Peter R. Ganz

Endothelial cell surface actin serves as a binding site for plasminogen, tissue plasminogen activator and lipoprotein(a)

One of the mechanisms by which endothelial cells (ECs) regulate fibrinolysis is through the regulated assembly of proteins such as plasminogen, tissue plasminogen activator (tPA) and urokinase (uPA) on their membrane surface. Receptors for many of these fibrinolytic factors have been isolated and characterized. A unique 45u2003kD plasminogen receptor present on ECs derived from vein vasculature has been identified and resolved into two plasminogen binding components. One component consists of the unique 45u2003kD plasminogen receptor (pIu2003=u20036.3) whereas the other component (pIu2003=u20035.1) is identified as the cytoskeletal protein, actin. Immunofluorescent studies of isolated ECs confirm the presence of actin on their extracellular surface. This observation is consistent with a number of other recent reports of actin externally localized on other cell types. In vitro studies using purified actin confirm that plasminogen binds to actin both saturably and with relatively high affinity. Competition studies with lysine indicated that the binding was largely kringle‐dependent, and when binding of tPA to actin was assessed, it also bound to actin with 70–80% of binding inhibited by lysine. Lipoprotein(a), which shows homology with plasminogen, also interacted with actin. Addition of plasminogen and low‐density lipoproteins inhibited Lp(a) binding to actin in a dose‐dependent fashion. Moreover, in competition with tPA, partial inhibition of plasminogen binding to actin was also observed. In experiments using anti‐actin antibodies added in excess to cultured ECs, binding of plasminogen was inhibited by 45%, tPA binding was inhibited by 46% and Lp(a) binding was reduced by 56%, confirming actin as a binding site for these various ligands whilst attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing binding not only of plasminogen, but also of tissue plasminogen activator and Lp(a) to the surface of human endothelial cells.

Incorporation of plasticizer into red cells during storage

The development of flexible plastic blood bags has permitted effective blood component production and therapy. However, the plasticizer di(2‐ ethylhexyl)phthalate (DEHP), whose toxicity in humans is still undefined, is known to leach from the plastic into stored blood. Despite the availability of bags made of plastics not using DEHP, the collection and storage of red cells is still done in DEHP plasticized packs, and in fact the storage life for red cells has recently been increased up to 49 days using new anticoagulant‐preservative solutions. We examined the relationship between DEHP and stored red cells. We found that 28 percent of available 14C‐DEHP binds immediately to sites in both the membrane and cytosol fractions of the red cells, and that the total amount and distribution of 14C‐DEHP does not change significantly over 7 days. When red cell concentrates were stored with or without DEHP, using either plastic (polyolefin) bags not containing DEHP or glass, definite reduction in the osmotic stability of the red cells was found in the absence of DEHP. Plasma‐free hemoglobin levels were 90.3 mg per dl after 35 days of storage in plastic packs containing DEHP and 181.7 mg per dl in the polyolefin bags. The advantages of improved in vitro stability of red cells stored in plastics containing DEHP must be weighed against the potential hazards of patient exposure to DEHP.

Sorting of glycoprotein B from human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco.

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.

Biologically active human GM-CSF produced in the seeds of transgenic rice plants

Rice flour is a well-known and characterized source of pharmaceutical ingredients, which are gluten-free and incorporated in many drug delivery applications such as excipient starch. To further exploit this uniqueness, the synthetic capacity of rice endosperm tissue, the basis of rice flour, was extended by genetic transformation. Recombinant human GM-CSF, a cytokine used in treating neutropenia and with other potential clinical applications, has been expressed in transgenic rice seeds using a rice glutelin promoter. Rice seeds accumulated human GM-CSF to a level of 1.3% of total soluble protein. The rice seed-produced human GM-CSF was found to be biologically active when tested using a human cell line TF-1. Use of rice as a host plant offers not only attractive features of safe production in seeds but also self-containment of foreign genes, as rice is primarily a self-pollinated crop plant.

