Age-Related Differences in the Glycosylation of Anticoagulant Protein C.

Abstract

Thromboembolic events can result from procoagulant stimuli and the inability of native anticoagulant mechanisms to pacify thrombin generation. In the pediatric population, although thrombosis occurs in the presence of major coagulant insults, children are relatively resistant to onset of thrombotic complications. A key aspect contributing to developmental differences in the young is that coagulation factors and inhibitors have been shown to exist in different plasma concentrations and developmental forms compared to adults. Increasing evidence indicates that protein C (PC) is a major factor that, upon activation, controls coagulation by shutting down thrombin generation. In adults, PC exists as either 4 N-linked glycan (alpha-PC) or 3 N-linked glycan (beta- PC) proteins that differ in the rate at which their corresponding activated forms react with coagulation factor Va. Our previous work has shown that although plasma PC concentrations are vastly reduced in newborn plasma, inactivation of factor Va is similar to adults, suggesting a potential difference in PC molecules during development. To assess the potential for age-related PC molecule variation, we performed carbohydrate analyses to determine the relative glycoform composition of PC in newborns compared to adults. Venous plasma from either adults or umbilical cords of healthy full-term babies was collected following the ethical guidelines of our institution. Samples of plasma were run on non-reducing SDS polyacrylamide gel (7.5%) electrophoresis, followed by transfer onto PVDF blotting membranes. Blots were probed with polyclonal sheep anti-human PC, followed by anti-sheep IgG-alkaline phosphatase conjugate and then alkaline phosphatase substrate to visualize the protein bands. Relative intensity of alpha-PC and beta-PC bands was determined by laser densitometry. Analyses revealed that significant age differences existed in PC glycoform composition between adults and newborns. While the mass ratio of alpha-PC to the lower molecular weight band of beta PC was 2.3 to 1 for the adult plasma protein, newborns were observed to have an alpha-PC to beta-PC ratio of 8.8 to 1. Thus, an almost 4-fold increase in the circulating alpha-PC to beta-PC ratio was observed in newborns compared to adults. As a confirmation that the difference in migration between the alpha-PC and beta-PC bands was due to glycosylation, removal of N-glycans by treatment of newborn and adult plasma with N-glycosidase F prior to electrophoresis resulted in single bands that now migrated at the same rate for both age groups. We have demonstrated that relative expression of the 2 major PC glycoforms varies widely during development. Given that alpha-PC in its activated form has a much faster rate for factor Va inactivation compared to beta-PC, its increased appearance in newborn plasma may play a role in protecting neonates from thrombosis, despite reduced plasma inhibitor concentrations.