A first-in-human phase I trial of locally delivered human plasmin for hemodialysis graft occlusion.

BACKGROUND: Hemodialysis (HD) grafts often fail because of 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 five 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 min via criss-crossed pulse-spray catheters within the graft. The primary efficacy endpoint was > or =50% thrombolysis, as determined by comparison of pre-plasmin and 30-min post-plasmin fistulograms. RESULTS: Of 31 subjects who received study drug (safety population), one 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 four patients were not related to the study drug. There was a dose-response relationship for the primary efficacy endpoint, all five 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 1-24 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.

Safety of plasmin in the setting of concomitant aspirin and heparin administration in an animal model of bleeding.

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.

Age-related changes in porcine corticosteroid-binding globulin (pCBG) as determined by an enzyme-linked immunosorbent assay.

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.

Distribution between protein-bound and free forms of plasma cortisol in the gilt and fetal pig near term.

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.

Affinity purification of von Willebrand factor using ligands derived from peptide libraries.

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.

Chemically derived peptide libraries: a new resin and methodology for lead identification.

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.

A highly purified antithrombin III concentrate prepared from human plasma fraction IV-1 by affinity chromatography.

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.

Purification and immunolocalization of ovine placental retinol-binding protein.

A retinol-binding protein (RBP), synthesized and secreted by ovine allantois in vitro, was purified from culture medium. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical molecular masses of about 23,000 Da as determined by two-dimensional PAGE under reducing conditions. Thirty-one of the first 34 N-terminal amino acids of the purified protein were sequenced and shown to have complete homology with bovine placental and bovine plasma RBP. The ultraviolet absorption spectrum and fluorescence excitation and emission spectra of the purified ovine placental RBP indicated the presence of bound retinol. Metabolic labeling studies demonstrated that the protein was synthesized by placental membranes. Using antiserum to bovine placental RBP, ovine placental RBP was immunolocalized in trophectoderm of 13-day-old blastocysts and trophectodermal cells of the chorion, endodermal cells lining the allantois, and ectodermal cells lining the amnion of 23-, 45-, and 53-day-old conceptuses. Results from this study suggest that ovine placental membrane epithelia synthesize and secrete RBP. Transport, storage, and metabolism of retinol mediated by placental RBP may be essential for normal embryonic development during pregnancy.

Differentiation of Streptococcus uberis from Streptococcus parauberis by polymerase chain reaction and restriction fragment length polymorphism analysis of 16S ribosomal DNA.

Streptococcus uberis type II has been proposed recently as a separate species designated Streptococcus parauberis (A. M. Williams and M. D. Collins, J. Appl. Bacteriol. 68:485-490, 1990). Differentiation of S. parauberis from S. uberis has been possible only by DNA-DNA hybridization or 16S rRNA sequencing, since the biochemical and serological characteristics of the two species are indistinguishable. A simple and reliable technique was developed for differentiating S. parauberis (S. uberis type II [ATCC 13386]) from S. uberis (S. uberis type I [ATCC 9927, ATCC 13387, and ATCC 27958]) by restriction fragment length polymorphism (RFLP) analysis of 1.4-kb 16S ribosomal DNA (rDNA). Oligonucleotide primers complementary to 16S rRNA genes were used to amplify 16S ribosomal gene fragments from genomic DNA by polymerase chain reaction. The 1.4-kb 16S rDNA fragment was digested with ScaI, NspI, DdeI, and AvaII restriction endonucleases. Restriction fragments produced by all four restriction endonucleases were characteristic for each species. RFLP analysis of 16S rDNA from 24 "S. uberis" isolates obtained from mammary secretions of dairy cows indicated that all 24 isolates were indeed S. uberis.

The retinol-binding protein of the expanding pig blastocyst: molecular cloning and expression in trophectoderm and embryonic disc.

Retinol-binding protein (RBP) is a major secretory product of the porcine conceptus. Using an oligonucleotide probe corresponding to a highly conserved region of all known mammalian RBP, we have isolated an apparently full-length cDNA clone for porcine conceptus RBP from a cDNA library constructed from pig conceptuses collected between days 13-17 of pregnancy. The cDNA was 937 base-pairs in length and coded for a protein whose inferred amino-terminal sequence was identical to that reported for both porcine conceptus RBP and porcine serum RBP. Its length was consistent with the size (approximately 1 kilobase) of the RBP message in porcine conceptuses. Porcine conceptus RBP and human serum RBP share 91% amino acid sequence identity. The inferred differences in sequence were evenly distributed throughout the length of the polypeptide. RBP mRNA was detectable within the trophoblast of day 11 porcine conceptuses by in situ hybridization with a 618-basepair 35S-labeled probe corresponding to the 3' end of porcine RBP. Silver grain density was distributed relatively uniformly over the trophoblast and the inner cell mass. Western blot analysis of conceptus culture medium demonstrated that the conceptuses of cattle (on day 19) and sheep (on day 15) as well as pigs secrete RBP during early pregnancy. Secretion of large quantities of RBP by the trophoblast of preimplantation pig conceptuses suggests important roles for vitamin A and RBP near the time of conceptus elongation.