DDAVP-induced release of von Willebrand factor from endothelial cells in vitro: the effect of plasma and blood cells.

The vasopressin analogue 1-deamino-8-D-arginine vasopressin (DDAVP) causes an immediate, transient rise in plasma levels of von Willebrand factor (vWF) after its administration. Although it is recognized that vascular endothelial cells play an essential role in this process, the molecular basis of the response is not understood. We have investigated the phenomenon using human umbilical vein endothelial cells as an in vitro model. When normal individuals were stimulated with DDAVP, plasma from blood samples collected subsequently caused the release of vWF from cultured endothelial cells over a 24 h period (22-46% increase over baseline), compared to control plasma (5-17%). DDAVP added directly to the endothelial cells produced no increase in vWF release. When whole blood was treated in vitro with DDAVP, and the plasma subsequently added to endothelial cells, a significant increase in vWF secretion was found. Peripheral blood mononuclear cells were then tested. In the presence of DDAVP, an increased response occurred. Further fractionation of these cells showed that monocytes were largely responsible, causing an increased vWF release of 162% at 2 h. These observations were reinforced by finding that the supernatants of monocytes incubated with DDAVP were also effective in causing increased vWF release (118% compared to 58% for the control sample). Our studies suggest that DDAVP plays an indirect role in causing the release of vWF from endothelial cells, and that peripheral blood monocytes may act as intermediary target cells, which then produce factor(s) acting directly on endothelial cells.

Biological activity of human granulocyte-macrophage colony stimulating factor is maintained in a fusion with seed glutelin peptide

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5 end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.

Identification of Novel Factor VIII Inhibitor Epitopes using Synthetic Peptide Arrays

Objectives: Mapping the antibody‐binding sites on the factor VIII (FVIII) protein opens the prospect of studying the development of FVIII inhibitors and the alteration of inhibitor specificities over time. This paper describes a novel approach to the mapping of FVIII antibody‐binding sites. Methods: Immobilized synthetic peptide arrays covering 80% of the complete 2351 amino acid sequence of factor VIII (FVIII) were used to determine epitope specificity of 6 alloantibodies and 3 autoantibodies inhibitory to FVIII activity. This detailed assessment was carried out using a modified enzyme‐linked immunosorbent assay with plasma from normal persons or hemophilia A patients without inhibitors as negative controls. Results: Antibody‐combining sites could be differentiated in both a qualitative and quantitative manner and were patient‐specific. Highly reactive peptides were restricted to specific sites in the Al‐A3 and Cl‐C2 domains and were not proximal to known proteolytic cleavage sites. Free peptides incubated in vitro with the plasmas of 3 patients significantly reduced residual inhibitor titers in a dose‐dependent manner. Conclusion: This technique permits the study of the development and specificity of FVIII inhibitors, can detect and differentiate between inhibitory and noninhibitory antibodies using immobilized or free peptides respectively, permits correlation of antibody‐combining sites with inhibition of FVIII activity and provides a basis for the development of inhibitor adsorption or neutralization technology.

Identification of an endothelial cell surface protein that binds plasminogen

To identify and characterize endothelial cell surface components that bind plasminogen, we used ligand-blotting to study binding of plasminogen to sodium dodecyl sulphate solubilized extracts of human umbilical vein endothelial cells. It was observed that glu-plasminogen bound predominantly to a 45 kDa endothelial cell polypeptide. The interaction of labelled glu-plasminogen with this polypeptide was reversible and specific as the binding could be inhibited by both excess cold lysine and unlabelled glu-plasminogen but not by unrelated proteins. Binding of glu-plasminogen to cell extracts prepared from endothelial cells that had been pretreated with proteinase K was significantly reduced indicating that the 45 kDa polypeptide is a cell-surface protein. The cell-surface localization of the 45 kDa polypeptide was also indicated by the positive interaction of glu-plasminogen with membrane fractions of endothelial cells. Lys-plasminogen also interacted with the 45 kDa polypeptide in a specific manner and reversibility experiments indicated that lysplasminogen could also displace the bound glu-plasminogen. Since binding of plasminogen to the 45 kDa endothelial cell surface polypeptide was very similar to plasminogen binding to intact endothelial cells, we propose that the 45 kDa protein represents one of the major receptors for plasminogen on human endothelial cells.

