Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx.

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Lactadherin, a milk protein, contains discoidin-type lectin domains with homology to the phosphatidylserine-binding domains of blood coagulation factor VIII and factor V. We have found that lactadherin functions, in vitro, as a potent anticoagulant by competing with blood coagulation proteins for phospholipid binding sites [J. Shi and G.E. Gilbert, Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites, Blood 101 (2003) 2628-2636]. We wished to characterize the membrane-binding properties that correlate to the anticoagulant capacity. We labeled bovine lactadherin with fluorescein and evaluated binding to membranes of composition phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine, 4:20:76 supported by 2 mum diameter glass microspheres. Lactadherin bound saturably with an apparent KD of 3.3+/-0.4 nM in a Ca++ -independent manner. The number of lactadherin binding sites increased proportionally to the phosphatidylserine content over a range 0-2% and less rapidly for higher phosphatidylserine content. Inclusion of phosphatidylethanolamine in phospholipid vesicles did not enhance the apparent affinity or number of lactadherin binding sites. The number of sites was at least 4-fold higher on small unilamellar vesicles than on large unilamellar vesicles, indicating that lactadherin binding is enhanced by membrane curvature. Lactadherin bound to membranes with synthetic dioleoyl phosphatidyl-L-serine but not dioleoyl phosphatidyl-D-serine indicating stereoselective recognition of phosphatidyl-L-serine. We conclude that lactadherin resembles factor VIII and V with stereoselective preference for phosphatidyl-L-serine and preference for highly curved membranes.

Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a ␤-hairpin turn, and also by net positive charge and specific interactions with phospho-L-serine. , and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipidlimiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced ϳ20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced ϳ5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipidbinding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.Factor VIII is a phosphatidyl-L-serine (PS) 1 binding cofactor (1, 2) for the vitamin K-dependent serine protease, factor IXa, that also binds to PS-containing membranes (3, 4). The membrane-bound factor VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation (5). The importance of this enzyme complex is illustrated by hemophilia, a disease in which a deficiency of either factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Factor IXa gains more than 100,000-fold greater efficiency in activating factor X by assembling with factor VIIIa on a PS-containing membrane than when free in solution (6). We have recently found that the predominant effect of PS-containing membranes on the factor VIIIa-factor IXa complex is to increase the k cat by more than 1000-fold (7). These membranes also increase the affinity of factor IXa for factor VIIIa and for factor X. The central importance of the membrane binding function of factor VIII motivates studies to define the membrane-binding motif.Factor VIII, with M r 280,000, is homologous to another procoagulant protein, factor V, in amino acid sequence (8 -10) and in function, as a membrane-bound enzyme cofactor (5, 11-13). The proteins share a repeating domain structure of A1-A2-B-A3-C1-C2 in which the A domains are homologous with ceruloplasmin, the B domain is unique to each protein, and the C domains are homologous with discoidin I, a phospholipid-binding lectin (14), and with a murine milk fat globule membrane ...

Lactadherin, a glycoprotein of the milk-fat globule membrane, contains tandem C domains with homology to discoidin-type lectins and to membrane-binding domains of blood-clotting factors V and VIII. We asked whether the structural homology confers the capacity to compete for the membrane-binding sites of factor VIII and factor V and to function as an anticoagulant. Our results indicate that lactadherin competes efficiently with factor VIII and factor V for binding sites on synthetic phosphatidylserine-containing membranes with half-maximal displacement at lactadherin concentrations of 1 to 4 nM. Binding competition correlated to functional inhibition of factor VIIIa-factor IXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherin was an efficient inhibitor of the prothrombinase and the factor Xase complexes regardless of the degree of membrane curvature and the phosphatidylserine content. Lactadherin also inhibited the factor VIIa-tissue factor complex efficiently whereas annexin V was less effective. Because the inhibitory concentration of lactadherin was proportional to the phospholipid concentration, and because lactadherin was not an efficient inhibitor in the absence of phospholipid, the major inhibitory effect of lactadherin relates to blocking phospholipid sites rather than forming inhibitory protein-protein complexes. Lactadherin was also an effective inhibitor of a modified whole blood prothrombin time assay in which clotting was initiated by dilute tissue factor; 60 nM lactadherin prolonged the prothrombin time 150% IntroductionLactadherin is a 47 000-Da molecular weight (MW) glycoprotein of milk-fat globules. It has also been known as PAS-6/7, indicating the 2 glycosylation variants, 1 bovine-associated mucoprotein, BA-46, P47, and MFG-E8. 2 Lactadherin has a domain structure of EGF1-EGF2-C1-C2 in which EGF indicates epidermal growth factor homology domains, and the C domains share homology with the discoidin family, including the lipid-binding C domains of blood coagulation factor VIII and factor V. 2 The second EGF domain displays an Arg-Gly-Asp motif, 3 which binds to the ␣ v ␤ 3 and ␣ v ␤ 5 integrins. 1,[4][5][6] The second C domain binds to phospholipids. 6 In milk-fat globules, lactadherin lines the surface of the phospholipid bilayer that surrounds the central triglyceride droplet, apparently stabilizing the bilayer. 7 Lactadherin decreases the symptoms of rotavirus infection in infants, possibly by binding to rotavirus particles via carbohydrate moieties. 8 In tissue sections, lactadherin is found localized on the apical portion of secretory epithelium in the breast. 7 Abundant expression by breast carcinoma tissue makes lactadherin a potential target for antigen-guided radiation therapy. 9 Lactadherin is also found on the apical surface of epithelia in the biliary tree, the pancreas, and sweat glands 7 and is synthesized by aortic medial smooth muscle cells. 10 Function in these tissues remains unknown. Lactadherin has been identified as a zona pellucida-binding protein on the a...

