The phospholipid-binding plasma protein beta2-glycoprotein I (beta2-GPI) is the primary antigen r... more The phospholipid-binding plasma protein beta2-glycoprotein I (beta2-GPI) is the primary antigen recognized by the circulating autoantibodies in patients with the "anti-phospholipid syndrome" (APS). Although heparin is routinely used in the treatment and prophylaxis of APS patients, the primary heparin-binding site within beta2-GPI has not been identified. More importantly, how heparin exerts its beneficial effects in vivo in APS patients has not been deduced at the molecular level. Using an expression/site-directed mutagenesis approach, we now show that the positively charged site that resides in the first domain of beta2-GPI is not the primary heparin-binding site. Rather it is the second positively charged site located within the fifth domain of the protein that also binds to phospholipids. Lys(284), Lys(286), and Lys(287) in this domain are essential for the interaction of beta2-GPI with heparin. These data indicate that beta2-GPI binds to heparin in a relatively specific manner even though the affinity for the interaction is rather low. Lys(317) resides in the center of the high affinity phospholipid-binding site. Surprisingly, heparin at concentrations that can be achieved in vivo during anticoagulation therapy greatly enhances the plasmin-mediated cleavage of the Lys(317)-Thr(318) site in beta2-GPI. Because the cleaved form cannot bind to phospholipids effectively, the combined actions of heparin and plasmin result in a diminished ability of beta2-GPI to recognize phospholipids. This, in turn, decreases the prothrombotic activity of the endogenous circulating anti-beta2-GPI antibodies in the patients. Thus, heparin exerts its beneficial effects in APS patients by at least two distinct mechanisms.
Angiotensinogen exists in two distinct redox forms in plasma, the oxidized sulfhydryl-bridge form... more Angiotensinogen exists in two distinct redox forms in plasma, the oxidized sulfhydryl-bridge form and the reduced, unbridged, free thiol form. The oxidized form of angiotensinogen compared to the free thiol form preferentially interacts with renin resulting in increased generation of angiotensin. The predictive potential of the ratio of free-thiol to oxidized angiotensinogen in the plasma for pre-eclampsia was first suggested by the Read group in ref 10. We propose an improved method for determining the ratio and validate the method in a larger cohort of pregnant women. Plasma samples from 115 individuals with pre-eclampsia and from 55 healthy pregnant control subjects were collected sequentially over a 2 year period. Using two distinct enzyme-linked immunosorbent assays (ELISAs) the plasma levels of total and free thiol angiotensinogen were quantified. The oxidized angiotensinogen plasma level is derived by subtracting the level of free thiol, reduced angiotensinogen from the total angiotensinogen levels in the plasma. The relative proportion of free thiol angiotensinogen, expressed as a percentage of that observed with an in-house standard, is significantly decreased in pre-eclamptic patients (70.85% ± 29.49%) (mean ± SD) as compared to healthy pregnant controls (92.98 ± 24.93%) (mean ± SD) p ≤ 0.0001. The levels of total angiotensinogen did not differ between the two groups. Patients with pre-eclampsia had substantially lower levels of free thiol angiotensinogen compared to healthy pregnant controls, whilst maintaining similar total angiotensinogen levels in the plasma. Hence, elevated levels of plasma oxidized angiotensinogen may be a contributing factor to hypertension in the setting of pre-eclampsia.
Transmembrane tryptase (TMT)/tryptase is a mem- brane-bound serine protease stored in the secreto... more Transmembrane tryptase (TMT)/tryptase is a mem- brane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranu- late. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only
The antiphospholipid syndrome (APS) is an important cause of acquired thrombo- philia. It is char... more The antiphospholipid syndrome (APS) is an important cause of acquired thrombo- philia. It is characterized by the core clinical manifestations of thrombosis, ei- ther venous or arterial, and in women it can also be associated with recurrent fetal loss. The detection of persistently elevated levels of antiphospholipid anti- bodies (aPL Abs) is a requisite laboratory feature for the diagnosis to be made. The dominant antigenic targets in APS are beta 2-glycoprotein I (2-GPI) and pro- thrombin. There is an accumulating body of experimental evidence that suggests that specific subgroups of aPL Abs may directly contribute to disease pathogene- sis. This review critically examines the experimental evidence underlying the various propositions made to explain how these antibodies may predispose to dis- ease in humans. Furthermore, it also ex- amines the evidence relating to the immu- nologic mechanisms that may contribute to the breakage of peripheral tolerance in this disorder. Delineating the strengths and limitations of the experimental evi- dence accumulated thus far will hopefully stimulate further experimentation toward achieving the ultimate goal of precisely defining the dominant pathogenic mecha- nisms operational in APS. This may pave the way for the development of improved therapies. (Blood. 2007;109:422-430)
The aim of the present study was to evaluate the urea resistance and binding characteristics of a... more The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-beta 2-glycoprotein I (anti-beta 2GPI) antibodies using standard anticardiolipin (aCL) and anti-beta 2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-beta 2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-beta 2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D(o)) corresponding to the same optical density was defined as residual activity (RA = 100 D/D(o)). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; ...
