We determined the in vivo molar concentrations of active tissue plasminogen activator (t-PA), active plasminogen activator inhibitor type 1 (PAI-1), and t-PA/PAI-1 complex. t-PA activity was measured in plasma stabilized by immediate acidification. PAI-1 activity and t- PA/PAI-1 complex antigen were measured in citrated plasma; these measurements were corrected for the loss in PAI-1 activity and increase in complex that occurs in unacidified plasma samples due to the continued reaction between t-PA and PAI-1 after the sample was drawn. To convert t-PA and PAI-1 activity measurements into molar concentrations we determined the specific molar activity of t-PA and PAI-1 in vivo: 4.48 x 10(13) IU/mol. Of 72 subjects studied, 13 had less than 150 pmol/L active PAI-1; in these individuals 33% +/- 21% of their t-PA was active and the molar ratio of active t-PA to active PAI- 1 was 0.20 +/- 0.13. In the 11 subjects with greater than 500 pmol/L active PAI-1, 1.5% = 1.1% of the t-PA was active and the molar ratio of active t-PA to active PAI-1 was 0.0043 +/- 0.0036. Overall, the fraction of active t-PA declined exponentially as a function of the active PAI-1 concentration. During the day, the percentage of total t- PA that was active increased from 12% at 8:00 AM to 31% at 8:00 PM, while the molar ratio of active t-PA to active PAI-1 increased from 0.05 to 0.22 from morning to evening (n = 12).

The effect of body condition per se on plasma IGFs and IGF-binding proteins (IGFBPs) and the whole-body metabolic responses to recombinant DNA-derived bovine GH (rbGH) in both the fed and the fasted state were determined in lean and dietary obese sheep (n = 6/group). Sheep at zero-energy balance and equilibrium body weight were injected s.c. for 12 days with 100 micrograms/kg rbGH immediately before their morning feeding. Before GH treatment, fasting plasma concentrations of insulin (17.0 +/- 1.9 vs 7.5 +/- 0.7 microU/ml), IGF-I (345 +/- 25 vs 248 +/- 10 ng/ml), glucose (52.6 +/- 1.1 vs 48.3 +/- 0.7 mg/dl), and free fatty acid (FFA) (355 +/- 45 vs 229 +/- 24 nmol/ml) were greater (P < 0.05) and those of GH (1.1 +/- 0.2 vs 2.6 +/- 0.3 ng/ml) were lower (P < 0.05) in obese than in lean sheep. Fasting concentrations of IGF-II and glucagon were not affected (P > 0.05) by obesity. GH concentrations were increased equivalently by 6-9 ng/ml in lean and obese sheep during GH treatment. GH caused an immediate and a marked fivefold increase in the fasting insulin level in obese sheep but only minimally affected insulin concentration in lean sheep. The increment in fasting glucose during GH treatment was greater (P < 0.05) in obese (8-12 mg/dl) than in lean (2-5 mg/dl) sheep. Frequent measurements in the first 8 h after feeding and injection of excipient (day 0) or the first (day 1) sixth (day 6) and twelfth (day 12) daily injection of GH showed that prandial metabolism in both groups of sheep was affected minimally by GH. However, GH treatment on day 1 (not days 6 or 12) acutely attenuated the feeding-induced suppression of plasma FFA in both groups of sheep and this effect was significantly greater in obese than in lean sheep. Although obese sheep were hyposomatotropic, the basal and GH-induced increases in plasma IGF-I concentrations were greater (P < 0.05) in obese than in lean sheep. Plasma IGF-II was unaffected by obesity and was not increased by GH stimulation. Western ligand blotting showed that IGFBP-3 accounted for approximately 50-60% of the plasma IGF-I binding capacity in sheep respectively both before and during GH treatment. Basal plasma levels of IGFBP-2 were lower (P < 0.05) and those of IGFBP-3 greater (P < 0.05) in obese compared with lean sheep. GH increased the level of IGFBP-3 equally in lean and obese sheep, but suppressed the expression of IGFBP-2 more (P < 0.05) in lean than in obese sheep. We concluded that the diabetogenic-like actions of GH in sheep were exaggerated markedly by obesity, and were expressed more during the fasted than the fed states. The effects of GH stimulation on the endocrine pancreas may be selective for beta-cells and preferentially enhanced by obesity. GH regulation of IGF-I and the IGFBPs differs in lean and obese sheep.

We have standardized the measurement of plasminogen activator inhibitor type 1 (PAI-1) activity in plasma. One-chain tissue-type plasminogen activator (t-PA; EC 3.4.21.31; final activity, 5 int. units/mL) was incubated with plasma (final dilutions 1:4 to 1:40) in phosphate buffer (pH 7.4, ionic strength = 0.15) for 15 min at 37 degrees C, followed by acidification and measurement of residual t-PA activity by an amidolytic method. The PAI-1 activity assay was 98% specific for PAI-1 activity in samples from both pregnancy and nonpregnancy, and varied linearly with added plasma volume when the percent inhibition of t-PA was between 8% and 50%. For the standardized method, analytical recovery was 93 +/- 5%, the detection limit was 1.6 arbitrary units per milliliter (1 arb. unit of PAI-1 activity = inhibition of 1 int. unit of t-PA activity), and total imprecision was 10.2 (SD 0.7) arb. units/mL (CV = 7%, n = 20). The average PAI-1 activity in 10 healthy individuals drawn between 0800 and 1000 hours was 23.9 +/- 15.4 arb. units/mL. Compared with the standardized assay, two of three previously described assays underestimated PAI-1 activity in plasma by 77% and 85%, respectively.

SummaryThe purpose of this study was to determine whether different regions of the rabbit vascular system show variations in the rate of plasminogen activator (PA) secretion. To start, we evaluated the time course, dose response and adrenergic specificity of PA release. Infusion of 1 µg/kg of epinephrine stimulated a 116 ± 60% (SD) increase in PA activity that peaked 30 to 60 s after epinephrine administration. Infusion of 1 µg/kg of norepinephrine, isoproterenol and phenylephrine had no effect on PA activity. Pretreatment with phentolamine, an alpha adrenergic antagonist, blocked the release of PA by epinephrine while pretreatment with the beta blocker propranolol had no effect. This suggests that PA release in the rabbit was mediated by some form of alpha receptor.Significant arterio-venous differences in basal PA activity were found across the pulmonary and splanchnic vascular beds but not the lower extremity/pelvic bed. After stimulation with epinephrine, PA activity increased 46% across the splanchnic bed while no change was seen across the lower extremity/pelvic bed. We conclude that several vascular beds contribute to circulating PA activity in the rabbit, and that these beds secrete PA at different rates under both basal and stimulated conditions.