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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.jtcvs.2004.08.020 Byline: Eric A. Williams (a), Richard J. Ing (b), Justin P. Hart (c), James Jaggers (d), Frank H. Kern (a)(b), Damian M. Craig (d), Salvatore V. Pizzo (c) Abstract: Cytokine dysregulation contributes to the systemic inflammatory response after cardiopulmonary bypass. Clearance of cytokine binding proteins may be important in the resolution of inflammation. Our aim was to determine whether the cytokine binding protein [alpha].sub.2-macroglobulin and its soluble receptor were upregulated in endotracheal aspirates from infants and children undergoing cardiopulmonary bypass. Author Affiliation: (a) Departments of Pediatrics, Duke University Medical Center, Durham, NC (b) Anesthesiology Duke University Medical Center, Durham, NC (c) Pathology Duke University Medical Center, Durham, NC (d) Surgery Duke University Medical Center, Durham, NC Article History: Received 22 April 2004; Revised 7 July 2004; Accepted 18 August 2004 Article Note: (footnote) Eric A. Williams, MD, is a National Institute of Child Health and Human Development Fellow of the Pediatric Scientist Development Program (National Institute of Child Health and Human Development Grant Award K12-HD00850). Salvatore V. Pizzo, MD, PhD, is funded by National Heart, Lung, and Blood Institute HL 24066.

Malignant ovarian epithelial cells appear to have an abundance of both urinary-type plasminogen activator and its receptor, whereas normal ovarian epithelium has urinary-type plasminogen activator receptor but little or no urinary-type plasminogen activator. Urinary-type plasminogen activator transforms plasminogen into plasmin, a potent enzyme that can break down the molecules that make up the matrixes in which cells are embedded. This process is a known means of tissue invasion and metastasis in several different kinds of cancer. Both ovarian cell cultures and tumor samples were studied. In cancerous ovarian epithelial cells, the activator and the receptor were found together on the cell membrane of the cancerous cells and were most abundant at the boundary between cancer cells and adjacent tissues. In normal ovarian epithelial cells, the receptor was found on cell membranes in similar amounts to cancerous cells, but no activator was found in combination with it.

Scarring is a highly prevalent and multifactorial process, yet no studies to date have attempted to distinguish pathologic from nonpathologic scarring.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP782on their cell surface with antibody directed against the COOH-terminal domain of GRP78 up-regulates and activates p53 causing decreased cell proliferation and up-regulated apoptosis.  In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, AktThr308and AktSer473kinase activities and reduces phosphorylation of FOXO, and GSK3β.  This treatment also suppresses activation of ERK1/2, p38 MAPK, and MKK3/6; however, it up-regulates MKK4 activity.  JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis.  Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX, and BAK expression as well as cleaved caspases-3, -7, -8, and -9.  Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects.  We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Cell surface expression of glucose-regulated protein 78 (GRP78) occurs in several types of cancer; however, its role in the behavior of primary cutaneous melanoma is not well studied. The association of cell surface GRP78 with other proteins such as MTJ1 stimulates cell proliferation. In this study, we characterized the pattern of expression of GRP78 and MTJ1 in invasive primary cutaneous melanomas and analyzed the relationships between the pattern of expression and various clinicopathological parameters. We found two patterns of GRP78 expression in invasive primary cutaneous melanoma. One pattern showed a gradual fading of protein expression from superficial to deeper levels within the same tumor. The second pattern of expression showed a similar fading with an abrupt regaining of expression at the deep invasive edge of the melanoma. These two distinct patterns of GRP78 expression correlated with both patient survival and depth of tumor invasion. A moderate MTJ1 expression was found to be associated with decreased patient survival; however, no significant associations were observed between patterns of GRP78 and MTJ1 expression. Our study (1) describes two distinct patterns of GRP78 in invasive primary cutaneous melanoma, (2) inversely correlates regain of GRP78 expression with patient survival, and (3) suggests a modifying effect of MTJ1 on GRP78 in enhancing tumor aggressiveness.

α‐2M*‐mediated antigen delivery leads to cross‐presentation and improved cell‐mediated immunity, suggesting that α‐2M* may have applications for vaccines based on weakly immunogenic subunits. α2M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen‐delivery vehicle, α2M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that α2M* encapsulation may also enhance antigen‐specific CTL immunity. In this study, we demonstrate that α2M*‐delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA257–264(SIINFEKL) demonstrated up to 25‐fold greater IFN‐γ and IL‐2 secretion when treated in vitro with α2M*‐OVA compared with soluble OVA. The frequency of IFN‐γ‐producing cells was increased ∼15‐fold, as measured by ELISPOT. Expansion of the OVA‐specific CD8+T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA‐specific cell‐mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by α2M*‐OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α2M*‐OVA. These α2M*‐OVA‐immunized mice demonstrated increased protection against a s.c.‐implanted, OVA‐expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that α2M*‐mediated antigen delivery elicits specific CTL responses suggests the cross‐presentation of antigen onto MHC class I. These results support α2M* as an effective antigen‐delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

CpG oligodeoxynucleotides (ODN) stimulate the immune system and are under evaluation as treatments and vaccine adjuvants for infectious diseases, cancer, and immune system disorders. Although they have shown promising results in numerous clinical trials, the ultimate use of CpG ODN‐based therapeutics may hinge on improved pharmacokinetics and reduced systemic side‐effects. CpG ODN efficacy and potency might be enhanced greatly by packaging them into particles that protect them from degradation and specifically target them for uptake by immune‐competent cells. The plasma proteinase inhibitor α2‐macroglobulin (α2M) binds numerous biologically active macromolecules, including cytokines, chemokines, and growth factors, and can modulate their activity. Molecules bound to α2M are protected from interactions with neighboring macromolecules and are targeted for receptor‐mediated uptake by immune‐competent cells. Here, we report that activated α2M (α2M∗) binds CpG ODN and enhances their immunostimulatory properties significantly. Murine macrophages treated with α2M∗‐ODN complexes respond more rapidly and produce a greater cytokine response than induced by free CpG ODN. Using human PBMC, α2M∗‐ODN complexes exhibit fourfold enhanced potency and 15‐fold greater efficacy for stimulating production of inflammatory cytokines. α2M∗ targets delivery of CpG ODN specifically to immune‐competent cells, which endocytose the complexes sixfold more rapidly than free CpG ODN. CpG ODN bound to α2M∗ are also protected from degradation by nucleases. This novel targeting technology may improve CpG ODN‐based therapeutics by increasing efficacy at reduced doses, thus reducing side‐effects and cost.