Characterization of protein production by ovine placental membranes: identification of a placental retinol-binding protein.

Ovine chorion, allantois, and amnion from days 23, 26, 28, 35, 45, 53, 62, and 72 and yolk sac from day 23 of pregnancy were isolated by dissection and cultured for 24 h in modified minimum essential medium in the presence of [35S] methionine to characterize in vitro synthesis and release of proteins. Proteins synthesized and released into medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Patterns of protein production by these isolated membranes remained relatively unchanged from days 23-72 with the exception of the products of yolk sac, which regress by day 35 of pregnancy. In general, chorion was the source of a number of basic-to-neutral proteins; allantois and amnion were the sources of more acidic proteins; and yolk sac was the source of serum-like proteins. A major low mol wt acidic protein, D4, was produced by all membranes and present in fetal membrane fluids. Protein D4 consisted of three isoelectric variants (isoelectric point 5.3-6.1) with identical mol wts (23,000 +/- 800) and was shown to react immunologically with antibovine placental retinol-binding protein (RBP) serum. Hence, protein D4 is a putative ovine placental RBP. The present study is the first to characterize and compare protein production by isolated ovine chorionic, allantoic, amniotic, and yolk sac membranes from day 23 through midpregnancy and identify the major low mol wt acidic protein produced by each membrane as a placental RBP.

Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro.

Mature uteroferrin (Uf; Mr = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a Mr of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man6-4GlcNac2 oligosaccharides found on mature Uf.

Purification and characterization of bovine placental retinol-binding protein.

A major low mol wt acidic protein, 3B3, produced from cultures of day 29-90 bovine allantoic membranes (in the presence of [3H]leucine or [35S]methionine) and from day 29-60 allantoic fluid, has been purified. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical mol wt (23,200 +/- 900) when analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequence analysis of 3B3 isolated from allantoic fluid on the first 43 amino acids showed that 3B3 had 93% and 91% homology with rabbit and human plasma retinol-binding protein (RBP), respectively. The UV absorption spectrum and the fluorescence excitation and emission spectra of purified 3B3 from both sources indicated the presence of bound retinol. Rabbit antiserum was raised against placental RBP (3B3) isolated from allantoic membrane culture medium. Placental RBP was immunoprecipitated from radiolabeled allantois and chorion culture medium and was detected in allantoic membrane culture medium and allantoic fluid by Western blotting. These results suggested that bovine placental membranes secrete RBP into allantoic fluid and that placental RBP may play important roles in vitamin A metabolism in the developing embryo.

N-glycosylated and unglycosylated forms of caprine trophoblast protein-1 are secreted by preimplantation goat conceptuses.

Goat conceptuses secrete caprine trophoblast protein-1 (cTP-1) which is related antigenically to the abundant embryonic interferon-alpha II of sheep and cattle. Antiserum to ovine and bovine TP-1s immunoprecipitated three molecular weight classes (23,000, 21,000 and 17,000, each with two isotypes) of cTP-1 from goat conceptus culture medium. Cultures which contained tunicamycin resulted in a shift in the Mr = 23,000 and Mr = 21,000 forms to a Mr of 17,000. The Mr = 23,000 and 21,000 forms, but not the Mr = 17,000 form, bound to Concanaval in A-Sepharose and were eluted under conditions selective for glycoproteins bearing complex-type oligosaccharide(s). Thus cTP-1 is a mixture of glycosylated and unglycosylated polypeptides.

Immunocytochemical localization of BP, a conceptus secretory protein, in trophectoderm during pig trophoblast elongation.

Tissue sections of periimplantation pig conceptuses (days 9-15 of pregnancy) were incubated with antiserum to the basic protein (BP), a major secretory protein of filamentous pig blastocysts. Bound antibody was detected by the peroxidase-antiperoxidase method. BP was restricted to trophectoderm in conceptuses which had made the transition from a spherical to an ovoid shape having a diameter of greater than 5 mm (day 11). Tubular (day 11, 10-20 mm) and filamentous (day 11-15) conceptus trophectoderm contained BP. These results suggest that BP synthesis commences at the time of rapid trophoblast growth.

Plasma cortisol distribution in the pig from birth to six weeks of age.

Plasma levels of cortisol and percent distribution of cortisol among protein-bound (corticosteroid-binding globulin (CBG) and albumin) and unbound forms were measured in naturally born/conventionally reared pigs from birth to 6 weeks of age. Total cortisol and percent unbound cortisol were highest in pigs at birth and decreased (p less than 0.01) in a linear fashion over the sampling period. Percent CBG-bound cortisol was lowest on days 3-21 relative to the peak value seen on day 42. However, actual CBG-bound cortisol was not different after day 1. Percent albumin-bound cortisol was highly correlated with percent CBG-bound cortisol (r = -0.90; p less than 0.001). These results suggest that a rapid shift in cortisol distribution from unbound and albumin-bound forms to that which is bound to CBG occurs by approximately day 28 of age in the neonatal pig.

Immunolocalization and endocytosis of the uterine secretory protein, uteroferrin, in pre-implantation pig trophectoderm on day 11 of pregnancy.

