Potency assays for Anistreplase: comparison of the fibrin plate assay and a 96-well plate assay.

Several production lots of Anistreplase (Eminase) were assayed for potency by either two fibrin plate assays or a clot lysis assay performed in 96-well microtiter plates. The 96-well plate assay yielded comparable data to the fibrin plate assays and had the advantage of greater efficiency with respect to both time and reagents. As a result the newer method appears to be a suitable alternative to the fibrin plate assays for lot release of Anistreplase.

A collaborative study to establish a U.S. reference for tissue plasminogen activator (t-PA).

In 1987 the Second International Standard for tissue plasminogen activator (t-PA) was established by the World Health Organization following an international collaborative study. At that time, the Center for Biologics Evaluation and Research (CBER) decided to establish a national reference t-PA to be used in lot release potency testing of Alteplase, a licensed t-PA biological or of other t-PAs in development. A candidate recombinant t-PA (rt-PA) preparation was donated by Genentech, Inc. (South San Francisco, California) for this purpose and a collaborative study was launched to calibrate this material against the 2nd I.S. Four laboratories (including the Center for Biologics Evaluation and Research (CBER) and three manufacturers) participated in the study to establish the potency of the rt-PA preparation using a clot lysis assay. The results indicate that the potency of the U.S. reference for t-PA is 2900 international units (IU) per vial.

igh sialic acid content slows prourokinase turnover in rabbits.

The clearance of natural and recombinant prourokinase (proUK) from the blood of rabbits was studied by means of a double-isotope method which allowed the differential removal of two distinct proUK species to be monitored when simultaneously administered to an individual animal. In initial experiments, proUK expressed in different cell lines contained between 0 and 2.5 molecules of sialic acid per molecule of protein. A slight trend toward slower clearance of proUK with higher sialic acid content was observed but rate differences were not statistically significant. Recombinant proUK produced in CHO cells grown in flow reactors, contained unusually high levels of sialic acid in excess of 3 moles/mole protein. Controlled exposure to immobilized neuraminidase was used to remove sialic acid from this protein in defined amounts. The clearance of the parent material was biphasic with average alpha and beta half-lives of 1.7 min and 16.7 min respectively. The AUC of the parent material was only slightly lowered upon removal of 30% of the original sialic acid. Species with 60% or 90% removal of sialate were much more rapidly cleared from the circulation respectively yielding AUCs equal to 56% and 41% of that observed with the parent material. Thus proUK containing 2.5-3.5 sialic acid molecules per molecule of protein turned over significantly more slowly in rabbits than did less sialylated proUK. The clearance rate was relatively insensitive to sialic acid content between 0 and 1.5 sialic acid residues per proUK molecule.

Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types.

Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between 125I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase.

A linear amino acid sequence involved in the interaction of t-PA with its endothelial cell receptor.

Endothelial cell receptors for tissue plasminogen activator (t-PA) have been demonstrated recently, and we have sought to identify a region of the t-PA molecule involved in its interaction with these receptors on human umbilical vein endothelial cells. Of three monoclonal antibodies against various regions of t-PA, one directed against the finger region inhibited 125I-t-PA binding to the cells. Synthetic peptides corresponding in amino acid sequences to segments from within the finger region were constructed, and one of these inhibited t-PA binding. This peptide corresponded to residues 7 through 17 of t-PA. The inhibition by this peptide was specific as other peptides from the finger region were inactive. The inhibitory peptide also did not affect the binding of another fibrinolytic ligand, urokinase, to the cells. Although a role for other regions of t-PA in binding to endothelial cells cannot be excluded, the results implicate a short span of linear amino acid sequence within the finger region in the interaction of t-PA with endothelial cells.

Turnover of tPA in rabbits: influence of carbohydrate moieties.

The turnover of tissue plasminogen activator (tPA) was studied in rabbits using a double-label technique which allowed the comparison of various tPA derivatives with a standard tPA in individual animals. Purified recombinant tPA (Alteplase) was labelled with 125I and used as a standard for each experiment. Various tPA preparations which lacked specific carbohydrate structures were labelled with 131I (in separate experiments) and injected along with the 125I-tPA standard into rabbits. The clearance of standard tPA was biphasic with an average T 1/2 alpha and T 1/2 beta of 0.85 min and 12 min respectively. Type II tPA which lacks a portion of carbohydrate associated with Type I tPA as well as desialated tPA demonstrated a longer T 1/2 beta than standard tPA.

Nutritional requirements of human malignant (leukemic) cell lines: implications for adjuvant therapy.

