scholarly journals Functional and binding studies of the roles of prothrombin and beta 2- glycoprotein I in the expression of lupus anticoagulant activity

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 2878-2892 ◽  
Author(s):  
P Permpikul ◽  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Calcium Ions ◽  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Protein A ◽  
Inhibitory Effect ◽  
Factor X ◽  
Glycoprotein I ◽  
Normal Plasma ◽  
Beta 2 ◽  
Factor X Activation

Abstract We present functional and binding data relevant to the reported roles for prothrombin and beta 2-glycoprotein I (beta 2GPI) in the expression of lupus anticoagulant activity. In a purified system containing human prothrombin, Xa, Va, and a rate-limiting concentration of phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles, the preliminary incubation of vesicles with protein A separated IgG preparations from 10 lupus anticoagulant plasmas, calcium, and prothrombin enhanced the inhibitory effect of all IgG preparations upon thrombin generation. Experiments in a purified factor X activation system provided supporting data that a similar preliminary incubation with prothrombin enhanced the inhibitory effect of many of the IgG preparations upon factor X activation. However, we could not obtain unequivocal evidence that prothrombin was an obligatory cofactor for lupus anticoagulant IgG to inhibit procoagulant phospholipid function, because lupus anticoagulant IgG separated by protein A chromatography contained traces of prothrombin. The binding of many IgG preparations to immobilized PS was enhanced by prothrombin when calcium ions were present. beta 2GPI enhanced binding of many of the IgG preparations to immobilized PS both in the presence and absence of calcium, yet beta 2GPI failed to enhance the ability of the IgG preparations to inhibit phospholipid function in purified prothrombin and factor X assays. Moreover, the IgG preparations prolonged the dilute Russell's viper venom time (dRVVT) of beta 2GPI-depleted normal plasma. Nine of 10 IgG preparations bound to prothrombin on Western blots in the absence of calcium and phospholipid, whereas no preparation bound to beta 2GPI. Passage of five citrated lupus anticoagulant plasmas through a prothrombin affinity column in the absence of added calcium and phospholipid removed most of the activity prolonging the dRVVT of normal plasma, and IgG in the pass-through plasma no longer bound to PS in the presence of prothrombin and calcium ions. IgG in prothrombin column eluates had strikingly enhanced specific lupus anticoagulant activity and also specific PS binding activity in the presence of prothrombin and calcium ions. Thus, lupus anticoagulant plasmas were shown to contain IgG binding to prothrombin, in the absence of calcium ions and phospholipid, which could also, in the presence of calcium ions and prothrombin, bind to PS and express lupus anticoagulant activity.

A GENERAL MECHANISM FOR LUPUS ANTICOAGULANTS

1987 ◽  
Author(s):  
V Pengo ◽  
M J Heine ◽  
P Thiagarajan ◽  
s s Shapiro
Keyword(s):  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Protein A ◽  
Coagulation Factors ◽  
Phosphatidyl Serine ◽  
General Mechanism ◽  
Factor X ◽  
Lupus Anticoagulants ◽  
Calcium Dependent ◽  
Anionic Phospholipids

Although- a number of observations have implied that lupus anticoagulants have immunologic specificity towards anionic. phospholipids, thereby prolonging phospholipid-dependent coagulation tests, this assumption has been directly demonstrated in only one patient with a monoclonal IgM paraprotein. We have tested the generality of this hypothesis directly by isolating five IgG lupus anticoagulants from patients with lupus-like syndromes and/or thrombosis. IgG lupus anticoagulant fractions were isolated free of other plasma proteins and free of contaminating phospholipid by adsorption to and elution from cardiolipin-cholesterol-dicetylphosphate liposomes , followed by chromatography on protein A-Sepharose. Cardiolipin liposomes, but not phosphatidylcholine liposomes, were capable of removing all, or nearly all, lupus anticoagulant activity from patient plasma. Anticardiolipin and lupus anticoagulant activity were both present in acidic fractions on isoelectric focusing. F(ab’)2 fragments retained lupus anti coagulant activity and bound to cardiolipin in an ELISA assay. The affinity-purified IgG preparations reacted with cardiolipin, phosphatidyl serine , phosphatidylinositol and phosphatidic acid, but not with phosphatidylcholine or phosphatidyl ethanol amine, and inhibited calcium-dependent binding of prothrombin and of factor X to phosphatidy1serine-coated surfaces. These data demonstrate a general mechanism for the action of lupus anticoagulants: antibodies that have immunologic specificity towards anionic phospholipids, thereby blocking the calcium-mediated binding of vitamin K-dependent coagulation factors to coagulation-active phospholipid surfaces.

