Background: Hemodialysis (HD) grafts often fail due to stenosis at the venous anastomosis and thrombotic occlusion. Percutaneous management relies on thrombolysis with plasminogen activators, mechanical removal of thrombus and angioplasty of the stenotic lesion. Objectives: This report describes a phase I trial using Plasmin (Human) TAL 05-00018, a direct-acting fibrinolytic agent, to evaluate safety and, secondarily, to establish effective thrombolytic dosing. Patients/methods: Six cohorts of 5 patients with acute HD graft occlusion documented by angiography were treated with escalating dosages of plasmin (1, 2, 4, 8, 12, and 24 mg) infused over 30 minutes via criss-crossed pulse-spray catheters within the graft. The primary efficacy endpoint was >/=50% thrombolysis, as determined by comparison of pre- and 30-minute post-plasmin fistulograms. Results: Of 31 subjects who received study drug (safety population), 1 withdrew and 30 completed the trial (evaluable for efficacy). There was no significant change in plasma alpha-2 antiplasmin or fibrinogen concentration, major bleeding did not occur and there were no deaths. Serious adverse events in 4 patients were not related to study drug. There was a dose-response relationship for the primary efficacy endpoint, all 5 subjects receiving 24 mg achieving >75% lysis. Conclusions: This first phase I study of Plasmin (Human) TAL 05-00018, infused into thrombosed HD grafts, documents safety at dosages of 1 to 24 mg and an effective thrombolytic dosage of 24 mg. The results establish a foundation for further clinical study of catheter-based plasmin administration in thrombotic disorders.

Plasmin is a direct thrombolytic which has been shown to have a strikingly favorable benefit to risk profile in comparison with plasminogen activators, notably tissue plasminogen activator (t-PA). As heparin is known to increase the risk of hemorrhage when co-administered with a plasminogen activator, we asked whether adjunct antithrombotic agents such as aspirin and heparin would affect the safety of plasmin. Three groups of rabbits were administered plasmin at a dose (4 mg kg-1) designed to induce significant decreases in antiplasmin, fibrinogen and factor (F)VIII, to about 25, 40 and 40%, respectively, of baseline values, but not cause prolongation of the ear puncture bleeding time. In a blinded and randomized trial, the results show that an intravenous aspirin bolus plus heparin administered as a bolus followed by a maintenance continuous infusion did not significantly prolong the bleeding time during plasmin infusion. These data indicate that in the rabbit, concomitant use of aspirin plus heparin does not affect the safety of a therapeutic dose of plasmin.

The objectives of this study were to develop an assay for the direct measure of porcine corticosteroid-binding globulin (pCBG) and to confirm age-related changes in plasma pCBG concentration. Isolation and purification of pCBG from plasma was performed by affinity chromatography and HPLC-DEAE anion exchange techniques. Analysis by SDS-PAGE revealed two polypeptides (54 and 59 kDa) having similar amino acid homology (>50%) to previously reported sequences of seven mammalian species for the first 33 amino acids. Porcine CBG (20 ng/well) was immobilized to microtiter plates and standards or samples added along with rabbit antiserum developed against the purified pCBG. Goat anti-rabbit IgG-alkaline phosphatase conjugate was added followed by p-NPP substrate. The resultant color development was read at 405 nm. Intra- and interassay coefficients of variation (n=26) of a pooled sample were 10 and 15%, respectively. Age-related changes (P<0.001) in plasma pCBG concentration (n=203) from day 3 through 168 of age confirmed that, in the pig, changes seen in the percent distribution of cortisol among protein bound and free forms around day 28 of age are associated with an increase in CBG concentration.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.

Thirty-five time-dated pregnant gilts were used to document plasma levels of total and free cortisol, corticosteroid-binding globulin (CBG) binding capacity, and percent distribution of cortisol among protein-bound (CBG and albumin) and free forms in the fetal pig during the last 24 days of gestation. Plasma from fetal pigs on days 110-114 of gestation (gestation length 114 days) had significantly higher levels of total cortisol (p < 0.01), percent albumin-bound and free cortisol (p < 0.10), and free cortisol concentration (p < 0.05) compared to samples on days 90, 100 and 105. Fetal plasma CBG binding capacity increased (p < 0.05) linearly from day 100 to 114. Fetal pigs located in the cervical region of the uterus had lower (p < 0.05) total and free cortisol and higher (p < 0.05) albumin and total protein concentrations compared to fetuses in the middle and oviductal regions. Total, percent free and free cortisol concentrations in maternal plasma on days 105-114 were greater (p < 0.10) than that measured on days 12-100 of gestation. These results suggest that the developmental patterns of plasma cortisol and CBG in the prenatal pig are directly related and highly similar to those of another precocious species, the sheep.

We have developed a new resin for peptide synthesis that can be used to synthesize and evaluate directly combinatorial peptide libraries for binding target proteins. Fidelity of the peptide synthesis using this hydrophilic resin is comparable to polystyrene-based resins. Peptide libraries synthesized on this resin were probed by a two color PEptide Library Immunostaining Chromatographic ANalysis (PELICAN) technique for sequences binding the serine protease Factor IX zymogen. This PELICAN technique readily distinguishes between beads interacting with the reagents for target detection (blue beads) from those beads specific for the target protein itself (red beads). Validation of the PELICAN technique, as well as purification of Factor IX from plasma, is demonstrated utilizing this resin.

The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6)(M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.

We describe an improved method for large-scale purification of antithrombin III (AT-III) from human plasma involving heparin affinity chromatography of redissolved fraction IV-1 paste, viral inactivation by heating, followed by a second heparin affinity column. The characteristics of a new heparin affinity resin and the ability to extrapolate process behavior from small-scale (20 ml) to large-scale (40 liter) columns are described. This supports the use of the small-scale column for process optimization and validation studies in compliance with current regulatory requirements for biological products. The process has been characterized by analytical techniques including sodium dodecyl sulfate (SDS), reducing SDS, and nondenaturing polyacrylamide gel electrophoresis; laser desorption time-of-flight mass spectroscopy, and electrospray mass spectroscopy. These results demonstrate that greater than 95% of the protein in the final products is AT-III, which is greater than 95% active as defined by thrombin inhibition.

Uteroferrin is an iron-containing, progesterone-induced, acid phosphatase that is secreted in large amounts by the uterine endometrium of pigs. During pregnancy, it transports iron across the chorioallantois (placenta) for use in fetal hematopoiesis. In this paper, it is reported that uteroferrin synthesized by cultured endometrial explants possesses N-linked, high-mannose, oligosaccharide chains that contain 6- phosphomannose units. The latter is regarded as a possible recognition marker whereby acid hydrolases are targeted to the lysosome. On uteroferrin, however, the majority of the phosphate is in single diester linkages between the mannose and a covering N-acetylglucosamine. It is suggested that uteroferrin is a lysosomal enzyme that has assumed a role in iron transport and metabolism and is secreted because the covering N-acetylglucosamine is not removed.