The association of human coagulation factors VIII, IXa and X with phospholipid vesicles involves both electrostatic and hydrophobic interactions

Blood coagulation factor X (FX) is converted to its active form (FXa) by a membrane bound multi-protein enzyme complex, comprised of factor VIII (FVIII), factor IXa (FIXa) and FX. Characterization of the molecular forces involved in the association of these proteins with phospholipids is crucial to understanding how these proteins bind to the lipid milieux of physiological membranes. In this report, the molecular forces involved in the association of FVIII, FIXa or FX with phospholipid vesicles (PLV) were characterized by ligand affinity chromatographic analyses. Treating FVIII-affinity columns with agents that disrupt electrostatic interactions caused elution of 15.2% of the total bound PLV, while agents that disrupt hydrophobic interactions caused elution of 84.8% of the total bound PLV. These results demonstrate that the association of PLV with FVIII is primarily hydrophobic. In contrast, the association of PLV with FIXa or FX is largely the result of electrostatic forces. This was established by observing that 71.3% and 78.9% of the total bound PLV was eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt electrostatic interactions. Of the total bound PLV, 28.7% and 21.2% were eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt hydrophobic interactions. These data demonstrate that hydrophobic forces play a heretofore unrecognized role in the association of PLV with FIXa or FX.

Pharming vaccines for hepatitis and cytomegalovirus: Towards the development of multivalent and subunit vaccines for oral delivery of antigens

A plant based high fidelity vaccine production system is being developed with emphasis on producing antigens capable of being orally delivered in multivalent or subunit plant packets. Plant-based edible vaccines may provide an attractive, safe and inexpensive alternative to conventional vaccine production. Edible plant tissues are not normally antigenic in nature. However, foreign antigens from common infectious organisms like hepatitis-B virus (HBV) can be produced along with naturally occurring storage proteins in DNA-transformed plants. Upon administration via the oral route, these transgenic plant tissues may mobilize the protective humoral and mucosal immune responses to challenge the natural infectious agent. When tobacco, carrot and rice plants were transformed with the truncated version of the HBV nucleocapsid gene expression construct, non-infective hepatitis B viral core particles were observed via electron microscopy. A second plant codon-optimised HBV expression construct was designed that included the extensin signal sequence for augmented HBV particle accumulation. Upon transformation of tobacco plants with the codon-optimised construct, over 4 times more transgenic plants with high levels of expression of the HBV nucleocapsid protein were generated in comparison with a similar vector containing the unmodified wild-type HBV gene codon sequence. Further analysis via Western blotting confirmed the presence of the viral antigen in the total protein extracts from transgenic tobacco leaves and seeds. Electron microscopy showed that the expressed protein self-assembled into viral-like particles of 25–30xa0nm in diameter. To develop an edible subunit vaccine in plant seeds, a third plant transformation construct was used for the synthesis of the human cytomegalovirus glycoprotein B (HCMV gB) subunit. The gB protein derived from tobacco seeds retained critical structural features including epitopes for neutralizing antibodies and was targeted to the protein storage vesicles of tobacco seed endosperm. Two different strains of mice were orally immunized with tobacco seeds containing low concentrations of HCMV gB, with varying dosages, but without adjuvant. No anti-gB response was detected in intestinal or serum samples. However, a systemic immune response to normal tobacco seed proteins was observed in both strains of mice. While higher expression levels of antigens in seeds must be achieved, seeds may provide an effective and immunostimulatory vehicle for delivering edible vaccines to the intestinal mucosa. One of the outstanding challenges includes defining optimum conditions of antigen presentation, dosage and immunization schedules that will induce strong mucosal and/or systemic immune responses in heterogeneous populations. Here we review the different strategies being employed to produce specific oral antigens in plant tissues.