P-Selectin Expression and Platelet Function Are Developmentally Regulated

Platelets have numerous well-defined roles during adult hemostasis, however little is known about whether these functions are developmentally regulated. In the adult, P-selectin is constitutively expressed in megakaryocytes and localized to the membrane of alpha granules. Activation of platelets results in granule secretion and P-selectin surface exposure allowing for recruitment of leukocytes to sites of vascular injury and the formation of leukocyte-platelet aggregates. Here, we examined the expression and function of P-selectin in platelets during murine embryogenesis. Platelets were isolated from timed pregnant outbred-ICR mice at embryonic day 12.5 (E12.5), E15.5, postnatal day 1 (PN1), PN7, PN21, and were assayed for thrombin induced integrin activation and P-selectin translocation. While no significant differences were seen in the ability of embryonic platelets to be activated by thrombin, P-selectin surface expression after thrombin stimulation was significantly reduced in embryonic compared to adult platelets (E15.5 p=.0006, E12.5 p=.0004, PN1 p=.01, n=3, unpaired t-test). Surprisingly, P-selectin expression began to appear on the surface of activated platelets only after birth, approaching 50% and 75% of maternal expression at PN7 and PN21, respectively. No significant difference was seen in VEGF secretion after thrombin activation of E12.5, E15.5 and adult platelets (n=5), suggesting that the lack of P-selectin surface expression was not due to a loss of alpha granule secretion. To further characterize expression of P-selectin, real-time qPCR was performed on mRNA harvested from platelets at E12.5, E15.5, PN1 and adult stages. P-selectin transcript levels were significantly lower in embryonic and PN1 platelets, suggesting that P-selectin expression is transcriptionally regulated. Consistent with this concept, Imagestream analysis indicates that P-selectin protein is absent in megakaryocytes derived from the fetal liver. P-selectin expression on platelets is an important mediator of platelet-leukocyte interactions; therefore, we assayed the ability of platelets to bind to leukocytes in embryonic whole blood. Preliminary data suggest that embryonic platelets do not form leukocyte-platelet aggregates upon ex vivo stimulation with AYPGKF, a Par4 agonist. Taken together, our data indicate that P-selectin expression is developmentally regulated in primary mouse platelets. The absence of P-selectin expression in embryonic platelets correlates with an impaired ability to interact with leukocytes.