Hypothesis for control of von Willebrand factor multimer size by intra-molecular thiol-disulphide exchange.
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ADAM Proteins↗
Biomedical subjects
P J Hogg
Publications and source records attributed to P J Hogg.
Allosteric disulfide bonds in thrombosis and thrombolysis.
Allosteric disulfide bonds control protein function by mediating conformational change when they undergo reduction or oxidation. The known allosteric disulfide bonds are characterized by a particular bond geometry, the -RHStaple. A number of thrombosis and thrombolysis proteins contain one or more disulfide bonds of this type. Tissue factor (TF) was the first hemostasis protein shown to be controlled by an allosteric disulfide bond, the Cys186-Cys209 bond in the membrane-proximal fibronectin type III domain. TF exists in three forms on the cell surface: a cryptic form that is inert, a coagulant form that rapidly binds factor VIIa to initiate coagulation, and a signaling form that binds FVIIa and cleaves protease-activated receptor 2, which functions in inflammation, tumor progression and angiogenesis. Reduction and oxidation of the Cys186-Cys209 disulfide bond is central to the transition between the three forms of TF. The redox state of the bond appears to be controlled by protein disulfide isomerase and NO. Plasmin(ogen), vitronectin, glycoprotein 1balpha, integrin beta(3) and thrombomodulin also contain -RHStaple disulfides, and there is circumstantial evidence that the function of these proteins may involve cleavage/formation of these disulfide bonds.
Allosteric Site↗
Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase.
N-dansylhomocysteine (DnsHCys) is quenched on S-nitrosation. The product of this reaction, N-dansyl-S-nitrosohomocysteine, is a sensitive, direct fluorogenic substrate for the denitrosation activity of protein disulfide isomerase (PDI) with an apparent K(M) of 2 microM. S-nitroso-BSA (BSA-NO) competitively inhibited this reaction with an apparent K(I) of 1 microM. The oxidized form of DnsHCys, N,N-didansylhomocystine, rapidly accumulated in cells and was reduced to DnsHCys. The fluorescence of DnsHCys-preloaded human umbilical endothelial cells and hamster lung fibroblasts were monitored as a function of extracellular BSA-NO concentration via dynamic fluorescence microscopy. The observed quenching of the DnsHCys fluorescence was an indirect measure of cell surface PDI (csPDI) catalyzed denitrosation of extracellular S-nitrosothiols as decrease or increase in the csPDI levels in HT1080 fibrosarcoma cells correlated with the rate of quenching and the PDI inhibitors, 5,5'-dithio-bis-3-nitrobenzoate and 4-(N-(S-glutathionylacetyl) amino)phenylarsenoxide inhibited quenching. The apparent K(M) values for denitrosation of BSA-NO by csPDI ranged from 12 microM to 30 microM. Depletion of membrane N(2)O(3) with the lipophylic antioxidant, vitamin E, inhibited csPDI-mediated quenching rates of DnsHCys fluorescence by approximately 70%. The K(M) for BSA-NO increased by approximately 3-fold and V(max) decreased by approximately 4-fold. These findings suggest that csPDI catalyzed NO released from extracellular S-nitrosothiols accumulates in the membrane where it reacts with O2 to produce N(2)O(3). Intracellular thiols may then be nitrosated by N2O3 at the membrane-cytosol interface.
Animals↗
Control of von Willebrand factor multimer size by thrombospondin-1.
Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury. Only the very large vWF multimers are effective in promoting platelet adhesion in flowing blood. A protein disulfide bond reductase in plasma reduces the average multimer size of vWF secreted by endothelial cells. This activity has been isolated from human endothelial cell conditioned medium and shown to be the trimeric glycoprotein, thrombospondin-1 (TSP-1). Incubation of purified TSP-1 with vWF resulted in formation of thiol-dependent complexes of TSP-1 and vWF, generation of new thiols in vWF, and reduction in the average multimer size of vWF. The ratio of the concentrations of TSP-1 and vWF in plasma reflected with average multimer size of vWF. The higher the plasma TSP-1/vWF molar ratio, the smaller the average vWF multimer size. In addition, administration of TSP-1 to mice resulted in reduction in the average multimer size of plasma vWF. Interaction of TSP-1 with vWF is mediated by TSP-1 type 1 properdin domains and the vWF A3 domain. These results indicate that TSP-1 regulates the multimeric size and therefore hemostatic activity of vWF.
