Decreased Dense Granule Volume in Neonatal Platelets.

Two models of biogenesis have been proposed for the development of platelet granules. One model postulates that granule contents develop in the maturing megakaryocyte via a direct biosynthetic pathway from the golgi, whereas the second model is thought to involve the multivesicular body for sorting and targeting granular constituents via an endosomal pathway. While these models may appropriately apply to the mechanism of the development of platelet alpha granules, dense granule biosynthesis is not readily explained. In animal studies and in human cultured megakaryocyte experiments, molecular markers (CD63 and serotonin) have been reported to appear concomitant with alpha granule formation, yet the characteristic density of the delta granule is not present until later in the maturation of the megakaryocyte. This suggests that dense granule vesicles must be “loaded” for a fully functioning platelet. Our evaluations of platelets obtained from umbilical cord blood would suggest that circulating platelets may become fully functional after birth via a mechanism that loads specific vesicles. The neonate is known to have a transient platelet dysfunction at the time of birth which has been explained in the literature as being due to 1) a diminished response to physiologic agonists, 2) defective platelet dense granule secretion, and/or 3) ineffective mobilization of intracellular calcium in platelets. Studies to define the transient platelet dysfunction in the neonate have been limited. We and others have found that the adenine content of platelets obtained from umbilical cord blood is significantly less than seen in adults. We have found that platelets obtained from umbilical cord blood have an average of 0.79 ± 0.08 DG/PL (n=62), which is significantly decreased compared to the adult population and that the ATP:ADP ratio in these platelets is 1.95 (n=61), consistent with established adult normal range ratio values (1.4–1.95). The actual measured adenine content was found to be decreased in the neonatal platelets (ATP = 2.76 ± 0.53 mmol/1011 PL; ADP=0.96±0.19 mmol/1011 PL). The normal adenine nucleotide DG contents have been reported to be 3.5–7 mmol/1011 PL for ATP and 1.9–3.7 mmol/1011 PL for ADP in adults. Using the uranaffin reaction technique to classify the four types (I–IV) of dense granules present in neonatal platelets, we have found a significant difference in the percentage of each type than is found in adult controls. We have employed an image analysis technique (Blood, 90: 140a, 1997) to calculate the mean volume of dense granules in neonatal platelets. We report that not only do neonatal platelets have a deficiency of dense granules, but that these granules are also extremely small in size having an average volume of 6.45 X 105 nm3 (n =36) (normal adult = 8–12 X 106 nm3). These results would suggest that the neonatal platelet has an incomplete incorporation of constituents (i.e., calcium, ADP) into the dense granules at the time of birth and that an unknown mechanism exists that induces dense vesicle “loading” at some time after birth. These observations may prove useful in pursuit of understanding the mechanism of platelet dense granule biosynthesis.