Basic fibroblast growth factor (FGF-2) is important in development, wound healing and angiogenesis. The human plasma proteinase inhibitor α2-macroglobulin (α2M) binds to and regulates the biological activity of various growth factors, including FGF-2. FGF-2 binds specifically and saturably to native α2M and conformationally modified α2M (α2M∗); however, the KD for FGF-2 binding to α2M∗ is 10-fold lower. This study investigates the biochemical nature of the interaction between FGF-2 and α2M∗ and localizes a possible FGF-2 binding site in the α2M subunit. FGF-2 binding to α2M∗ was not affected by shifts in pH between 6.5 and 10; however, increasing temperature decreased the KD for this interaction. The binding affinity of FGF-2 for α2M∗ also increased with increasing ionic strength. These results are consistent with the hypothesis that hydrophobic interactions predominate in promoting FGF-2 association with α2M∗. Consistent with this hypothesis, FGF-2 bound to a glutathione S-transferase fusion protein containing amino acids 591–774 of the α2M subunit (FP3) and to a hydrophobic 16-amino-acid peptide (amino acids 718–733) within FP3. Specific binding of FGF-2 to the 16-amino-acid peptide was inhibited by excess transforming growth factor-β1. When the 16-amino-acid peptide was chemically modified to neutralize the only two charged amino acids, FGF-2-binding activity was unaffected, supporting the predominant role of hydrophobic interactions. FGF-2 presentation to signalling receptors is influenced by growth factor binding to heparan sulphate proteoglycans (HSPGs), which is electrostatic in nature. Our results demonstrate that the interactions of FGF-2 with α2M∗ and HSPGs are biochemically distinct, suggesting that different FGF-2 sequences are involved.

Dendritic cells (DC) are potent antigen-presenting cells that govern the effector cell responses of the immune system. DC are thought to continuously develop from circulating progenitors in a process that is accelerated by inflammatory stimuli. However, the physiological signals that regulate the development of DC from precursor cells have not been well defined. Here we show that a serine protease acting viaprotease-activated receptor-2 (PAR-2) stimulates the development of DC from bone marrow progenitor cells cultured in granulocyte-macrophage colony-stimulating factor and IL-4. DC fail to develop in bone marrow cultures treated with soy bean trypsin inhibitor, a serine protease inhibitor, but this inhibition is overcome by a PAR-2 agonist peptide. DC do not spontaneously develop from the bone marrow of PAR-2-deficient mice, but can be stimulated to do so by inflammatory mediators. These results suggest that endogenous serine proteases stimulate DC development in vitro. Thus, serine proteases may help trigger adaptive immune responses in vivo.

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of alpha(2)-Macroglobulin (alpha(2)M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni(2+) nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block alpha(2)M*-induced signal transduction. In the current study, we employed alpha(2)-macroglobulin (alpha(2)M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to alpha(2)M* with high affinity (K(d) approximately 150 pm). A monoclonal antibody directed against Grp 78 both abolished alpha(2)M*-induced signal transduction and co-precipitated LRP. Ligand blotting with alpha(2)M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on alpha(2)M*-mediated signaling. We conclude that Grp 78 is essential for alpha(2)M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.

ABSTRACTDipeptidyl peptidase IV (DPP IV) (CD26) plays a critical role in the modulation and expression of autoimmune and inflammatory diseases. We recently reported that sera from patients with rheumatoid arthritis and systemic lupus erythematosus contained low levels of DPP IV and high titers of anti-DPP IV autoantibodies of the immunoglobulin A (IgA) and IgG classes and found a correlation between the low circulating levels of DPP IV and the high titers of anti-DPP IV autoantibodies of the IgA class. Since streptokinase (SK) is a potent immunogen and binds to DPP IV, we speculated that patients with autoimmune diseases showed higher DPP IV autoantibody levels than healthy controls as a consequence of an abnormal immune stimulation triggered by SK released during streptococcal infections. We assessed this hypothesis in a group of patients suffering from acute myocardial infarction, without a chronic autoimmune disease, who received SK as part of therapeutic thrombolysis. Concomitant with the appearance of anti-SK antibodies, these patients developed anti-DPP IV autoantibodies. These autoantibodies bind to DPP IV in the region which is also recognized by SK, suggesting that an SK-induced immune response is responsible for the appearance of DPP IV autoantibodies. Furthermore, we determined a correlation between high titers of DPP IV autoantibodies and an augmented clearance of the enzyme from the circulation. Serum levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-a) and interleukin 6 (IL-6) increased significantly after 30 days of SK administration, while the levels of soluble IL-2 receptor remained unchanged during the same period, suggesting a correlation between the lower levels of circulating DPP IV and higher levels of TNF-a and IL-6 in serum in these patients.