Age-related macular degeneration (AMD) affects the region of the retina responsible for high reso... more Age-related macular degeneration (AMD) affects the region of the retina responsible for high resolution vision. It is a major cause of blindness in the ageing population. This is the first study to examine the association of redox modified, cysteine based, post-translational forms of beta 2-glycoprotein I (β2GPI) in the plasma of individuals with early and late stages of patients with AMD compared to controls. Exploration is also undertaken to assess whether the free thiol form of β2GPI versus the oxidized disulfide form have distinct functional properties in the setting of hydrogen peroxide mediated cell death of an immortalized human retinal pigment epithelium (RPE) cell line. We demonstrate β2GPI in the retina and choroid of patients with AMD. Free thiol β2GPI is shown to protect the immortalized human RPE cell line against H2O2 induced cell death, whereas the oxidized form of β2GPI and free thiol bovine serum albumin were not protective. Free thiol β2GPI levels were significantl...
β2-glycoprotein I (β2GPI) is the major autoantigen in the antiphospholipid syndrome. The central ... more β2-glycoprotein I (β2GPI) is the major autoantigen in the antiphospholipid syndrome. The central importance of understanding β2GPI physiology from the perspective of the rheumatologist is that it forms the foundation for understanding the pathophysiology underlying autoantibody generation, and the diverse mechanisms by which anti-β2GPI antibodies in complex with β2GPI may predispose an individual to the antiphospholipid syndrome clinical phenotype. This review examines some of the latest novel findings in this area.
The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of lig... more The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). Beta2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, beta2GPI retains binding to FXI but not to phospholipids. Native beta2GPI, but not cleaved beta2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on beta2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of beta2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves beta2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of beta2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhib...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2002
Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the... more Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in beta(2)GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole beta(2)GPI containing single point mutations in the first domain. The binding to beta(2)GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40-43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-beta(2)GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms th...
Dapsone hypersensitivity syndrome is an idiosyncratic reaction to this drug and can present with ... more Dapsone hypersensitivity syndrome is an idiosyncratic reaction to this drug and can present with different clinical manifestations of varying severity. We describe a patient with disseminated intravascular coagulation (DIC) as an adverse reaction to dapsone. To the best of our knowledge, this is the first time it has been described in the literature. She presented with fever, rash and abdominal pain; she also had marked eosinophilia and features suggestive of oxidative haemolysis. Her course was complicated by DIC, splenic infarction and gastrointestinal bleeding. Extensive investigations did not reveal any alternative aetiology. She was initially treated with supportive measures and folic acid; steroids were administered later, following clinical deterioration. There was gradual improvement and the steroids were tapered. The patient recovered fully and remains well; her underlying chronic dermatologic condition is under satisfactory control with other medications.
Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restric... more Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restricted protease of unknown function that is retained on the outer leaflet of the plasma membrane when MCs are activated. We determined the nucleotide sequences of the Prss31 gene in different mouse strains and then used a Cre/loxP homologous recombination approach to create a novel Prss31(-/-) C57BL/6 mouse line. The resulting animals exhibited no obvious developmental abnormality, contained normal numbers of granulated MCs in their tissues, and did not compensate for their loss of the membrane tryptase by increasing their expression of other granule proteases. When Prss31-null MCs were activated with a calcium ionophore or by their high affinity IgE receptors, they degranulated in a pattern similar to that of WT MCs. Prss31-null mice had increased baseline airway reactivity to methacholine but markedly reduced experimental chronic obstructive pulmonary disease and colitis, thereby indicati...