scholarly journals Immunological specificity and mechanism of action of IgG lupus anticoagulants

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 69-76 ◽  
Author(s):  
V Pengo ◽  
P Thiagarajan ◽  
SS Shapiro ◽  
MJ Heine
Keyword(s):  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Protein A ◽  
Coagulation Factors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor X ◽  
Lupus Anticoagulants ◽  
Calcium Dependent ◽  
Anionic Phospholipids ◽  
Coated Surfaces

Although observations have implied that lupus anticoagulants have immunologic specificity toward anionic phospholipids, this assumption has been directly demonstrated in only one patient with a monoclonal IgM paraprotein. We tested the generality of this hypothesis directly by isolating five IgG lupus anticoagulants from patients with lupuslike syndromes and/or thrombosis. IgG lupus anticoagulant fractions were isolated free of other plasma proteins and free of contaminating phospholipid by adsorption to and elution from cardiolipin-cholesterol- dicetyl phosphate liposomes, followed by chromatography on protein A- Sepharose. Cardiolipin liposomes, but not phosphatidylcholine liposomes, were capable of removing all, or nearly all, lupus anticoagulant activity from patient plasma. The affinity-purified IgG preparations reacted with cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid, but not with phosphatidylcholine or phosphatidylethanolamine, and inhibited calcium- dependent binding of prothrombin and of factor X to phosphatidylserine- coated and to cardiolipin-coated surfaces. F(ab')2 fragments retained lupus anticoagulant activity and bound to cardiolipin in an enzyme- linked immunosorbent assay (ELISA). Anticardiolipin and lupus anticoagulant activity were both present in acidic fractions on isoelectric focusing. These data strongly suggest that most, if not all, lupus anticoagulants are antibodies that have immunologic specificity towards anionic phospholipids, thereby blocking the calcium- mediated binding of vitamin K-dependent coagulation factors to coagulation-active phospholipid surfaces.

Procoagulant Effect of Anti–β2-Glycoprotein I Antibodies With Lupus Anticoagulant Activity

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3814-3819
Author(s):  
V. Pengo ◽  
T. Brocco ◽  
A. Biasiolo ◽  
P. Rampazzo ◽  
P. Carraro ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Antiphospholipid Antibody ◽  
Antiphospholipid Antibody Syndrome ◽  
Anticoagulant Treatment ◽  
Oral Anticoagulant Treatment ◽  
Glycoprotein I ◽  
Procoagulant Effect

Prothrombin time (PT) is routinely used to monitor oral anticoagulant treatment in patients with the antiphospholipid antibody syndrome (APS). The fact that PT is a phospholipid (PL)-dependent coagulation test raises the possibility that lupus anticoagulant (LA) might interfere with this test, thus complicating the control of anticoagulant treatment. The effect of 6 affinity-purified preparations of anti- (a)β2-glycoprotein I (GPI) antibodies with LA activity on the PT was tested. Instead of prolonging PT as expected, the aβ2-GPI antibodies reduced the PT of both normal plasma and anticoagulated plasma by a mean of 2.4 seconds and 5.6 seconds, respectively. This effect was also observed using other 5 commercially available preparations of thromboplastin. The aβ2-GPI–mediated reduction in PT was dose-dependent and was lost upon removal of β2-GPI. The failure of aβ2-GPI antibodies to express LA activity in PT was found to depend on the fact that calcium ions were added together with PL at the beginning of the assay. In fact, modification of the standard diluted Russell viper venom time (dRVVT) test by adding calcium ions together with PL resulted in a loss of aβ2-GPI anticoagulant activity. The procoagulant effect was not as evident in an assay that used stimulated monocytes as a source of thromboplastin. These results show that aβ2-GPI antibodies exhibit an ‘in vitro’ procoagulant effect in PT and an anticoagulant effect in dRVVT only when the interaction with their antigen and PL occurs in the absence of calcium ions.