Tissue factor (TF), the initiator of the extrinsic pathway of coagulation, binds plasminogen (Pg) with high affinity through an interaction between kringles 1-3 of Pg and the extracellular domain of TF. We investigated the binding of Pg type 1 (Pg 1) and Pg type 2 (Pg 2) to highly invasive, TF-expressing, 1-LN human prostate tumor cells and to TF isolated from 1-LN cell membranes. Pg 1, containing both N-linked and O-linked oligosaccharide chains, bound to isolated TF with high affinity, whereas Pg 2, containing only one O-linked oligosaccharide chain, did not bind to TF. Although Pg 1 and Pg 2 bind to 1-LN cells, only anti-TF antibodies inhibited the binding of Pg 1, suggesting that TF functions as the receptor for Pg 1 on 1-LN cells. Binding of Pg 1 to isolated TF was inhibited by 6-aminohexanoic acid and α-methylmannoside, suggesting that Pg 1 l-lysine binding sites and the biantennary, mannose-containing N-linked oligosaccharide chain are involved in this interaction. Binding of Pg 1 to 1-LN cells promoted activation by receptor-bound urinary-type Pg activator (u-PA) and initiated a Ca++ signaling cascade. In previous studies we demonstrated that the Pg 2 O-linked carbohydrate chain is essential for its binding to CD26 on 1-LN cells. The current studies suggest that Pg oligosaccharide chains regulate the binding of Pg 1 and Pg 2 to separate receptors on the cell surface.

The amino acid sequence of the monomeric α-macroglobulin (αM) from the American bullfrog, Rana catesbiana , was determined. The mature protein consisted of 1469 amino acid residues and shared sequence identity with other members of the αM family of protein. The central portion of the frog monomeric αM contained Cys residues positioned analogously to the Cys residues in human α 2 -macroglobulin (α 2 M), known to be involved in disulfide bridges. Additionally, the frog monomeric αM contained six Cys residues in a ∼60 residue COOH-terminal extension not present in previously characterized αMs. The spacing of the Cys residues and the overall sequence identity of this COOH-terminal extension were consistent with a trefoil motif. This is the first time a member of the trefoil factor family has been identified in the circulatory system. The "bait region" was located between Arg-Lys and contained mainly basic amino acid residues. The COOH-terminal receptor-binding domain was not exposed prior to proteolysis of this highly susceptible region. The proximity of the receptor-binding and trefoil domains implied that the trefoil domain is similarly concealed before bait region cleavage.

We have previously shown that antigens complexed to the receptor-recognized form of α2-macroglobulin (α2M*) demonstrate enhanced immune responsiveness mediated by the low density lipoprotein receptor-related protein LRP/CD91. Recently, we developed a proteinase-independent method to covalently bind antigens to α2M*. Given the potential applications of this chemistry, we analyzed the kinetics, thermodynamics, and pH dependence of this reaction. The incorporation of lysozyme into α2M* was a mixed bimolecular second-order reaction with a specific rate constant of 91.0 ± 6.9m−1s−1, 50.0 °C, pH 7.4. The activation energy, activation entropy, and Gibbs’ free energy at 50.0 °C were 156 kJ mol−1, 266 J mol−1K−1, and 70 kJ mol−1, respectively. The rate of incorporation increased as a function of pH from pH 5.0 to 7.0 and was unchanged thereafter. Furthermore, the reaction between α2M* and lysozyme was irreversible. The data are consistent with a two-step mechanism. In the first step, α2M* reforms its thiol ester bond, entering a reactive state that mimics the proteolytically induced “nascent state.” In the rate-limiting second step, the reformed bond quickly undergoes nucleophilic attack by lysozyme. The kinetic equations derived in this study are the basis for optimizing the formation of stable α2M*·antigen complexes.

Apolipoprotein E (ApoE) is a 34‐kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca2+stores. This cascade is inhibited by pretreatment with receptor‐associated protein and Ni2+, and it is mediated by a pertussis toxin‐sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor‐related protein. A peptide derived from the receptor‐binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE‐mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE‐dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.

A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. α2-Macroglobulin (α2M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized α2M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating α2M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo α2M oxidation, we measured the degree of oxidative α2M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid α2M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median α2M level than OA, while having only half the α2M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded α2M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma α2M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as α2M.

The fibroblast growth factor (FGF) family has an important role in processes such as angiogenesis, wound healing, and development in which precise control of proteinase activity is important. The human plasma proteinase inhibitor α2-macroglobulin (α2M) regulates cellular growth by binding and modulating the activity of many cytokines and growth factors. These studies investigate the ability of native and activated α2M (α2M*) to bind to members of the FGF family. Both α2M and α2M* bind specifically and saturably to FGF-1, -2, -4, and -6, although the binding to α2M* is of significantly higher affinity. Neither α2M nor α2M* bind to FGF-5, -7, -9, or -10. FGF-2 was chosen for more extensive study in view of its important role in angiogenesis. It was demonstrated that FGF-2 binds to the previously identified TGF-β binding site. The α2M* inhibits FGF-2–dependent fetal bovine heart endothelial cell proliferation in a dose-dependent manner. Unexpectedly, α2M* does not affect FGF-2–induced vascular tubule formation on Matrigel basement membrane matrix or collagen gels. Further studies demonstrate that FGF-2 partitions between fluid-phase α2M* and solid-phase Matrigel or collagen. These studies suggest that the ability of α2M* to modulate the activity of FGF-2 is dependent on an interplay with extracellular matrix components.

Both plasminogen (Pg) activation and matrix metalloproteinases (MMPs) are involved in the proteolytic degradation of extracellular matrix components, a requisite event for malignant cell metastasis. The highly invasive 1-LN human prostate tumour cell line synthesizes and secretes large amounts of Pg activators and MMPs. We demonstrate here that the Pg type 2 (Pg 2) receptor in these cells is composed primarily of the membrane glycoprotein dipeptidyl peptidase IV (DPP IV). Pg 2has six glycoforms that differ in their sialic acid content. Only the highly sialylated Pg 2γ, Pg 2δ and Pg 2ε glycoforms bind to DPP IV via their carbohydrate chains and induce a Ca2+ signalling cascade; however, Pg 2ε alone is also able to significantly stimulate expression of MMP-9. We further demonstrate that the Pg-mediated invasive activity of 1-LN cells is dependent on the availability of Pg 2ε. This is the first demonstration of a direct association between the expression of MMP-9 and the Pg activation system.