The phospholipid-binding plasma protein beta2-glycoprotein I (beta2-GPI) is the primary antigen r... more The phospholipid-binding plasma protein beta2-glycoprotein I (beta2-GPI) is the primary antigen recognized by the circulating autoantibodies in patients with the "anti-phospholipid syndrome" (APS). Although heparin is routinely used in the treatment and prophylaxis of APS patients, the primary heparin-binding site within beta2-GPI has not been identified. More importantly, how heparin exerts its beneficial effects in vivo in APS patients has not been deduced at the molecular level. Using an expression/site-directed mutagenesis approach, we now show that the positively charged site that resides in the first domain of beta2-GPI is not the primary heparin-binding site. Rather it is the second positively charged site located within the fifth domain of the protein that also binds to phospholipids. Lys(284), Lys(286), and Lys(287) in this domain are essential for the interaction of beta2-GPI with heparin. These data indicate that beta2-GPI binds to heparin in a relatively specific manner even though the affinity for the interaction is rather low. Lys(317) resides in the center of the high affinity phospholipid-binding site. Surprisingly, heparin at concentrations that can be achieved in vivo during anticoagulation therapy greatly enhances the plasmin-mediated cleavage of the Lys(317)-Thr(318) site in beta2-GPI. Because the cleaved form cannot bind to phospholipids effectively, the combined actions of heparin and plasmin result in a diminished ability of beta2-GPI to recognize phospholipids. This, in turn, decreases the prothrombotic activity of the endogenous circulating anti-beta2-GPI antibodies in the patients. Thus, heparin exerts its beneficial effects in APS patients by at least two distinct mechanisms.
Angiotensinogen exists in two distinct redox forms in plasma, the oxidized sulfhydryl-bridge form... more Angiotensinogen exists in two distinct redox forms in plasma, the oxidized sulfhydryl-bridge form and the reduced, unbridged, free thiol form. The oxidized form of angiotensinogen compared to the free thiol form preferentially interacts with renin resulting in increased generation of angiotensin. The predictive potential of the ratio of free-thiol to oxidized angiotensinogen in the plasma for pre-eclampsia was first suggested by the Read group in ref 10. We propose an improved method for determining the ratio and validate the method in a larger cohort of pregnant women. Plasma samples from 115 individuals with pre-eclampsia and from 55 healthy pregnant control subjects were collected sequentially over a 2 year period. Using two distinct enzyme-linked immunosorbent assays (ELISAs) the plasma levels of total and free thiol angiotensinogen were quantified. The oxidized angiotensinogen plasma level is derived by subtracting the level of free thiol, reduced angiotensinogen from the total angiotensinogen levels in the plasma. The relative proportion of free thiol angiotensinogen, expressed as a percentage of that observed with an in-house standard, is significantly decreased in pre-eclamptic patients (70.85% ± 29.49%) (mean ± SD) as compared to healthy pregnant controls (92.98 ± 24.93%) (mean ± SD) p ≤ 0.0001. The levels of total angiotensinogen did not differ between the two groups. Patients with pre-eclampsia had substantially lower levels of free thiol angiotensinogen compared to healthy pregnant controls, whilst maintaining similar total angiotensinogen levels in the plasma. Hence, elevated levels of plasma oxidized angiotensinogen may be a contributing factor to hypertension in the setting of pre-eclampsia.
Transmembrane tryptase (TMT)/tryptase is a mem- brane-bound serine protease stored in the secreto... more Transmembrane tryptase (TMT)/tryptase is a mem- brane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranu- late. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only
The antiphospholipid syndrome (APS) is an important cause of acquired thrombo- philia. It is char... more The antiphospholipid syndrome (APS) is an important cause of acquired thrombo- philia. It is characterized by the core clinical manifestations of thrombosis, ei- ther venous or arterial, and in women it can also be associated with recurrent fetal loss. The detection of persistently elevated levels of antiphospholipid anti- bodies (aPL Abs) is a requisite laboratory feature for the diagnosis to be made. The dominant antigenic targets in APS are beta 2-glycoprotein I (2-GPI) and pro- thrombin. There is an accumulating body of experimental evidence that suggests that specific subgroups of aPL Abs may directly contribute to disease pathogene- sis. This review critically examines the experimental evidence underlying the various propositions made to explain how these antibodies may predispose to dis- ease in humans. Furthermore, it also ex- amines the evidence relating to the immu- nologic mechanisms that may contribute to the breakage of peripheral tolerance in this disorder. Delineating the strengths and limitations of the experimental evi- dence accumulated thus far will hopefully stimulate further experimentation toward achieving the ultimate goal of precisely defining the dominant pathogenic mecha- nisms operational in APS. This may pave the way for the development of improved therapies. (Blood. 2007;109:422-430)
The aim of the present study was to evaluate the urea resistance and binding characteristics of a... more The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-beta 2-glycoprotein I (anti-beta 2GPI) antibodies using standard anticardiolipin (aCL) and anti-beta 2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-beta 2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-beta 2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D(o)) corresponding to the same optical density was defined as residual activity (RA = 100 D/D(o)). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; ...