The anti-factor VIII (fVIII) C2 domain monoclonal antibody ESH8 inhibits fVIII activity only when fVIII is bound to von Willebrand factor (vWf). However, ESH8 binds with similar affinity to fVIII and fVIII⅐vWf complex, and it does not affect the kinetics of thrombin cleavage at positions 372 and 740 within the fVIII heavy chain and at 1689 within the light chain. The latter is required for fVIII release from vWf. We showed that ESH8 reduced the initial rate of thrombin-activated fVIII (fVIIIa) release from vWf by 4.3-fold compared to that in the absence of antibody. The complex of vWf⅐fVIII⅐ESH8 was activated, and the rate constant determined for fVIIIa dissociation from vWf was 4 ؋ 10. We constructed a mathematical model incorporating the measured rates for fVIIIa release from vWf and for inactivation of heterotrimeric fVIIIa due to the spontaneous loss of the A2 subunit and found that the decreased release rate is sufficient to explain our experimentally observed inhibition of fVIII activity by ESH8. We hypothesize that the slowed rate of fVIIIa release from vWf in the presence of ESH8 allows time for inactivation of unstable fVIIIa prior its participation in the formation of the factor Xase complex. The relevance of these findings is illustrated by our observation that reduction of fVIIIa release from vWf represents an additional mechanism of fVIII inhibition by an anti-C2 domain antibody (epitope 2218 -2307) from a hemophilia A patient. This rare antibody binds to a more amino-terminal epitope than other human anti-C2 inhibitors, resulting in its lack of inhibition of fVIII binding to vWf but not to phospholipid. These two fVIII ligands therefore bind to C2 sites which do not overlap completely.

Neutrophils (polymorphonuclear leukocytes[ IntroductionCirculating polymorphonuclear leukocytes (PMNs), the first line of defense against invasion by pathogenic bacteria, influence the balance between humoral and cell-mediated immunity in the early stages of the immune response by synthesizing and releasing immunoregulatory cytokines. 1 Under pathologic conditions PMNs may injure normal tissue by releasing reactive oxygen intermediates, toxic cationic proteins, and degradative enzymes. 2 Because 10 million new granulocytes are released into the bloodstream from the bone marrow every minute, an equal number must be removed within a relatively short period to maintain a proper balance in cell numbers. 3 Therefore, regulating the number of PMNs in the circulation and prompt removal of senescent PMNs may be important for maintaining normal immune function and preventing tissue injury.Complete absence of endothelial P-selectin results in neutrophilia, indicating that P-selectin is involved in removal of PMNs 4-6 from the circulation. Furthermore, expression of P-selectin on hepatic endothelia promotes phagocytosis of PMNs by Kupffer cells. 7 Circulating Fas ligand is involved in mediating neutropenia, 8 suggesting a pathway for PMN senescence. However, questions remain concerning the sites of P-selectin expression and of PMN sequestration by macrophages and the timing and location of the Fas/Fas ligand (FasL) stimulation toward apoptosis, although interactions between leukocytes and endothelia have been reviewed. 9,10 Material and methods Antibodies and drugsAntibody (Ab) HIS48 to granulocytes, polyclonal Ab to P-selectin, CD61 monoclonal Ab (MoAb) to platelet, and annexin V-biotin to phosphatidylserine (PS) were from Pharmingen (San Diego, CA). Abs to Fas and FasL were from Wako (Osaka, Japan) and Pharmingen. ED1 and ED2 Abs to the macrophage-related antigens were from Serotec (Kidlington, Oxford, United Kingdom). A control Ab to rat IgG, lipopolysaccharide (LPS), low molecular weight heparin (LMWH), and fucoidin, antagonists to P-selectin, and gadolinium chloride (GdCl 3 ), an agent used to block the function of Kupffer cells, were from Sigma (St Louis, MO). Proteinase K and biotin-16-2Ј-deoxyuridine-5Ј-triphosphate (biotin-16-dUTP) were from Boehringer (Mannheim, Germany). Terminal deoxynucleotidyl transferase (TdT) was from Gibco BRL (Gaithersburg, MD). Other Abs, including biotinylated goat anti-mouse Ig, biotinylated goat anti-rabbit Ig, goat Ig to mouse IgG, goat Ig to rabbit IgG, mouse peroxidase-antiperoxidase (PAP) Ab complex, rabbit PAP Ab complex, and peroxidase-conjugated streptavidin, were from Dako (Carpinteria, CA). Experimental designIn the first experiment, 7-to 9-week-old male Wistar rats (Nippon Biological Supply, Tokyo, Japan) were injected with LPS (0.1 mg/kg intravenously [IV]) or sterile saline, and perfusion fixed at 1, 3, 6, 12, and 24 hours after injection. In the second experiment, animals were treated with anti-P-selectin Ab (2.0 mg/kg, IV) or LMWH (70-125 IU/mg, 30 mg/kg, IV), or fucoidi...

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.