Cellular binding of receptor-recognized forms of α2-macroglobulin (α2M*) is mediated by the low-density lipoprotein receptor related protein (LRP) and the α2M signaling receptor (α2MSR). In nonmalignant cells, ligation of α2MSR promotes DNA synthesis and cellular proliferation. Here, we report that insulin treatment of highly metastatic 1-LN human prostate carcinoma selectively increases α2MSR expression and binding of α2M* to 1-LN cells. α2M* induces transient increases in intracellular calcium and inositol 1,4,5-trisphosphate in insulin-treated 1-LN cells, consistent with activation of α2MSR. Inhibition of signaling cascades activated by insulin blocks upregulation of α2MSR. By contrast, α2M* does not bind to nor induce intracellular signaling in PC-3 cells, even though 1-LN cells were subcloned from PC-3 cells. We suggest that α2M* behaves like a growth factor in these highly malignant cells. The 1-LN metastatic phenotype may result, in part, from aberrant expression of α2MSR, indicating the possible involvement of α2M* in tumor progression.

We have studied insulin-dependent regulation of macrophage α2-macroglobulin signaling receptors (α2MSR) and low density lipoprotein receptor-related protein/α2M receptors (LRP/α2MR) employing cell binding of 125I-α2M*, inhibition of binding by receptor-associated protein (RAP) or Ni2+, LRP/α2MR mRNA levels, and generation of second messengers. Insulin treatment increased the number of α2M* high (α2MSR) and low (LRP/α2MR) affinity binding sites from 1,600 and 67,000 to 2,900 and 115,200 sites per cell, respectively. Neither RAP nor Ni2+blocked the binding of125I-α2M* to α2MSR on insulin- or buffer-treated cells, but they both blocked binding to LRP/α2MR. Insulin significantly increased LRP/α2MR mRNA levels in a dose- and time-dependent manner. Insulin-augmented125I-α2M* binding to macrophages was severely reduced by wortmannin, LY294002, PD98059, SB203580, or rapamycin. The increase in α2MSR receptor synthesis was reflected by augmented generation of IP3and increased [Ca2+]ilevels upon receptor ligation. Incubation of macrophages with wortmannin, LY294002, PD98059, SB203580, rapamycin, or antibodies against insulin receptors before insulin treatment and α2M* stimulation significantly reduced the insulin-augmented increase in IP3and [Ca2+]ilevels. Pretreatment of cells with actinomycin D or cycloheximide blocked the synthesis of new α2MSR. In conclusion, we show here that insulin coordinately regulates macrophage α2MSR and LRP/α2MR, utilizing both the PI 3-kinase and Ras signaling pathways to induce new synthesis of these receptors.

Exposure of humans to beryllium dusts can induce a specific form of chronic pneumonitis that consists mainly of noncaseating granulomas in the lungs. Multiple studies have documented both genetic and immune components of chronic berylliosis. Much work has focused on T cells and their reactivity in berylliosis, but less work has focused on the end effector cells in granulomatous inflammation, macrophages. Because macrophages must become activated to form granulomas, and they become activated by responding to numerous immunomodulatory signals, we investigated the effects of beryllium (BeCl2) on a central signal transduction pathway in macrophages, increases in intracellular calcium ([Ca2+]i). Exposure of cultured murine peritoneal macrophages to low, non‐toxic concentrations induced successive spikes or oscillations in [Ca2+]i. Concentrations as low as 5 nM induced significant increases in [Ca2+]i. The source of the increased [Ca2+]iwas exclusively extracellular in that increases in [Ca2+]icould be completely blocked by chelating extracellular Ca2+, were inhibited by the Ca2+channel blocker verapamil, and exposure of macrophages to BeCl2had no effect on IP3concentrations. DNA synthesis, a Ca2+‐sensitive function, was enhanced in dividing 1LN cells and induced de novoin quiescent macrophages. Furthermore, BeCl2enhanced DNA synthesis in the absence of coexposure to the protein kinase C activator phorbol myristate acetate. These data support the hypothesis that beryllium toxicity is in part the result of altered Ca2+metabolism in mononuclear phagocytes consequent to reversible opening of plasma membrane channels. J. Leukoc. Biol.65: 786–791; 1999.

The catabolism of streptokinase (SK) and polyethylene glycol derivatives of SK (PEG-SK) were studied in mice. The clearance and catabolism of SK:plasmin (SK:Pm) and PEG-SK:Pm activator complexes were also investigated. Native 125I-SK cleared rapidly (t1/2= 15 minutes) from the circulation. with the majority of the ligand accumulating in the liver and gastrointestinal (Gl) tract and a substantial fraction also localizing in the kidneys. SK, which was removed from the plasma by the liver, was secreted into bile and then the Gl tract. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) demonstrated that 125I-SK recovered from liver and bile was homogeneous and of the same molecular weight (mol wt ~50,200) as native SK. PEG-125I-SK cleared slowly (t1/2greater than 200 minutes), with more than 80% of the preparation localizing in liver and Gl tract. The PEG-125I-SK secreted into the bile was also intact. The bile containing 125I-SK was incubated with stoichiometric amounts of plasminogen and electrophoresed under nondenaturing conditions. This study demonstrated that the secreted SK was able to form SK:Pg complexes. SDS-PAGE also showed activation of 125l-Pg that was incubated with recovered bile containing the SK. 125l-SK:Pm catabolism was also studied. In these experiments, the mol wt ~42,000 fragment obtained when SK is cleaved by plasmin was found in the bile. This fragment of 125I-SK was not recovered as part of a complex with plasmin, consistent with our previous observations that catabolism of SK:Pm involves transfer of the plasmin to plasma proteinase inhibitors while SK is catabolized independently. By contrast, when PEG-125l-SK:Pm was injected into mice, only intact PEG-125I-SK was found in the bile, consistent with our previous observations that the PEG derivatization blocks its degradation by plasmin.

