The long-term goal is to establish whether a relationship exists between levels of endothelial cell-produced fibrinolytic proteins and the pathogenesis of hemorrhagic disorders in premature and low-birth-weight(LBW) infants. Specifically, we will determine whether variations in the synthesis, activity, distribution and types of plasminogen activators (PAs: tissue-type, t-PA; urokinase-type, u-PA) produced/secreted by primary and subcultured human umbilical vein endothelial cells (HUVECs) from individual cords, can be correlated with hemorrhagic disorders (IVH-GMH) in the respective infants from which each cord was derived. The Mr 115K and 70K t-PA, and Mr 95K and 54K u-PA forms will be studied. PA activity in serum-free conditioned medium will be assayed following SDS-PAGE, using 125 I-fibrin- coated wells in the absence/presence of specific anti-PA IgGs. The synthesis, activity and distribution (cells vs. media) of total free (following nucleophilic dissociation of PA-inhibitor complexes) immunobead isolated, radiolabeled PA antigens will be analyzed by SDS-PAGE, under non-reduced/reduced conditions. These analysis will permit the simultaneous measurement of: total free radiolabeled Mr 70K t-PA and Mr 54K u-PA antigen synthesis; activity of total free immunobead-isolated PAs; and identify the molecular composition (single- and two-chain forms) of the isolated PAs. In addition, t-PA and u-PA cDNA probes will be used to quantitate poly(A+) RNA levels corresponding to these genes in these HUVECs, using RNA dot blot and Northern blot analysis. cDNA probes will also be used to determine whether induction of PA synthesis (hormones/agents) is coincident with increases in PA mRNAs and is similar/different in HUVECs from neonates with IVH-GMH vs. """"""""normals"""""""". Finally, euglobulin lysis time (fibrin plate) and PA antigen content (IRMA) of cord blood will be measured as potential early indicators of increased fibrinolysis. In concert, these studies will provide new insights into PA synthesis/levels in """"""""normal"""""""" fetal HUVECs, demonstrate whether PA synthesis is regulated or affected at the translational or transcriptional levels and establish whether abnormalities in regulation of PA synthesis in cultured HUVECs from premature and LBW infants can be correlated with hemorrhagic tendencies in these infants.