Age-related macular degeneration (AMD) affects the region of the retina responsible for high reso... more Age-related macular degeneration (AMD) affects the region of the retina responsible for high resolution vision. It is a major cause of blindness in the ageing population. This is the first study to examine the association of redox modified, cysteine based, post-translational forms of beta 2-glycoprotein I (β2GPI) in the plasma of individuals with early and late stages of patients with AMD compared to controls. Exploration is also undertaken to assess whether the free thiol form of β2GPI versus the oxidized disulfide form have distinct functional properties in the setting of hydrogen peroxide mediated cell death of an immortalized human retinal pigment epithelium (RPE) cell line. We demonstrate β2GPI in the retina and choroid of patients with AMD. Free thiol β2GPI is shown to protect the immortalized human RPE cell line against H2O2 induced cell death, whereas the oxidized form of β2GPI and free thiol bovine serum albumin were not protective. Free thiol β2GPI levels were significantl...
β2-glycoprotein I (β2GPI) is the major autoantigen in the antiphospholipid syndrome. The central ... more β2-glycoprotein I (β2GPI) is the major autoantigen in the antiphospholipid syndrome. The central importance of understanding β2GPI physiology from the perspective of the rheumatologist is that it forms the foundation for understanding the pathophysiology underlying autoantibody generation, and the diverse mechanisms by which anti-β2GPI antibodies in complex with β2GPI may predispose an individual to the antiphospholipid syndrome clinical phenotype. This review examines some of the latest novel findings in this area.
The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of lig... more The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). Beta2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, beta2GPI retains binding to FXI but not to phospholipids. Native beta2GPI, but not cleaved beta2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on beta2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of beta2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves beta2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of beta2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhib...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2002
Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the... more Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in beta(2)GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole beta(2)GPI containing single point mutations in the first domain. The binding to beta(2)GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40-43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-beta(2)GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms th...
Dapsone hypersensitivity syndrome is an idiosyncratic reaction to this drug and can present with ... more Dapsone hypersensitivity syndrome is an idiosyncratic reaction to this drug and can present with different clinical manifestations of varying severity. We describe a patient with disseminated intravascular coagulation (DIC) as an adverse reaction to dapsone. To the best of our knowledge, this is the first time it has been described in the literature. She presented with fever, rash and abdominal pain; she also had marked eosinophilia and features suggestive of oxidative haemolysis. Her course was complicated by DIC, splenic infarction and gastrointestinal bleeding. Extensive investigations did not reveal any alternative aetiology. She was initially treated with supportive measures and folic acid; steroids were administered later, following clinical deterioration. There was gradual improvement and the steroids were tapered. The patient recovered fully and remains well; her underlying chronic dermatologic condition is under satisfactory control with other medications.
Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restric... more Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restricted protease of unknown function that is retained on the outer leaflet of the plasma membrane when MCs are activated. We determined the nucleotide sequences of the Prss31 gene in different mouse strains and then used a Cre/loxP homologous recombination approach to create a novel Prss31(-/-) C57BL/6 mouse line. The resulting animals exhibited no obvious developmental abnormality, contained normal numbers of granulated MCs in their tissues, and did not compensate for their loss of the membrane tryptase by increasing their expression of other granule proteases. When Prss31-null MCs were activated with a calcium ionophore or by their high affinity IgE receptors, they degranulated in a pattern similar to that of WT MCs. Prss31-null mice had increased baseline airway reactivity to methacholine but markedly reduced experimental chronic obstructive pulmonary disease and colitis, thereby indicati...
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Papers by Steven Krilis