Christian W. Heegaard

Interactions of Multiple Heparin Binding Growth Factors with Neuropilin-1 and Potentiation of the Activity of Fibroblast Growth Factor-2

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000–1,600,000 m–1 s–1). These results suggest that Npn-1 possesses a “heparin” mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.

Immunohistochemical localization of urokinase‐type plasminogen activator, type‐1 plasminogen‐activator inhibitor, urokinase receptor and α2‐macroglobulin receptor in human breast carcinomas

We have investigated the localization of urokinase‐type plasminogen activator (u‐PA), type‐1 plasminogen‐activator inhibitor (PAI‐1), u‐PA receptor (u‐PAR) and α2‐macroglobulin‐receptor/low‐density‐lipoprotein‐receptor‐related protein (α2MR/LRP) in human breast tumors by immunohistochemical methods. Frozen sections of 133 primary breast carcinomas, 6 ductal carcinomas in situ and 33 lymph‐node metastases were stained with monoclonal antibodies. Formalin‐fixed sections of 15 primary tumors and 2 lymph‐node metastases were stained with polyclonal antibodies. In primary tumors, u‐PA and PAI‐1 immunoreactivities were intense in macrophages and mast cells, and moderate in benign and malignant epithelial cells as well as in myofibroblasts and endothelial cells. A sub‐group of poorly differentiated tumors showed particularly strong staining of stromal fibroblasts. u‐PA immunoreactivity was also present in lymphocytes. α2MR/LRP and u‐PAR immunoreactivities were intense in macrophages, but apart from these cells, α2MR/LRP was found only in fibroblasts, and u‐PAR only in tumor cells located peripherally in tumor‐cell clusters and glands and some myofibroblasts in the adjacent stroma. Lymph‐node metastases showed staining for u‐PA and PAI‐1 both of cancer cells and of stromal fibroblasts, also staining for u‐PA of lymphocytes. Similarly to some of the poorly differentiated primary tumors, approximately half of the metastases showed very strong staining of stromal fibroblasts, and extracts of these metastases had higher u‐PA and PAI‐1 levels, as determined by ELISA, than extracts of metastases without this staining pattern. α2MR/LRP was present only in fibroblasts and u‐PAR only in some tumor cells. The presence of u‐PA, PAI‐1, α2MR/LRP and u‐PAR was controlled biochemically by immunoblotting analyses, ligand‐blotting analyses, and direct and reverse zymography. The spatial distribution and the variation in concentration of the various components of the plasminogen‐activation system point to a complex, multifunctional role for the 4 proteins in and/or during the development and spread of breast cancer.

Interconversions between active, inert and substrate forms of denatured/refolded type-1 plasminogen activator inhibitor

The latent form of type-1 plasminogen activator inhibitor (PAI-1) acquires inhibitory activity by denaturation followed by refolding. We show here that the reactions of denatured/refolded PAI-1 with plasminogen activators are affected by low concentrations of SDS, which may remain after using SDS for denaturation. Without SDS, the active fraction of denatured/refolded PAI-1 comprised around 60%. Increasing SDS concentrations led to conversions to an inert form without inhibitory activity; then to a substrate form, that is being cleaved proteolytically in the reactive centre by the activators without complex formation, and finally to a second inert form. The first two conversions were associated with changes of the reactivity with monoclonal antibodies and of the thermal stability, respectively. Our results define clearly different interconvertible forms of denatured/refolded PAI-1, distinguish these from the latent and the reactive-centre-cleaved forms, and provide conditions for reproducibly producing reactive-centre-cleaved PAI-1 and PAI-1/activator complexes.

Lactadherin Detects Early Phosphatidylserine Exposure on Immortalized Leukemia Cells Undergoing Programmed Cell Death

Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS‐binding protein, which tethers apoptotic cells to macrophage integrins.

Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure

Summary.  Background: Platelet membrane phosphatidylserine (PS) is considered to be essential for hemostasis and thrombosis, but the in vivo topography of platelet PS has not been characterized. We hypothesized that platelet PS exposure would be identified on adherent platelets at the site of vascular injury and that blockade of PS would impede hemostasis and thrombosis. Objective: To localize and estimate the extent of platelet PS exposure and evaluate the impact of PS blockade in vivo. Methods: Lactadherin, a PS‐binding milk protein, was utilized together with annexin V to detect both partial and complete membrane PS exposure on platelets in a mouse model of thrombosis and to evaluate the functional need for PS. Preliminary experiments were performed with synthetic membranes and with purified platelets. Results: The number of lactadherin‐binding sites on synthetic membranes was proportional to PS content, whereas annexin V required a threshold of 2.5–8% PS. Approximately 95% of thrombin‐stimulated platelets exposed PS, but the quantity was below the threshold for annexin V binding at physiologic Ca2+ concentrations. In mice, most adherent and aggregated platelets on the walls of ferric chloride‐treated mesenteric veins exposed low levels of PS, rather than having complete exposure. In mice, blockade of PS with lactadherin inhibited platelet prothrombinase and factor Xase activity, and prolonged tail bleeding time and the time to carotid artery thrombosis. Conclusions: In vivo PS exposure contributes to both hemostasis and thrombosis. In this model of vascular injury, most platelets exhibit partial rather than complete PS exposure.