The Interaction of altered lipids or proteins with the several scavenger receptors (SR) on macrophages can lead to disparate results in both gene expression and cell function. However, the molecular bases of signaling induced by SR ligation have remained obscure. Here we report that maleylated‐bovine serum albumin (maleyl‐BSA) binds a low‐affinity SR, initiating POP2hydrolysis, [Ca2+J spikes, phospholipase A2(PLA2) activation, nuclear factor‐κB (NF‐κB) binding to its cognate nucleotide and tumor necrosis factor α (TNF‐α) gene transcription. We recently reported that oxidized low‐density lipoprotein (ox‐LDL), which binds another macrophage SR, induced pertussis‐toxin‐sensitive hydrolysis of PIP2and elevations in [Ca2+]i[J. Biol. Chem. 270, 3475–3478, 1995]. By contrast, maleyl‐BSA‐initiated events were not pertussis toxin‐sensitive and produced less [Ca2+]ispiking than ox‐LDL. Furthermore, maleyl‐BSA led to binding of NF‐κB to its cognate nucleotide and TNF‐α gene transcription, whereas ox‐LDL suppressed these events. Collectively, this data suggests that maleyl‐BSA and ox‐LDL bind to distinct SR on murine macrophages, initiate distinct signal transduction pathways, and produce different functional effects. J. Leukoc. Biol. 60: 784–792; 1996.

Low-density lipoprotein receptor-related protein (LRP)/α2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including α2-macroglobulin–proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, andPseudomonasexotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4°C (Kd= 1.8 nM) and competes for the binding of a receptor-recognized form of α2-macroglobulin (α2M*) (Ki= 3 nM). α2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd= 5 nM). LDL can also completely inhibit the binding of α2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with α2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as α2M*. In addition, the ability of α2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.

Transcatheter electrocoagulation of the pulmonary artery was used to successfully create experimental pulmonary infarcts in 15 of 16 dogs. The animals were evaluated from one to 13 weeks by chest radiography, computed tomography (CT), angiography, and pathologic examination. The abnormal densities in the dogs' lungs cleared in two to three weeks. The majority of the chest radiographs were normal in two weeks, and all were normal after three weeks. Prior to sacrifice, CT showed abnormal densities in the lungs in only two of eight animals. CT proved to be more sensitive than chest radiography for showing the densities in the lungs due to pulmonary infarction.

Arterial occlusion by direct-current transcatheter electrocoagulation (TCEC) has been developed experimentally and used successfully in clinical situations. The major shortcoming of the technique has been the lengthy time necessary for occlusion of larger arteries. In the present study we attempted to decrease the time necessary for TCEC occlusion of the renal and femoral arteries of heparinized mongrel dogs. The results showed that the occlusion time could be significantly decreased, either by increasing the TCEC current from 15 ma to 30 or 60 ma, or by using a Meditech balloon catheter to occlude the artery during TCEC. The combination of higher current and simultaneous balloon occlusion consistently produced permanent occlusion of arteries up to 7 mm in diameter in 15 minutes or less. Balloon occlusion with TCEC is significantly faster than without this modification and makes the technique of TCEC more applicable for human use and for continued experimental study than any previous refinements.

Previous work has shown that localized electrocoagulation of blood vessels using direct current applied by catheter placed electrodes is feasible. In vitro and in vivo experiments snowed that clot size and thrombosis of vessels are directly related to the product of the amount and duration of the current. Surgically created splenic hemorrhage was well controlled in 9 of 10 animals. The vessels remained permanently occluded after electrocoagulation in 8 of 9 animals. Pathological studies of all the vessels showed intimal damage at the sites of occlusion. These experiments indicate that clot formation may be a product of both intimal damage and platelet attraction to the positive electrode. The technique has advantages over embolization since no foreign material is injected and a localized clot is produced at the tip of the guidewire. There are certain disadvantages and further refinements must be developed to establish transcatheter electrocoagulation as a useful clinical technique for vessel occlusion.

The sensitivity of methionine residues to oxidation is a mechanism by which many proteins, including plasma proteinase inhibitors, may be oxidatively inactivated. Much evidence suggests that methionine oxidation and concurrent losses of protein activity not only occur widely in living systems but are physiologic, homeostatic processes. Neutrophils, macrophages and other leukocytes secrete large quantities of powerful oxidants at sites of inflammation and may readily bring about methionine oxidative inactivation of proteins. In particular, oxidation of proteinase inhibitors may favorably alter the proteinase‐antiproteinase balance to facilitate tissue remodeling and protection from invading organisms. Leukocyte‐mediated inhibitor oxidation also appears to regulate local immunosuppressive activity. Pathophysiologic processes such as emphysema and rheumatoidal disease involve derangements of these homeostatic mechanisms.

The relationship between inter‐α inhibitor (IαI) and urinary proteinase inhibitor (UPI) was examined by comparing purified UPI with a proteolytic fragment of IαI (l'), and by demonstrating that inflammatory cells produce similar fragments under physiologic conditions. Purified I', derived by chymotrypsin digestion of IαI, was similar to UPI in apparent molecular weight (68,000‐69,000), amino acid composition, immunoreactivity, and inhibitory activity against trypsin, chymotrypsin, and neutrophil elastase. The production of similar inhibitory fragments by murine peritoneal macrophages, human neutrophils, and a murine mast cell line was quantified. Neutrophils were most efficient at proteolyzing IαI. Comparison of the pattern of IαI degradation by neutrophil preparations with that by pure enzymes, suggested that both elastase and cathepsin G mediate neutrophil proteolysis of IαI. These proteinases may thus be responsible for inflammation‐related increases in UPl‐like inhibitor levels in vivo.