Uteroferrin is a progesterone-induced, iron-binding glycoprotein secreted by the glandular epithelium of the pig endometrium. Evidence is presented that maternal uteroferrin is present in trophectoderm of preimplantation pig blastocysts on day 11 of pregnancy. Although [35S]-methionine was not incorporated into uteroferrin during in vitro culture of blastocysts, solubilized tissue extracts from 10-20-mm-diameter blastocysts contained uteroferrin by western blotting with monospecific antiserum to uteroferrin. Uteroferrin was detected in the apical and basolateral cytoplasm of trophectoderm by immunocytochemistry of paraffin-embedded blastocysts. Immunostaining was excluded from cells of the endoderm and the inner cell mass. Furthermore, blastocysts internalized fluorescein-labeled uteroferrin from medium during in vitro culture in a temperature-dependent manner. Fluorescent label was located in apical and basolateral cytoplasm in a punctate distribution, and clustered in the supranuclear region of trophectoderm. Addition of a threefold excess of unlabeled uteroferrin to culture medium did not inhibit uptake. These results suggest that the pre-implantation pig blastocyst actively endocytoses uteroferrin from glandular secretions in utero. Uptake was restricted to trophectoderm.

Isolation, characterization and immunocytochemical localization of bovine trophoblast protein-1.

Bovine trophoblast protein-1 (bTP-1) was isolated to 90% purity from culture medium of Day 18-20 conceptuses incubated in vitro (in the presence of L-[3H]leucine) by a combination of Sephacryl S-200 gel filtration chromatography and O-(diethylaminoethyl) (DEAE) anion-exchange high-performance liquid chromatography (DEAE-HPLC). The radiolabeled protein had an Mr of 21,200 +/- 800 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had three isoelectric variants (pI 5.7-6.5) by two-dimensional SDS-PAGE. DEAE-HPLC-enriched bTP-1 cross-reacted with anti-o TP-1 serum on Western blots. A monospecific antiserum against bTP-1 was produced by excising the bTP-1 polypeptide band from preparative SDS-PAGE gels. Antiserum reacted with a single polypeptide with an Mr of 21,200 as determined by Western blotting of SDS-PAGE-separated conceptus medium proteins and by immunoprecipitation from L-[35S]methionine-labeled medium proteins followed by SDS-PAGE and autoradiography. Bovine trophoblast protein-1 was localized by immunocytochemistry in the cytoplasm of both mono- and binuclear trophectoderm cells of Day 20 bovine conceptuses, indicating that it is a product of the trophoblast.

Purification, characterization, and immunocytochemical localization of the major basic protein of pig blastocysts.

The major basic protein (BP) synthesized and secreted by elongating pig blastocysts was purified from medium of Day 14-17 conceptus cultures. Sequential ion-exchange and gel-filtration chromatographies resulted in isolation of BP as a single polypeptide of Mr = 43,100 or 42,800 under denaturing or native conditions, respectively. BP was found to be a glycoprotein by incorporation of [3H] glucosamine and susceptibility to N-glycopeptidase F. Two BP polypeptides were produced by N-glycopeptidase F (Mr = 39,800 and 36,300). Antiserum to BP immunoprecipitated radiolabeled BP from blastocyst culture medium. BP was not detected in medium from 1-2 mm diameter spherical (Day 10) blastocysts but was found in medium from 3-5 mm spherical (Day 10) and filamentous (less than 50 cm, Day 12) conceptuses, suggesting that BP synthesis and secretion began at the initiation of trophoblast expansion. With immunocytochemical procedures, BP was located in the apical cytoplasm of trophectoderm cells of Day 11 expanding (5-7 and 10-20 mm) blastocysts. These results suggest that trophoblast epithelium secrete BP apically toward the uterine lumen and that BP may play a role in maternal-fetal interactions during the peri-implantation period.

Characterization of protein production by bovine chorionic and allantoic membranes.

Bovine allantoic (A) and chorionic (C) membranes from Days 29, 32, 36, and 40 of pregnancy were isolated by dissection and cultured in a modified minimum essential medium in the presence of L-[35S]methionine to characterize in vitro synthesis and release of proteins. Membranes were also cultured in the presence of the glycosylation inhibitor tunicamycin. Proteins synthesized and released into the medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography of dried gels. Stained gels were used to analyze protein from allantoic fluids. Percent incorporation of the radiolabeled amino acid into nondialyzable protein was higher for A than for C (A = 8.0 +/- 1.2 vs. C = 6.4 +/- 0.5 per 200 mg tissue) but not significantly different. C released significantly more total protein (nonradioactive) into the medium (57.0 +/- 3 vs. 9.6 +/- 0.6 micrograms/ml). Of the 25 proteins analyzed, 19 appeared to be produced primarily by one membrane or the other. In general, C was the source of a number of basic-to-neutral glycosylated proteins and A was the source of a number of more acidic glycosylated proteins. Many but not all proteins synthesized by A were present in allantoic fluid. The present study is the first to characterize protein production by isolated chorionic and allantoic membranes and to demonstrate that both tissues contribute to the production of secretory conceptus proteins.