Adult↗
Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid.
Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 microM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.
Adjuvants, Immunologic↗
Identification and characterisation of a platelet GPIb/V/IX-like complex on human breast cancers: implications for the metastatic process.
The glycoprotein (GP) Ib /V/IX receptor complex is an important adhesion molecule, originally thought to be unique to the megakaryocytic lineage. Recent evidence now indicates that GPIb /V/IX may be more widely expressed. In this study we report the presence of all subunits of the complex on four breast cancer cell lines, and 51 / 80 primary breast tumours. The surface expression of GPIb /V/IX was confirmed by flow cytometry, and by immunoprecipitation of biotin surface-labelled tumour cells. Western blotting of cell lysates under reducing conditions revealed that tumour cell-GPIb alpha had a relative molecular weight of 95 kDa as compared to 135 kDa on platelets. Despite the discrepant protein size, molecular analyses on the tumour cell-GPIb alpha subunit using RT-PCR and DNA sequencing revealed 100% sequence homology to platelet GPIb alpha. Tumour cell-GPIb /V/IX was capable of binding human von Willebrand factor (vWf), and this binding caused aggregation of tumour cells in suspension. Tumour cells bound to immobilised vWf in the presence of EDTA and demonstrated prominent filapodial extensions indicative of cytoskeletal reorganisation. Furthermore, in a modified Boyden chamber assay, prior exposure to vWf or a GPIb alpha monoclonal antibody, AK2, enhanced cell migration. The presence of a functional GPIb /V/IX-like complex in tumour cells suggests that this complex may participate in the process of haematogenous breast cancer metastasis.
Blotting, Western↗
Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase.
Disulphide bonds in secreted proteins are considered to be inert because of the oxidizing nature of the extracellular milieu. An exception to this rule is a reductase secreted by tumour cells that reduces disulphide bonds in the serine proteinase plasmin. Reduction of plasmin initiates proteolytic cleavage in the kringle 5 domain and release of the tumour blood vessel inhibitor angiostatin. New blood vessel formation or angiogenesis is critical for tumour expansion and metastasis. Here we show that the plasmin reductase isolated from conditioned medium of fibrosarcoma cells is the glycolytic enzyme phosphoglycerate kinase. Recombinant phosphoglycerate kinase had the same specific activity as the fibrosarcoma-derived protein. Plasma of mice bearing fibrosarcoma tumours contained several-fold more phosphoglycerate kinase, as compared with mice without tumours. Administration of phosphoglycerate kinase to tumour-bearing mice caused an increase in plasma levels of angiostatin, and a decrease in tumour vascularity and rate of tumour growth. Our findings indicate that phosphoglycerate kinase not only functions in glycolysis but is secreted by tumour cells and participates in the angiogenic process as a disulphide reductase.
Angiostatins↗
Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.
Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).
Antibodies↗
Presence of closely spaced protein thiols on the surface of mammalian cells.
It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.
Animals↗
Reduction of von Willebrand factor by endothelial cells.
The haemostatic activity of plasma von Willebrand factor (vWF) is a function of multimer size. Only the large vWF multimers are effective in promoting platelet adhesion to a site of vascular injury. We observed that the conditioned medium of cultured human umbilical vein, human microvascular and bovine aortic endothelial cells contained an activity which reduced the average multimer size of plasma or purified vWF. The average multimer size of vWF produced endogenously by human umbilical vein endothelial cells was similarly reduced following secretion. The reducing activity was ablated by pre-treatment with heat or the thiol blocking agents. iodoacetamide, N-ethylmaleimide or E-64, but not by a range of specific serine-, cysteine-, aspartic-, or metalloproteinase inhibitors. Reduction in vWF multimer size was associated with formation of new thiols in vWF and there was no evidence for additional proteolytic processing of vWF. The reducing activity was associated with a protein with an anionic pi that binds heparin and contains reactive thiol(s). These results suggested that the interchain disulfide bonds that link the vWF homodimers near the N-termini were being reduced by a vWF reductase secreted by endothelial cells. In support of this hypothesis, incubation of vWF with the protein reductants, protein disulfide isomerase and thioredoxin, resulted in formation of new thiols in vWF and reduction in the average multimer size of vWF. These findings may have consequences for control of vWF haemostatic activity.
