Jan T. Rasmussen

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.

Isolation and characterization of plasminogen and plasmin from bovine milk

Abstract A procedure was developed for the isolation of plasminogen and plasmin from bovine milk. Pure undegraded plasminogen with N-terminal amino acid sequence and mobility in SDS-PAGE similar to plasminogen isolated from bovine blood was obtained. Additionally, the preparation contained the proteolytically modified midi-plasmin, consisting of kringle 4,5 and the light chain with an apparent molecular weight of 50 k Da in unreduced SDS-PAGE, and resulting in two bands of 30 and 26 k Da after reduction. Some 15% of the amino acid sequence of plasminogen/midi-plasmin isolated from milk was determined. The partial amino acid sequence was identical to that previously reported for plasminogen isolated from bovine blood (J. Schaller et al., (1985). Eur. J. Biochem., 149, 267–278), and to the bovine liver plasminogen cDNA sequence (L. Berglund et al., (1995). Int. Dairy J., 5, 593–603). Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments revealed that immunoreactive plasminogen was associated with acid-precipitated casein, rennet-coagulated casein and casein micelles. Some was found in acid whey and to a lesser extent in rennet whey. The amount of plasminogen associated with the milk fat globule membrane was very low and reflected the presence of casein. By Western blotting, immunoreactive plasminogen was found in zones with apparent molecular weights of 85, 80 and 50 k Da in unreduced gels. These hands may represent two plasminogen forms (85 and 80 k Da) and midi-plasmin/midi-plasminogen (50 k Da). The total concentration of plasminogen in bovine milk was estimated to 1.5 μg/mL.

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).

Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia

Summary.  Background: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As2O3). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Methods: Lactadherin, a milk protein with stereospecific binding to phosphatidyl‐L‐serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. Results: Plasma procoagulant activity of NB4 and APL cells increased approximately 15‐fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As2O3. Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As2O3 and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80–85% of intrinsic FXase, FVIIa‐tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low‐level PS exposure. Conclusions: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity.

The in Vivo Toxicity of Hydroxyurea Depends on Its Direct Target Catalase

Hydroxyurea (HU) is a well tolerated ribonucleotide reductase inhibitor effective in HIV, sickle cell disease, and blood cancer therapy. Despite a positive initial response, however, most treated cancers eventually progress due to development of HU resistance. Although RNR properties influence HU resistance in cell lines, the mechanisms underlying cancer HU resistance in vivo remain unclear. To address this issue, we screened for HU resistance in the plant Arabidopsis thaliana and identified seventeen unique catalase mutants, thereby establishing that HU toxicity depends on catalase in vivo. We further demonstrated that catalase is a direct HU target by showing that HU acts as a competitive inhibitor of catalase-mediated hydrogen peroxide decomposition. Considering also that catalase can accelerate HU decomposition in vitro and that co-treatment with another catalase inhibitor alleviates HU effects in vivo, our findings suggests that HU could act as a catalase-activated pro-drug. Clinically, we found high catalase activity in circulating cells from untreated chronic myeloid leukemia, offering a possible explanation for the efficacy of HU against this malignancy.

Platelet apoptosis by cold‐induced glycoprotein Ibα clustering

Summary.  Background:  Cold‐storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα‐distribution indicative of receptor clustering and initiates thromboxane A2‐formation. GPIbα is associated with 14‐3‐3 proteins, which contribute to GPIbα‐signaling and in nucleated cells take part in apoptosis regulation.

A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and αv-integrin. Phagocytosis via the phosphatidylserine-lactadherin-αv-integrin pathway is the acknowledged route for removal of apoptotic innate cells by phagocytes. Design and Methods Endothelial cell phagocytosis of red blood cells was further explored using a more (patho)physiological approach. Red blood cells were exposed to oxidative stress, induced by tert-butyl hydroperoxide. After opsonization with lactadherin, red blood cells were incubated with endothelial cells to study erythrophagocytosis and examine cytotoxicity. Results Red blood cells exposed to oxidative stress show alterations such as phosphatidylserine exposure and loss of deformability. When incubated with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape (‘rounding up’). Increased expression of apoptotic markers indicated that marked erythrophagocytosis has cytotoxic effects. Conclusions Activated endothelial cells show significant phagocytosis of phosphatidylserine-exposing and rigid red blood cells under both static and flow conditions. This results in a certain degree of cytotoxicity. We postulate that activated endothelial cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.

Lactadherin binds to phosphatidylserine-containing vesicles in a two-step mechanism sensitive to vesicle size and composition.

Lactadherin binds to phosphatidylserine (PS) in a stereospecific and calcium independent manner that is promoted by vesicle curvature. Because membrane binding of lactadherin is supported by a PS content of as little as 0.5%, lactadherin is a useful marker for cell stress where limited PS is exposed, as well as for apoptosis where PS freely traverses the plasma membrane. To gain further insight into the membrane-binding mechanism, we have utilized intrinsic lactadherin fluorescence. Our results indicate that intrinsic fluorescence increases and is blue-shifted upon membrane binding. Stopped-flow kinetic experiments confirm the specificity for PS and that the C2 domain contains a PS recognition motif. The stopped-flow kinetic data are consistent with a two-step binding mechanism, in which initial binding is followed by a slower step that involves either a conformational change or an altered degree of membrane insertion. Binding is detected at concentrations down to 0.03% PS and the capacity of binding reaches saturation around 1% PS (midpoint 0.15% PS). Higher concentrations of PS (and also to some extent PE) increase the association kinetics and the affinity. Increasing vesicle curvature promotes association. Remarkably, replacement of vesicles with micelles destroys the specificity for PS lipids. We conclude that the vesicular environment provides optimal conditions for presentation and recognition of PS by lactadherin in a simple binding mechanism. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Structural characterization of bovine CD36 from the milk fat globule membrane

Bovine CD36 from milk fat globule membranes was characterized and a full-length CD36 cDNA of 2772 nucleotides was isolated from a bovine mammary gland cDNA library. The deduced protein sequence contains 472 amino acid residues with 82-84% identity to the amino acid sequences of CD36 from other species. Peptides corresponding to 43% of the protein were sequenced. All eight potential N-glycosylation sites were glycosylated and the carbohydrate compositions of the individual sites were determined.