Substantial evidence indicates that proteolytic degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Our previous work has demonstrated elevated secretion by cultured ovarian adenocarcinoma cells of two gelatinolytic metalloproteinases, a 72-kDa enzyme resembling matrix metalloproteinase 2 (MMP-2) and a 92-kDa enzyme resembling MMP-9 (Moseret al., Int. J. Cancer56, 552–559, 1994). To assess the potentialin vivorelevance of these enzymes, we have examined ovarian carcinoma ascites using gelatin substrate zymography. MMP species identical to those secreted from several well-characterized ovarian adenocarcinoma cell lines were found in the majority of ascites: MMP-2-like gelatinase (23 of 23 cases) and MMP-9-like gelatinase (18 of 23 cases), suggesting a prevalence of these species in the ovarian carcinoma microenvironment and their availability for tumor-associated proteolysis. The contribution of these proteinases to ovarian cancer invasion was further demonstrated by experiments measuring tumor cell-mediated proteolysis of native endothelial cell extracellular matrix (ECM) and tumor cell invasion of reconstituted basement membrane (Matrigel). These data showed that secretion of type IV collagenase activity by a series of independently isolated ovarian adenocarcinoma cell lines correlated well with the ability of these cells to proteolyze the ECM and invade the basement membrane. Furthermore, we have identified and characterized an ovarian carcinoma-associated gelatinase, the 72-kDa MMP found in conditioned media of the DOV 13 cell line, as MMP-2. This enzyme was identical to the previously described MMP-2 from other sources by Western blot, amino terminal sequence, and substrate specificity. Additionally, a large portion of the MMP-2 activity found in DOV 13 conditioned media is active without organomercurial treatment, suggesting that ovarian cancer cells have an endogenous activator of the zymogen. Together, these data suggest that ECM proteolysis mediated by tumor-associated proteinases plays an important role in the invasion and/or metastasis of ovarian carcinoma.

The catabolism of human tissue plasminogen activator (t-PA) was studied in mice. The clearance of t-PA labeled with iodine 125 was rapid (t1/2). The clearance of phenyl-methylsulfonyl-125l-t-PA, which is active site-inhibited, was identical to the active enzyme. Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the vast majority of 125l-t-PA injected into the circulation was present as free enzyme and not in a complex with inhibitors. The clearance of 125l-t-PA was unaltered by large molar excesses of several ligands of known clearance specificities, including macroalbumin, asialoorosomucoid, and diisopropylphosphorylthrombin and was also not altered in the presence of a 1,000-fold molar excess of unlabeled t-PA. Organ distribution studies demonstrated that the early rapid clearance of 125l-t-PA occurred in hepatocytes, followed by a later renal phase of clearance. The clearance of 125l-urokinase (UK) also was studied and was very similar in all aspects to the clearance of 126l-t-PA. These results suggest that both t-PA and UK are cleared from the circulation by unique nonsaturable processes localized in the liver that are independent of the proteinase active site.

Several recent investigations have demonstrated that matrix metalloproteinase-2 (MMP-2) binds to the cell surface and undergoes zymogen activation via a plasma membrane-associated activity. The purpose of this study was to determine if association of MMP-2 with the plasma membrane also modulates the catalytic efficiency of the active enzyme. Using density gradient centrifugation, we isolated the plasma membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA 432, previously described either to express MMP-2 or to express no gelatinolytic metalloproteinases, respectively. While DOV 13 cells contained plasma membrane-associated MMP-2 and OVCA 432 did not, both cell types were able to bind exogenous MMP-2. Furthermore, plasma membrane fractions from these cells significantly enhanced the rate of cleavage of [14C]gelatin I substrate by both MMP-2•tissue inhibitor of metalloproteinases-2 (TIMP-2) complex (2.5-8-fold) and TIMP-2-free MMP-2 (5.9-fold). This stimulatory activity was dose-dependent, soluble in Triton X-100, and abolished by trypsin treatment of the membranes, but was stable to heat treatment. Plasma membrane stimulation of MMP-2 resulted in a 3.8-4.6-fold increase in the catalytic efficiency of gelatinolysis. These data suggest that, in addition to promoting zymogen activation, cell surface binding of MMP-2 may regulate enzyme activity by increasing the rate of substrate cleavage. Via this mechanism, tumor cell types that do not express MMPs (such as OVCA 432) nevertheless may be able to utilize exogenous MMP-2 to mediate proteolysis associated with invasion and metastasis.

Six glycoforms of plasminogen 2 were isolated using a combination of lectin affinity chromatography and chromatofocussing, and the sialic acid content of each glycoform was determined. The kinetics of activation of each glycoform by tissue-type plasminogen activator were analyzed on a fibrin surface and in solution. The second-order rate constant (measured on a fibrin surface) decreased from 1.65 × 106M−1s−1to 3.77 × 104M−1s−1as the sialic acid content of the glycoforms increased from 1.3 mol/mol of protein to 13.65 mol/mol of protein. A similar correlation was noted for activation in solution. Each glycoform was converted to plasmin, and the inhibition constants for the reaction between α2-antiplasmin and plasmin glycoforms were determined. All overall Ki'values, reflecting the final essentially irreversible complex, were in the picomolar range. Sialic acid does not affect inhibition of plasmin byα2-antiplasmin; however, hypersialylated plasmin does not appear to have a kringle-dependent component to inhibition.

The α-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal β-cysteinyl–γ-glutamyl thiol ester bonds are an important structural feature of most α-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human α2-macroglobulin (α2M) and monomeric rat α1-inhibitor-3 (α1 I3). When employing NH3 as the nucleophile, the thiol ester in α1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, α2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between α2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for α2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native α2M is restored when the proteinase-inhibitory capacity is recovered.