Animals↗
Dependence on prothrombin for inhibition of activated protein C activity by lupus antibodies.
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Humans↗
Lupus anticoagulants form immune complexes with prothrombin and phospholipid that can augment thrombin production in flow.
Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid binding proteins, including prothrombin. We found that a murine monoclonal antiprothrombin antibody and 7 of 7 LA IgGs tested enhanced binding of prothrombin to 25:75 phosphatidyl serine:phosphatidyl choline vesicles in a concentration-dependent manner. We hypothesized that enhanced binding of prothrombin to phospholipid in the presence of LA IgG might result in increased thrombin production when reactions are performed in flow. Thrombin production by purified prothrombinase components was measured in a phospholipid-coated flow reactor. The flow reactor was incubated with prothrombin, calcium ions, and the IgGs and then perfused with prothrombin, calcium ions, the IgGs, factor Va, and factor Xa. A murine monoclonal antiprothrombin antibody and 4 of 6 LA IgGs from patients with a history of thrombosis increased thrombin production up to 100% over control in the first 15 minutes. In summary, LA IgGs concentrate prothrombin on a phospholipid surface that can augment thrombin production by prothrombinase in flow. These observations suggest that LA might propagate coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall.
Animals↗
An Arg/Ser substitution in the second epidermal growth factor-like module of factor IX introduces an O-linked carbohydrate and markedly impairs activation by factor XIa and factor VIIa/Tissue factor and catalytic efficiency of factor IXa.
Factor IXR94S is a naturally occurring hemophilia B defect, which results from an Arg 94 to Ser mutation in the second epidermal growth factor (EGF)-like module of factor IX. Recombinant factor IXR94S was activated by factor XIa/calcium with an approximately 50-fold reduced rate and by factor VIIa/tissue factor/phospholipid/calcium with an approximately 20-fold reduced rate compared with wild-type factor IX. The apparent molecular mass of the light chain of factor IXaR94S was approximately 6 kD higher than that of plasma or wild-type factor IX, which was not corrected by N-glycosidase F digestion. This result indicated the presence of additional O-linked carbohydrate in the mutant light chain, probably at new Ser 94. The initial rate of activation of factor X by factor IXaR94S in the presence of polylysine was 7% +/- 1% of the initial rate of activation of factor X by plasma factor IXa, and the kc/Km for activation of factor X by factor IXaR94S/factor VIIIa/phospholipid/calcium was 4% +/- 1% of the kc/Km for activation of factor X by plasma factor IXa/factor VIIIa/phospholipid/calcium. The reduced efficiency of activation of factor X by factor IXaR94S in the tenase enzyme complex was due to a 58-fold +/- 12-fold decrease in kcat with little effect on Km. In conclusion, the R94S mutation had introduced an O-linked carbohydrate, which markedly impaired both activation by factor XIa and turnover of factor X in the tenase enzyme complex.
Amino Acid Substitution↗
Microheterogeneity of beta-2 glycoprotein I: implications for binding to anionic phospholipids.
