consequent to the high leucocyte count, and speculated that ecto-NTPDase CD39 on leucocytes may be involved. Here we have investigated the association between ADP-induced platelet aggregation, ADP metabolism and expression of ecto-NTPDase CD39 on leucocytes in patients with leucocytosis. Six patients with leucocytosis were compared with six normal controls. Platelet aggregation was measured using platelet counting. ADP metabolism was analysed by HPLC. CD39 on leucocytes from each volunteer and patient was measured by flow cytometry and is presented as the CD39 fluorescence index (CD39FI, the sum of the product of CD39 median fluorescence and cell number for each leucocyte subtype). Compared with the controls, all patients displayed markedly reduced platelet aggregation to ADP in whole blood, markedly enhanced metabolism of ADP to AMP in whole blood, and increased leucocyte CD39FI. The increased CD39FI was due to either a high number of CD39+ve lymphocytes or a high number of CD39+ve neutrophils. In contrast, the measures of aggregation and ADP metabolism performed in platelet-rich plasma from the patients were similar to those obtained for the controls. There was an inverse correlation between ADP-induced aggregation in whole blood and CD39FI, and between the time taken to achieve complete removal of ADP from blood and CD39FI. For two patients with very high CD39FI (60,000 cf 1500 for controls) ADP-induced aggregation was abolished. Reduced aggregation, enhanced ADP metabolism and a raised CD39FI returned to normal in one patient following successful chemotherapy. It is concluded that ADP-induced platelet aggregation in leucocytosis is reduced as a result of enhanced ADP metabolism due to raised levels of leucocyte-associated CD39.