Streptokinase, an extracellular protein produced by Streptococci, is capable of activating the human fibrinolytic zymogen plasminogen. The rate of amidolytic activity of the plasminogen-streptokinase complex is greatly diminished by micromolar concentrations of ATP and heparin oligosaccharides. In addition, the plasminogen activator activity of the plasminogen-streptokinase complex is also inhibited by these effectors. ATP and heparin oligosaccharides show structural similarity, suggesting that the inhibition is caused by binding of these molecules to a common newly formed binding pocket in streptokinase, which appears after interaction with plasminogen. Addition of the bivalent cations Ca2+ and Mg2+ reverses the inhibition caused by ATP and heparin. In the presence of ATP and bivalent cations, the complex between plasminogen and streptokinase develops an autophosphorylating activity whose target is the sequence LTSRPAHG in the 4.5 kDa streptokinase N-terminal peptide, which is an early autolysis peptide. This streptokinase N-terminal peptide, which is essential for streptokinase activating activity, may serve, once phosphorylated, in mechanisms related to the pathogenicity of Streptococci. These studies suggest a critical role for plasminogen in regulating the activity of the streptokinase molecule.

Releasable tissue plasminogen activator (t-PA) and the fast inhibitor of t-PA were measured in 18 controls and a pedigree with venous thrombosis. The functional assay was performed by a technique that destroys the t-PA inhibitor when blood is drawn. It was found that activator and inhibitor levels varied widely in the control group. One patient demonstrated inhibitor levels, on two different occasions, of 2.82 and 3.54 IU of t-PA per milliliter of plasma, as compared with a releasable activator level of 1.87 IU/mL. The t-PA antigen levels of this patient and the remainder of the pedigree were essentially normal for all seven subjects. Thus, it is suggested that the previously reported fibrinolytic disorder in this pedigree represents an imbalance between activator and inhibitor levels rather than an actual deficiency of t-PA.(Arch Intern Med 1986;146:188-191)

Summary This case is the sixth report of colonic necrosis developing secondary to acute pancreatitis. In the previous cases there was no roentgen evidence of colonic inflammation or necrosis prior to surgery or autopsy. The initial radiographic findings in this 56-year-old woman consisted of a diffuse transverse colitis on both abdominal films and a hypaque enema. Subsequent radiographs revealed evidence of a pancreatic abscess and changes consistent with necrosis in the splenic flexure. A contrast enema is recommended in all patients suspected of having colonic manifestations of pancreatitis.

Direct binding of receptor-recognized α2-macroglobulin (α2M*) or a cloned receptor binding fragment from rat α1-macroglobulin (RBF) to human trabecular meshwork cells demonstrated two classes of cell surface binding sites. One class has an apparentKdof 5.0 nmand a receptor number of 31,800 receptors/cell. The other class has an apparentKdof 20 pmand a receptor number of 1600 receptors/cell. Binding studies of α2M* or RBF in the presence of a competitor for binding to low-density-lipoprotein receptor-related protein/α2M receptor (LRP/α2MR) called receptor-associated protein (RAP) show that only the lower affinity class of binding sites is susceptible to competition with RAP. Uptake studies demonstrate specific internalization and degradation of α2M* which is inhibitable by RAP. Exposure of the cells to α2M* and RBF (40 nm) is associated with mean increases of 171 and 210%, respectively, in the intracellular calcium concentration, which is not inhibitable by RAP or pertussis toxin. These studies present the first characterization of α2M* and RBF signaling in a primary human cell type and suggest a role for α2M* in the physiology of the eye.

We have expressed receptor-binding domains of human α 2-macroglobulin in Escherichia coli. Expression levels of both recombinants were quite high, but the human one was insoluble, probably forming inclusion bodies. The rat domain, which lacks the human disulfide, was produced in a soluble form and readily purified by two simple chromatographic steps. Purified recombinant rat α 1-macroglobulin receptor-binding domain was fully functional in binding to the α-macroglobulin receptor on human fibroblasts. This 142 residue domain should serve as an excellent template for analyzing the structural requirements for α-macroglobulin receptor ligation and dissecting the varied biological functions resulting from such ligation.

Transcatheter electrocoagulation using direct current has been shown to be an effective method for vessel occlusion. To date, the stainless steel anode has not been entirely satisfactory. Thus, an evaluation of bipolar platinum electrodes and a comparison of monopolar platinum and stainless steel electrodes have been performed. Experimental results have shown that both monopolar and bipolar platinum electrodes caused perforation in most cases. At the present time, we have not found a satisfactory replacement for the unipolar stainless steel anode.

Recently inhibitors of the serpin family were shown to form complexes with dichloroisocoumarine (DCI)-inactivated proteinases under native conditions (Enghild, J. J., Valnickova, Z., ThI., and Pizzo, S. V.(1994) J. Biol. Chem.269, 20159-20166). This study demonstrates that serpin-DCI/proteinase complexes resist dissociation when analyzed in reduced SDS-polyacrylamide gel electrophoresis. Previously, SDS-stable serpin-proteinase complexes have been observed only between serpins and catalytically active proteinases. The stability of these complexes is believed to result from an acyl-ester bond between the active site Ser195of the proteinase and the α-carbonyl group of the scissile bond in the reactive site loop. We have further analyzed the structure of the SDS-stable serpin-proteinase and serpin-DCI/proteinase complexes. The results of these studies demonstrate the presence of the COOH-terminal region of the serpin in both complexes. Since (i) modification of Ser195does not prevent formation of SDS-stable complexes and (ii) COOH-terminal peptides are present in both complexes, the previously described mechanism does not sufficiently explain the formation of SDS-stable complexes.