The toxicity of bovine α‐lactalbumin made lethal to tumor cells is highly dependent on oleic acid and induces killing in cancer cell lines and noncancer‐derived primary cells

A complex between α‐lactalbumin and oleic acid (C18:1, 9 cis) has been reported to be cytotoxic to cancer cells. We have prepared such complexes and tested their activity against both cancer cell lines and noncancer‐derived primary cells. Unexpectedly, some primary cell types were more sensitive to treatment than cancer cell lines. We found the complex to be cytotoxic to all of the tested cells, with a 46‐fold difference between the most sensitive and the least sensitive cell type. Oleic acid by itself exhibited a remarkably similar activity. The cell‐killing mechanisms of the complex and of oleic acid alone were examined by flow cytometry, testing for apoptosis‐ and necrosis‐ inducing activity. The T‐cell leukemia‐derived Jurkat cells primarily underwent cell death resembling apoptosis, whereas the monocytic leukemia‐derived THP1 cells adopted a more necrotic‐like cell death. Erythrocytes were sensitive to lysis by the complex and oleic acid. We conclude that oleic acid is cytotoxic by itself and that, in contrast to the literature, a complex of α‐lactalbumin and oleic acid has cytotoxic activity against primary cells, as well as cancer cells.

Measurement of phosphatidylserine exposure during storage of platelet concentrates using the novel probe lactadherin: a comparison study with annexin V

BACKGROUND: Annexin V binding to platelets (PLTs) is considered the gold standard for monitoring phosphatidylserine (PS) exposure. However, recent comparison of annexin V with the new calcium‐independent PS probe lactadherin revealed that annexin V requires a certain threshold of PS exposure (2%‐8%) for binding to occur. The aim of this study was to compare annexin V and lactadherin labeling of PLTs in PLT concentrates (PCs).

Very low density lipoprotein receptor from mammary gland and mammary epithelial cell lines binds and mediates endocytosis of M(r) 40,000 receptor associated protein.

We here report that the M r 40,000 receptor associated protein (RAP), previously found to bind to α2‐macroglobulin receptor/low density lipoprotein receptor related protein (α2MR/LRP) and glycoprotein 330 (gp330), binds to an M r, 105,000 membrane protein from bovine mammary gland, human mamma tumors and mammary epithelial cell lines. We have purified this protein from bovine and human sources. N‐terminal amino acid sequencing and immunoblotting analyses showed that the protein was identical or closely related to very low density lipoprotein receptor (VLDL‐R). Experiments with the human mamma carcinoma cell line MCF‐7 showed that this receptor was able to mediate an efficient endocytosis of RAP. These novel findings strongly suggest that RAP functions as a modulator of ligand binding to VLDL‐R, similarly to α2MR/LRP and gp330.

The plasminogen activation system in bovine milk: Differential localization of tissue-type plasminogen activator and urokinase in milk fractions is caused by binding to casein and urokinase receptor

We have analyzed the occurrence of components of the plasminogen activation system in bovine milk. Zymographic analyses showed that tissue-type plasminogen activator (t-PA) occurred in association with casein micelles, partially as a complex with type-1 plasminogen activator inhibitor (PAI-1), whereas urokinase-type plasminogen activator (u-PA) was confined to milk leukocytes. Whey contained a component with a plasminogen dependent proteolytic activity which was shown to be plasma prekallikrein (PPK). The u-PA in the milk leukocytes was shown to be bound to urokinase receptor (u-PAR). A purification to near-homogeneity of the bovine u-PAR was undertaken. Investigating the novel t-PA binding to casein micelles by ligand blotting and Sepharose immobilized casein, multimeric forms of kappa-casein and dimeric alpha s2-casein were identified as t-PA binding components. The kappa-casein gene and the fibrinogen gene are believed to have evolved from a common ancestor. Thus, the recent finding that casein enhances t-PA catalyzed plasminogen activation (Marcus, G., Hitt, S., Harvey, S.R. and Tritsch, G.L. (1993) Fibrinolysis 7, 229-236), and the observed t-PA/casein binding suggests that the casein micelle, which also contains plasminogen, may serve as a matrix for t-PA-catalyzed plasminogen activation in milk.

Plasminogen activators in bovine milk during mastitis, an inflammatory disease

We have investigated the content of plasminogen activators in bovine milk during mastitic inflammation induced by Staphylococcus aureus. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with fibrin agarose zymography and a coupled peptidyl anilide plasminogen activation assay of samples of whey prepared by acidification, we found that the level of tisue-type plasminogen activator (t-PA) in milk was increased immediately after infection and remained elevated during an experimental period of 42 days. The maximal increase was 10 to 20-fold. By zymography, we also demonstrated a strong increase in urokinase-type plasminogen activator (u-PA) associated with the bovine cells in the milk. By ligand blotting, we demonstrated an increase in the level of the urokinase-receptor (u-PAR) on the milk cells during inflammation. Plasma kallikrein was also detected as a plasminogen dependent proteolytic activity by zymography of whey samples. When analyzed in the presence of the t-PA in the milk, the plasma kallikrein lysis zone was strongly increased in mastitic whey, but when analyzed after separation from t-PA, its level was unaffected by mastitis; this could be ascribed to a t-PA dependent stimulation of plasma prekallikrein. These results suggest an important role for plasminogen activators in the inflammatory response during bovine mastitis. Using an enzyme-linked immunosorbent assay we measured the plasminogen/plasmin level during the inflammation, but found a less than 2-fold increase during the experimental period.