Differences in Thromboxane Production between Neonatal and Adult Platelets in Response to Arachidonic Acid and Epinephrine

NEONATAL PLATELET THROMBOXANE FORMATION 003 1-3998/84/1809-0823$02.00/0 PEDIATRIC RESEARCH Copyright O 1984 International Pediatric Research Foundation, Inc. 823 Vol. 18, No. 9, 1984 Prinred in (I.S.A. Differences in Thromboxane Production between Neonatal and Adult Platelets in Response to Arachidonic Acid and Epinephrine MARIE J. STUART, JON DUSSE, DAVID A. CLARK AND RONALD W. WALENGA Divisions of Pediatric Hematology-Oncology and Neonatology, Department of Pediatrics, State University of New York, Upstate Medical Center, Syracuse, New York 13210 Summary An impairment in platelet function is well recognized in the neonate, and includes abnormalities in the release of storage pool In this study, we have investigated the possible role of the pro- adenine nucleotides, and aggregation responses to a variety of aggregatory arachidonic acid (AA) metabolite thromboxane, in stimuli (2, 3, 10). A previous evaluation of exogenous [I4C]AA the impaired function of neonatal platelets. In platelet-rich metabolism in the platelet of the neonate did not suggest that plasma thromboxane production (measured by radioimmunoas- neonatal platelet dysfunction is related to any specific abnorsay of thromboxane B2) was not different between neonates and mality in this pathway (13). We observed an increased release of adults when stimulated by thrombin (at 0.1 or 1.0 U/ml) or endogenous AA from neonatal platelet membrane phospholipcollagen (70 pg/ml) although neonatal platelets produced de- ids, and decreased activity of neonatal platelet cyclooxygenase. creased thromboxane (TBXZ) postepinephrine stimulation. In These two effects counteracted each other resulting in similar response to 1 U/ml thrombin, adult and neonatal platelet-rich conversion of prelabeled arachidonic acid to TXBz in adult and plasmas produced mean values of 3.41 0.35 (SEM) and 3.11 f 0.49 pmol of TXB2/106 platelets, respectively. Production of neonatal platelets. The present study was undertaken to evaluate endogenous radioimmunoassayable TXB? production by neoTXB2 in response to 0.1 U/ml thrombin was not dissimilar natal platelets in response to a variety to physiologic stimuli. Our 0.46 pmol) and adults (1.04 0.38 between neonates (1.01 results provide evidence that although there is no difference in pmol). When collagen was used as the aggregating agent, TXB2 TXB2 production between adult and neonatal platelet-rich plasproduction was also not significantly different with values of 2.44 mas when thrombin and collagen are used as aggregating agents, 2 0.48 and 1.90 0.46 pmol/106 platelets produced by adult and neonatal TXB? production is decreased in response to epinephneonatal platelet-rich plasma, respectively. In response to 200 rine, and markedly increased in the presence of low doses of f 0.39 pmol pM epinephrine, adult platelets produced 1.03 exogenous arachidonic acid. No such enhanced response by TXB2/106platelets while neonatal platelet TXBz production was neonatal platelets to arachidonic acid was seen in a washed significantly decreased (0.15 0.04; P < 0.05). Thromboxane platelet system. Abnormalities in thromboxane production by production in response to AA, however, was markedly elevated neonatal platelets in response to epinephrine persisted in the in neonatal platelet-rich plasma. When 200 and 400 pM concen- washed platelet system. trations of AA were used as the aggregating stimuli, neonatal platelet rich plasma produced 3.17 f 0.77 and 8.0 f 1.47 pmol TXB2/106platelets, respectively. These values were significantly MATERIALS AND METHODS elevated P < 0.02 and < 0.005) when compared to mean values of 0.41 f 0.10 and 3.32 0.15 pmol in adult platelet-rich plasma. Prepartion of platelet-rich plasma or washed platelets. UmbilThis elevated thromboxane production was not, however, inher- ical cord blood samples were obtained at the time of delivery ent in neonatal platelets since when washed platelets were stud- from 16 neonates (nine males and seven females). All infants ied, results were reversed. Adult platelets produced more throm- weighed more than 2500 g and were born to healthy mothers boxane at all doses of AA evaluated. These results suggest that after normal full-term pregnancies. No mother had ingested the elevated response to exogenous AA observed in neonatal aspirin or other drugs known to affect platelet thromboxane platelet-rich plasma results from as yet undetermined plasma production within 2 weeks prior to delivery. Immediately after factors. The reported deficiencies in platelet function in the delivery, clamps were placed on the umbilical cord, and an 18newborn clearly do not result from deficient thromboxane pro- gauge needle was inserted into the umbilical vein near its placenduction poststimulation with the physiologic aggregating agents tal insertion. Blood was also obtained from 13 normal healthy collagen and thrombin. Moreover, our study introduces a new adults (six males and seven females) who had taken no medicaand possibly important difference between adult and neonatal tions for 2 weeks prior to evaluation. The study was approved plasma, namely, the differential response to exogenous arachi- by the Institutional Human Experimentation Committee. Following informed consent, blood was drawn into a plastic donic acid. syringe and immediately anticoagulated with 10% by volume of Abbreviations 0.1 M buffered citrate anticoagulant. After correction for hematocrit, PRP was prepared by centrifuging the citrated blood at AA, arachidonic acid 200 x g for 15 min. Platelet-poor plasma was obtained by TXB2, thromboxane Bz centrifugation of the remaining blood for 15 min at 1800 x g. PRP, platelet-rich plasma For the aggregation studies, PRP was adjusted with autologous Received December 2, 1983. platelet-poor plasma to a platelet count of 2.5 to 3 x 108/ml. This work was supported by United States Public Health Service Grant HD- Platelet counts were performed using a Technicon platelet ana14405 and by Clinical Research Grant 6-282 from The National Foundation, The lyzer. For the experiments using washed platelets, following the March of Dimes. Address correspondence to: Marie J. Stuart. Professor of Pediatrics, SUNY, washing procedure previously described (14), platelets were reUpstate Medical Center. 750 East Adams Street, Syracuse, NY 13210. suspended in Hanks' balanced salt solution containing 0.5 mM + + + + + + 824 STUART ET AL. calcium chloride at a concentration of 2.5 to 3 x 10"ml. Platelet aggregation was evaluated at 37°C using aliquots of 0.25 ml/ aggregation experiment. The aggregating agents used in PRP included thrombin ( 1 and 0.1 unitslml; Parke-Davis, Kalamazoo, MI), acid-soluble bovine collagen (70 pg/ml; Sigma), epinephrine (200 pM final concentration), and varying concentrations of arachidonic acid (200 pM, 400 pM, and 2 mM; Nucheck Prep, Elysian, MN). Aggregating agents evaluated in the washed platelet system included epinephrine (200 pM) and 10, 30, and 50 pM a (...truncated)