ABSTRACT. We have investigated the impaired secretion response of neonatal platelets. We compared the response of washed neonatal and adult platelets to thrombin and collagen, and to specific activators of calcium flux (inositol trisphosphate) and protein kinase C activation (oleoylacetyl glycerol). Neonatal platelets show no impairment ofaggregation, secretion of ['4C]serotonin or phosphorylation of saecific intracellular rotei ins in resDonse to thrombin. inositol trisphosphate, o; oleoyl-acetyl glycerol. ow ever; neonatal platelets have a markedly decreased response to collagen. To further evaluate this deficient response, we examined specific aspects of the collagen activation pathway. Collagen-platelet interaction as measured by adhesion of platelets to collagen-coated dishes showed no difference in adhesion of neonatal platelets compared to adult controls (20.1 f 11.6 versus 18.6 2 9.3%). The presence of GPIa/ Ila, a Mgz+-dependent collagen receptor, was evaluated by flow cytometric analysis of binding of fluorescein-tagged monoclonal antibody, 6F1 (directed against GPIaJIIa). There was no difference either in the percent of platelets that bound antibody (80 versus 81%) or in the mean fluorescence intensity of the adult and neonatal samples. Phosphoinositide hydrolysis was decreased in neonatal platelets in response to collagen but normal in response to thrombin. Neonatal platelets released more arachidonic acid than adult platelets in response to thrombin (29.5 f 3.2 versus 19.6 + 1.8%) but less than adult platelets in response to 10 pg/mL collagen (3.2 2 1.1 versus 9.3 + 3.0%). Thromboxane B2 production was also decreased in response to collagen (52.7 f 12.6 versus 101.4 + 18.7 ng/mL). These results suggest that the deficient collagen response in neonatal platelets may lie in transduction of the collagen signal to phospholipases Az and C . (Pediatr Res 27: 337-343,1990) Abbreviations AA, arachidonic acid IP3, inositol-1,4,5-trisphosphate OAG, oleoyl-acetyl glycerol PIPZ, phosphatidylinositol-4,5-bisphosphate MLC, myosin light chain 47 K, 47000 dalton protein TxAZ, thromboxane A2 TxBz, thromboxane Bz HBSS, Hanks' balanced salt solution clinical importance to those caring for sick and premature infants. The differences in clotting factor activity and fibrinolytic enzymes between cord blood and that of adult controls are well established (1,2). The abnormalities of neonatal platelet function are less completely understood. In the mid-1970s, differences in the in vitro responses of cord and newborn platelets compared to those from older subjects were identified (3-6). These abnormalities include a decreased aggregation response to certain agonists including ADP, epinephrine and collagen (3, 4), and decreased secretion of dense granule contents (5, 6). Neonatal platelets were shown to have decreased endogenous stores of dense granule contents, ADP, and serotonin, although Whaun demonstrated that their uptake and storage of radiolabeled serotonin was as effective as that of adult platelets (6). In addition,...
In vitro function of cord blood platelets from 35 premature infants (gestational age 32 +/- 3.2 wk) was compared with that of 12 full-term infants and 14 adult control subjects. In comparison with adult platelets, preterm platelets showed impaired aggregation, in response to thrombin, collagen, ADP, and U46619 [a stable analog of thromboxane A2 (TxA2)], and impaired [14C]serotonin secretion in response to collagen and U46619. The production of TxB2 (the stable TxA2 metabolite) in response to collagen was reduced in preterm platelets (30.2 +/- 5.5 ng/mL) compared with full-term (52.7 +/- 12.6 ng/mL) or adult control platelets (132.3 +/- 38.7 ng/mL). The deficient TxB2 production and U46619 response prompted further investigation of TxA2 receptor number and binding characteristics. Immunoblotting using an anti-TxA2 receptor antibody (anti-P2) identified a single, identical 55-kD band in solubilized membranes of control, full-term, and preterm platelets. Flow cytometry using anti-P2 produced histograms that did not differ between adults and neonates. Ligand binding studies using [3H]U46619 were carried out on 10 samples from each group. Scatchard analysis yielded a single class of binding sites with no significant difference among the Kd values (85 +/- 16 versus 99 +/- 12 versus 100 +/- 12 nM) or number of binding sites per platelet (1876 +/- 460 versus 2450 +/- 478 versus 2777 +/- 536) for preterm and full-term infants and adults. Therefore platelets of preterm infants show impaired TxA2 production and response. The poor response is not related to altered binding characteristics of the TxA2 receptor but may lie in the postreceptor signal transduction pathway.
SummaryLysosomal Associated Membrane Protein-2 (LAMP-2) is an inherent component of lysosomal granule membranes in diverse cell types, including platelets. We examined platelets for evidence of LAMP-2 in dense granule membranes as CD63 has previously been shown to be present in both lysosomal and dense granule membranes. Immunological techniques were used to examine the localization of LAMP-2 in control platelets and those from an individual with Hermansky-Pudlak syndrome (HPS), a condition characterised by platelet dense granule deficiency. Immunoblotting studies demonstrated that LAMP-2 was enriched in a dense granule preparation. Flow cytometry of thrombin-stimulated control platelets was consistent with biphasic surface expression of LAMP-2. The early expression was accompanied by dense granule, but minimal lysosomal granule, release. The late expression was accompanied by additional lysosomal granule release only. Thrombin stimulation of HPS platelets showed only late, lysosome-associated LAMP-2 expression. Immunoelectron microscopy indicated the presence of LAMP-2 in the membranes of serotonin-containing granules as identified by an anti-serotonin polyclonal antibody. These data indicate that LAMP-2 is present in the membranes of platelet dense granules in addition to lysosomal granules, and has a similar distribution to CD63.
Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.
Sex-related differences may be present during fetal lung growth and at the onset of surfactant synthesis. In this study we investigated the role of dihydrotestosterone (DHT) and estrogen (EST) on cell division and on labeled palmitate incorporation into disaturated phosphatidylcholine (DSPC) at various times of gestation. Using organ cultures of fetal rat lung from sexed littermates, it was shown that both DHT and EST reduce DNA synthesis only in tissue taken during the rapid growth phase from day 16 to 19 of gestation. From autoradiographic counts, epithelial cell division was most affected. Both hormones reduced DSPC synthesis in explants prepared at day 18, when levels are normally low. At day 19, DHT reduced palmitate incorporation into DSPC of female explants to the male level; subsequently DHT had no effect on any tissue. In contrast, the addition of EST stimulated DSPC synthesis 40% above controls in both male and female explants taken at day 20 only. The results suggest that sex differences seen in late fetal lung development may arise from the combined effects of slowed epithelial growth induced by these hormones followed by inhibition of DSPC synthesis by DHT and acceleration by EST at the crucial period when surfactant synthesis begins.
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