Incomplete Platelet Dense Granule Formation in Normal Neonates.
Authors: Kahr Walter H. , Baidya Shoma , Das Animitra , Toprak Ayca , Christensen Hilary , Meulman Fanny , Rand Margaret L. , Pluthero Fred G. , Chitayat David , Shah Prakeshkumar , Blanchette Victor S. 
Published: October 2019

Abstract
Platelets are important in maintaining hemostasis in newborns, where bleeding can arise from abnormal platelet function and/or thrombocytopenia. It is well established that plasma coagulation factor concentrations are lower in neonates compared to children and adults, but less is known about the development and function of neonatal platelets. It has been postulated that platelets from neonates, and to a greater extend from premature neonates, are dysfunctional due to low dense granule counts (Blood2006;108:331a), however, other studies have shown normal neonate platelet function. Our previous studies indicated a slightly decreased number of dense granules per platelet in neonates (Blood2005;106:4159–4156). We have now extended these studies to a larger cohort of 19 normal neonatal cord blood samples (gestational age 37.5–40 weeks) from planned Caesarean sections, which were analyzed under optimal sample handling conditions and compared to platelets from 10 children (age 8–10 years). 50 platelets from each subject were evaluated for dense granule content utilizing whole mount and thin section electron microscopy (EM) for the quantification of dense granules (detected via their electron-dense calcium content) and ultrastructural assessment. A subset of samples was tested via flow cytometry for P-selectin expression as a measure of platelet activation, and platelet structural integrity was also assessed using thin section EM. Our data revealed that platelets in neonatal cord blood had a mean dense granule count of 2.3 (SD=2.2) per platelet, compared to 4.4 dense granules per platelet (SD=2.7) in blood from older children; t-test comparisons showed the difference between these groups to be highly significant (P<0.001). Interestingly, 22% of cord blood platelets contained no measurable dense granules, whereas only 3% of platelets from older children where devoid of dense granules. We suspected that the mean dense granule counts of <1 per platelet in neonatal cord blood reported by others may have arisen due to high levels of platelet activation during sample acquisition or handling. In our samples platelet activation as measured by P-selectin expression was similar in both populations and did not exceed 7.5%, and platelet morphology as assessed by thin section EM was also comparable. Our studies confirm that neonatal cord blood platelets contain fewer recognizable dense granules than those found in older children. Two possible explanations for this observation are:
normal numbers of dense granules are present in neonatal platelets, but a subset cannot be detected via EM owing to insufficient calcium uptake; there are fewer dense granules in neonatal platelets owing to peculiarities in the development of megakaryocytes, where recent studies have suggested that dense granules originate by an active transport mechanism and move into proplatelets.
These possibilities point to the usefulness of studying fetal and neonatal megakaryopoiesis.