Considerable interest is currently focused on the interactions of beta-2 glycoprotein I (beta2GPI) and anti-phospholipid antibodies with anionic phospholipids in an attempt to understand the association between these antibodies and clinical diseases such as thrombosis. The interactions of beta2GPI and anionic phospholipids have only been characterized partially, and the physiological role of this glycoprotein remains uncertain. In this study we have explored in detail the physical and phospholipid-binding characteristics of a number of beta2GPI preparations. We have found (i) that perchloric acid-purification methods are damaging to beta2GPI during purification, (ii) that the dissociation constants of the various preparations for phosphatidylserine vary between 0. 1-2 microM and are considerably weaker than previously reported, (iii) that considerable differences in affinity of the various beta2GPI preparations for anionic phospholipids are obtained when comparing anionic phospholipids immobilized to a solid-phase versus phospholipid assembled in unilamellar vesicles, (iv) that the integrity of the fifth domain of beta2GPI is important for binding immobilized anionic phospholipid but not especially important in binding vesicular anionic phospholipid, and (v) that beta2GPI preparations with differing isoelectric species content bind anionic phospholipids differently, suggesting that varying glycosylation and/or protein polymorphisms impact upon phospholipid binding. These results highlight the importance of assessing the determinants of the interaction of beta2GPI with anionic phospholipids assembled in unilamellar vesicles.
Anions↗
Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin.
Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin.
Amino Acid Sequence↗
Redox control of exofacial protein thiols/disulfides by protein disulfide isomerase.
Protein disulfide isomerase (PDI) facilitates proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum and is secreted by cells and associates with the cell surface. We examined the consequence of over- or underexpression of PDI in HT1080 fibrosarcoma cells for the redox state of cell-surface protein thiols/disulfides. Overexpression of PDI resulted in 3.6-4. 2-fold enhanced secretion of PDI and 1.5-1.7-fold increase in surface-bound PDI. Antisense-mediated underexpression of PDI caused 38-53% decreased secretion and 10-33% decrease in surface-bound PDI. Using 5,5'-dithio-bis(2-nitrobenzoic acid) to measure surface protein thiols, a 41-50% increase in surface thiols was observed in PDI-overexpressing cells, whereas a 29-33% decrease was observed in underexpressing cells. Surface thiol content was strongly correlated with cellular (r = 0.998) and secreted (r = 0.969) PDI levels. The pattern of exofacial protein thiols was examined by labeling with the membrane-impermeable thiol reactive compound, 3-(N-maleimidylpropionyl)biocytin. Fourteen identifiable proteins on HT1080 cells were labeled with 3-(N-maleimidylpropionyl)biocytin. The intensity of labeling of 11 proteins was increased with overexpression of PDI, whereas the intensity of labeling of 3 of the 11 proteins was clearly decreased with underexpression of PDI. These findings indicated that secreted PDI was controlling the redox state of existing exofacial protein thiols or reactive disulfide bonds.
Base Sequence↗
Evidence for multiple enzyme site involvement in the modulation of thrombin activity by products of prothrombin proteolysis.
Kinetic evidence is presented for the interaction of prothrombin with several distinctive topological regions of the thrombin molecule. Modulations of thrombin catalytic activity on the protein substrates prothrombin and prethrombin 1 are demonstrated that involve the fragment 1 and fragment 2 portions. The inhibitory effects are demonstrably non-competitive. In addition to exhibiting non-competitive inhibition, fragment 2 is capable of enhancing proteolysis by thrombin; and therefore to react with a second region of the enzyme. On the basis of the crystallographic studies of the complex between fragment 2 and thrombin (Arni et al., Biochemistry 32 (1992) 4727), this activating site is proposed to be associated with exosite II. The allosteric switch between procoagulant and anticoagulant activities identified from studies by Di Cera (Dang et al., Proc. Natl. Acad. Sci USA 92 (1995) 5977) could be 'thrown' by a macromolecular effector that is generated during thrombin formation--a plausible mechanism for switching that deserves further investigation.
Allosteric Site↗
Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase.
Thrombospondin-1 is a matrix protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell alphav beta3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the alphav beta3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693-701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that protein disulfide isomerase, which is known to catalyze disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of approximately 2.2 fg of protein disulfide isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified protein disulfide isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the alphav beta3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for protein disulfide isomerase. These results suggest that endothelial cell derived protein disulfide isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.
Animals↗