The role of endothelium in the inhibition of human factor Xa was studied in a plasma environment. Human factor Xa can bind to and function on bovine aortic endothelium in a manner similar to that of bovine factor Xa. Approximately 70% of the bound factor Xa is subject to inhibition by plasma proteinase inhibitors, and the remaining 30% is irreversibly bound as part of a 125 Kd membrane-associated complex not subject to proteolytic degradation. The proportion reversibly bound and its rate of release do not alter with changes in calcium, citrate, heparin, or active proteinase inhibitor concentrations. The principal plasma proteinase inhibitor of human factor Xa was antithrombin III, which accounted for 60% to 65% of factor Xa released from endothelium, with a,-proteinase inhibitor inactivating 20% to 25% and a2-macroglobulin approximately 15%. All of the reversibly bound factor Xa was identified in complex with one of these three proteinase inhibitors. The thrombin active-site inhibitor hirudin was found to markedly accelerate the displacement of reversibly bound factor Xa from the endothelium and to associate specifically with factor Xa without a loss of activity toward chromogenic substrates, perhaps accounting for a novel mechanism of anticoagulation.

The binding of human fibrinogen degradation fragments D1, E, X, and Y, as well as fibrin fragment D1dimer, to mouse peritoneal macrophages was examined. A Scatchard plot of fragment D1binding was biphasic, suggesting two classes of receptors. Fragments D1, D1dimer, X, and Y in low concentrations bound to macrophages with high affinity (Kd= 23 to 73 × 10-11mol/L). Fragment E bound specifically but at a much lower level than the other fragments. Fragment D1was able to compete for the binding of radiolabeled fragments X and Y but not radiolabeled fragment E. These studies indicate that fragments D and E are recognized by separate receptor systems but that all of the fibrinogen degradation products that contain the D domain are recognized by the same receptor system.

The immunoregulatory plasma protein α1-microglobulin (α1-m) and the proteinase inhibitor α1-inhibitor-3 (α1I3) form a complex in rat plasma. In the present work, it was demonstrated that the α1I3•α1-m complex has no inhibitory activity, the bait region was not cleaved by low amounts of proteinases, and it was unable to covalently incorporate proteinases. The results also indicated that the thiolester bond of the α1I3•α1-m complex was broken. The α1I3•α1-m complex was cleared from the circulation much faster than native α1I3, with a half-life of approximately 7 min. Structurally, however, the α1I3•α1-m complex was similar to native α1I3rather than α1I3cleaved by proteinases. It is speculated that the role of α1-m is to destroy the function of α1I3by blocking the bait region and breaking the thiolester and causing its physical elimination by rapid clearing from the blood circulation. It is also possible that the formation of complexes between α1-m and α1I3may serve as a mean to regulate the function of α1-m since its complex with α1I3is taken up rapidly by cellular receptors for α-macroglobulins.

Evidence is presented suggesting that hepatocytes contain a receptor that binds glycoproteins specifically through fucose in α1→3 linkage to N-acetylglucosamine. Human lactoferrin, which contains this type of linkage, is rapidly cleared from the circulation of mice after intravenous injection, and greater than 90% of the injected material is found in hepatocytes. Binding of lactoferrin is mediated through its carbohydrate groups, since its clearance is prolonged after periodate oxidation or after its oligosaccharide groups are extensively degraded with glycosidases. In addition, glycopeptides from lactoferrin inhibit lactoferrin clearance. That lactoferrin clearance is mediated through binding to its fucosyl groups is suggested for several reasons. First, transferrin and asialotransferrin, whose oligosaccharide groups are essentially structurally identical to those of lactoferrin but devoid of fucose, are not cleared on intravenous injection. Second, when fucose is incorporated into asialotransferrin by α1→3 N-acetylglucosamine fucosyl transferase, the resulting fucosylated derivative is cleared rapidly. Neither mannan nor derivatives of orosomucoid that are cleared by binding to receptors for galactose, N-acetylglucosamine, or mannose, inhibit clearance of lactoferrin although clearance is inhibited by fucoidin. Finally, glycoproteins containing fucose in α1 → 2 linkage to galactose or α1 → 6 linkage to N-acetylglucosamine do not inhibit lactoferrin clerance by the liver. Since clearance of other glycoproteins, such as human lactoperoxidase, also appears to be mediated through binding to the same hepatocyte receptor as lactoferrin, it is concluded that the fucose-specific receptor studied here may fulfill other functions than binding lactoferrin. Preliminary studies with liver homogenates and detergent extracts of liver show binding in vitro.

Conditions were defined for the derivatization of recombinant tissue plasminogen activator (rt-PA) with polyethylene glycol (PEG) so as to retain functional activity as a possible means of producing a t-PA species with a prolonged circulating lifetime. Derivatives with a wide range of retention of activities were prepared by varying the concentration and species of activated PEG. The specific activities of the PEG-rt-PA derivatives were dependent on the method of assay. Assays using preformed fibrin gave higher estimates of retention of activity than assays using soluble components. Plasma elimination studies in mice and rats indicated prolonged circulating lifetimes for the radiolabeled PEG-rt-PA derivatives after a rapid clearance and distribution phase; however, the disappearance of functional activity was much more rapid than the disappearance of radiolabeled material. The PEG-rt-PA deriva- tives appeared to accumulate in tissues above their interstitial fluid concentrations and were rapidly inactivated, apparently by reaction with the plasma protease inhibitors. These results were consistent with the inactivation of the PEG-rt-PA derivatives in rat plasma in vitro. A somewhat longer half-life (t1/2) of the one derivative studied was observed in dogs (t1/2, 16 minutes) as compared with the rat (t1/2, five minutes). This was sufficient to confer thrombolytic activity upon the derivative (administered by bolus injection) in contrast to native rt-PA. The potential of PEG-modified rt-PA as a long-lived thrombolytic agent in humans will depend, however, on whether there will be a further extension of the t1/2 because of a reduction in clearance and/or a reduction in the rate of inactivation.