An enzyme-linked immunosorbent assay for urokinase-type plasminogen activator (u-PA) and mutants and chimeras containing the serine protease domain of u-PA.

An enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 +/- 0.6 ng/ml (mean +/- SD, n = 27). The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising AA 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with alpha 2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Propofol does not inhibit hypoxic pulmonary vasoconstriction in humans.

The influence of increasing doses of propofol (from 6 to 12 mg/kg/h by continuous infusion) on hypoxic pulmonary vasoconstriction was studied in 10 patients prior to thoracic surgery. All patients were intubated with a left-sided double-lumen endobronchial tube. Initial anesthesia and muscle relaxation were accomplished by administering fentanyl, droperidol, and pancuronium. After 100% oxygen ventilation of both lungs for 20 min in a lateral decubitus position, the nondependent lung was deflated and one-lung ventilation was started. The dependent lung was continuously ventilated with 100% oxygen. Twenty minutes after the start of one-lung ventilation, propofol at an IV infusion rate of 6 mg/kg/h was added to the anesthetic technique. Thirty minutes later it was increased to 10 mg/kg/h and another 15 min later to 12 mg/kg/h. Then the propofol infusion was stopped. Thirty minutes later, two-lung ventilation was restarted to compare initial values. No changes in venous admixture or PaO2 were observed during propofol infusion. There was no change in any respiratory or circulatory variables except systemic vascular resistance, which decreased significantly immediately after the propofol infusion commenced but returned to control values 15 min later for the rest of the observation period. After reestablishing two-lung ventilation, all variables did not differ from control values. In all patients, the hypoxic pulmonary vasoconstriction reflex was present after institution of one-lung ventilation and was not abolished after administration of propofol in doses from 6 to 12 mg/kg/h.