Metabolic requirements of malignant cell lines derived from patients with chronic myelogenous and acute lymphoblastic leukemias were compared to those of proliferating normal cells (mitogen-stimulated human lymphocytes) and circulating blasts from acute myeloblastic and acute lymphoblastic leukemias. Requirements were judged by degree of amino acid (AA) utilization in short-term cultures and assessed by the effect of selective AA deprivation on cell growth. Cell growth was measured by DNA synthesis and growth rate analysis. Six AAs (serine, threonine, methionine, valine, phenylalanine, and lysine) were appreciably utilized (52-87%) by IM-9, CEM, MOLT-4, and K-562 cells, but little or no utilization of these or any other AAs were noted in HSB cells, in leukemic blasts, or in mitogen-stimulated normal lymphocytes in short-term culture. Omission of lysine from culture media greatly inhibited cell growth (DNA synthesis by 91%), and cell density (by 83%) of IM-9 cells. However, omission of lysine, valine, serine, threonine, methionine, or phenylalanine had less of an effect on CEM and MOLT-4 cell lines. These observations demonstrate that under the conditions used the IM-9 cell line is uniquely dependent on extracellular lysine levels in contrast to the other cell lines studied. This suggests that human malignancies other than acute lymphoblastic leukemia which exhibits an obligate dependence on extracellular asparagine might be manageable by enzymatic degradation in vivo or by dietary restriction of indispensable AAs.

Binding of tissue plasminogen activator to human umbilical vein endothelial cells.

Binding of purified recombinant human tissue plasminogen activator to cultures of human umbilical vein endothelial cells (HUVEC) was studied in vitro. 125I-tPA was shown to bind to HUVEC in a specific, saturable, and dissociable manner. Scatchard analysis revealed a low affinity binding site with Keq = 4.2 X 10(6) M-1 and 1.2 X 10(7) sites per cell. Binding of tPA was inhibited by L-lysine, e-aminocaproic acid, and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone but not by carbohydrates including mannose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. Neat human plasma abrogates but does not totally inhibit binding of tPA to HUVEC. These results may indicate a role for endothelial cells in the removal of tPA from the circulation in vivo.

Turnover of human tissue plasminogen activator (tPA) in rabbits.

The turnover of purified tissue plasminogen activator (tPA) from two different manufacturers was compared in rabbits. The first was melanoma derived one-chain tPA and the second was recombinant two-chain tPA. No differences were noted between the two products. A biphasic disappearance curve was observed for the protein (125Iodine labelled). The first phase was extremely rapid with a T1/2 of 0.59-0.89 min; the secondary phase had a T1/2 of 10-12 min. tPA accumulated rapidly in the liver (44% at twenty min) and appeared to be degraded as demonstrated by the increase in plasma of low molecular weight material which was also TCA soluble. Fractionation of purified recombinant two-chain tPA on a Dupont GF-250 column yielded two peaks of protein (Peak 1 and Peak 2) and the turnover of each in rabbits was compared.

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement.

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.

Neutralization of vesicular stomatitis virus (VSV) by human complement requires a natural IgM antibody present in human serum.

Neutralization of VSV by human serum ws previously shown to involve C1, C2, C3, and C4 of the classical complement (C) pathway. All normal human sera tested were equivalently active in this regard. However, purified C1, C2, C3, and C4 were unable to mediate VSV neutralization. In the present studies an additional factor required for C-mediated neutralization was isolated from normal human serum and identified as a natural IgM antibody specific for a viral encoded antigen. Purified IgM bound to the virus and formed a complex that activated component C1. Normal serum concentrations of purified IgM, C1, C2, C3, C4 neutralized VSV to the same extent as normal serum. Purified IgM did not neutralize VSV alone or in conjunction with C1, C2, and C4. Inclusion of C3 resulted in full neutralization and C3b binding to the virus was demonstrated. Thus, normal human serum contains a natural antibody of the IgM class that is directed toward a viral antigen. The antibody facilitates neutralization by forming an immune complex that activates C1 and thus efficiently initiates the classical pathway at the viral surface. Neutralization occurs with C3b deposition on the viral envelope and probably results from a blanket of C protein that interferes with viral attachment to susceptible cells.

Antibody-independent neutralization of vesicular stomatitis virus by human complement. II. Formation of VSV-lipoprotein complexes in human serum and complement-dependent viral lysis.

Vesicular stomatitis virus (VSV) is efficiently neutralized by normal, nonimmune human serum without the participation of antibody. Neutralization is complement- (C) dependent and requires the early-acting components of the classical pathway, C1, C4, C2, and C3, but not later-acting C components. In further studies, normal human serum was found to markedly increase the density of a variable but significant proportion of virus-associated RNA and to markedly decrease the density of the remainder of virus-associated RNA. The RNA of increased density was found to be dense ribonucleocapsid cores released from VSV by C-dependent viral lysis mediated through the classical pathway. The released ribonucleocapsid cores found at the bottom of sucrose density gradient after incubation of VSV with human serum were resistant to degradation by proteolytic enzymes. The VSV-derived RNA found floating on the tops of sucrose density gradients performed on serum-treated VSV was infectious virus. The decreased density was due to binding of VSV to human serum lipoproteins (LP), primarily very low density lipoproteins (VLDL). Binding of VLDL to VSV required the presence of the viral envelope and the external glycoprotein, G. Despite the binding of LP to VSV, LP did not neutralize VSV, and LP-depleted sera were fully active in neutralizing VSV. Thus, LP do not represent an accessory factor for the C-dependent neutralization of VSV.