Functional Effects of Anticardiolipin Antibodies

Lupus ◽  
1996 ◽  
Vol 5 (5) ◽  
pp. 372-377 ◽  
Author(s):  
EN Harris ◽  
SS Pierangeli
Keyword(s):  
Lupus Anticoagulant ◽  
Protein C ◽  
Inhibitory Effect ◽  
Anticardiolipin Antibodies ◽  
Factor X ◽  
Free System ◽  
Immunoglobulin Preparations ◽  
Coagulation Protein ◽  
Factor X Activation

The ‘lupus anticoagulant’ phenomenon is the best documented functional effect of antiphospholipid (aPL) antibodies, occurring either by inhibition of the prothrombinase and/or Factor X activation reactions. Understanding the mechanism by which aPL antibodies inhibit phospholipid dependent coagulation reactions may yield important clues about their ‘thrombogenic effects’ in vivo. We conducted a series of studies to determine the specificity, diversity, and mechanism by which aPL antibodies inhibit phospholipid dependent reactions. Results showed that purified immunoglobulins with lupus anticoagulant and anti-cardiolipin activities were absorbed by negatively charged phospholipids and both activities were recovered from the phospholipid-antibody precipitate. Purified aPL antibodies inhibited the prothrombinase reaction in a plasma free system in which β2-glycoprotein 1 (β2-GP1) was absent. Affinity purified aPL antibodies had 25–50 times the inhibitory activity of immunoglobulin preparations. The phospholipid binding proteins, β2-GP1 and placental anticoagulant protein I (PAP I), independently inhibited the prothrombinase reaction, and when these proteins were combined with aPL, inhibition of the prothrombinase reaction was additive. Antibodies of syphilis had no inhibitory effect, partially accounted for by lack of specificity for phosphotidylserine (PS). Although aPL antibodies inhibited the protein C activation reaction, there was no correlation of these activities with inhibition of the prothrombinase reaction. Together, these results show that aPL exert their effects by interaction with negatively charged phospholipids, in particular phosphotidylserine, but lack of correlation between inhibition of the prothrombinase and protein C activation reactions, suggests that the nature of the coagulation protein is also important.

scholarly journals Lupus anticoagulant activity of some antiphospholipid antibodies against phospholipid bound beta 2 glycoprotein I.

1993 ◽  
Vol 46 (7) ◽  
pp. 665-667 ◽  
Author(s):  
D M Keeling ◽  
A J Wilson ◽  
I J Mackie ◽  
D A Isenberg ◽  
S J Machin
Keyword(s):  
Antiphospholipid Antibodies ◽  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Glycoprotein I ◽  
Beta 2

scholarly journals Immunological specificity and mechanism of action of IgG lupus anticoagulants

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 69-76 ◽  
Author(s):  
V Pengo ◽  
P Thiagarajan ◽  
SS Shapiro ◽  
MJ Heine
Keyword(s):  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Protein A ◽  
Coagulation Factors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor X ◽  
Lupus Anticoagulants ◽  
Calcium Dependent ◽  
Anionic Phospholipids ◽  
Coated Surfaces

Abstract Although observations have implied that lupus anticoagulants have immunologic specificity toward anionic phospholipids, this assumption has been directly demonstrated in only one patient with a monoclonal IgM paraprotein. We tested the generality of this hypothesis directly by isolating five IgG lupus anticoagulants from patients with lupuslike syndromes and/or thrombosis. IgG lupus anticoagulant fractions were isolated free of other plasma proteins and free of contaminating phospholipid by adsorption to and elution from cardiolipin-cholesterol- dicetyl phosphate liposomes, followed by chromatography on protein A- Sepharose. Cardiolipin liposomes, but not phosphatidylcholine liposomes, were capable of removing all, or nearly all, lupus anticoagulant activity from patient plasma. The affinity-purified IgG preparations reacted with cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid, but not with phosphatidylcholine or phosphatidylethanolamine, and inhibited calcium- dependent binding of prothrombin and of factor X to phosphatidylserine- coated and to cardiolipin-coated surfaces. F(ab')2 fragments retained lupus anticoagulant activity and bound to cardiolipin in an enzyme- linked immunosorbent assay (ELISA). Anticardiolipin and lupus anticoagulant activity were both present in acidic fractions on isoelectric focusing. These data strongly suggest that most, if not all, lupus anticoagulants are antibodies that have immunologic specificity towards anionic phospholipids, thereby blocking the calcium- mediated binding of vitamin K-dependent coagulation factors to coagulation-active phospholipid surfaces.

Procoagulant Effect of Anti–β2-Glycoprotein I Antibodies With Lupus Anticoagulant Activity

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3814-3819 ◽  
Author(s):  
V. Pengo ◽  
T. Brocco ◽  
A. Biasiolo ◽  
P. Rampazzo ◽  
P. Carraro ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Antiphospholipid Antibody ◽  
Antiphospholipid Antibody Syndrome ◽  
Anticoagulant Treatment ◽  
Oral Anticoagulant Treatment ◽  
Glycoprotein I ◽  
Procoagulant Effect

Abstract Prothrombin time (PT) is routinely used to monitor oral anticoagulant treatment in patients with the antiphospholipid antibody syndrome (APS). The fact that PT is a phospholipid (PL)-dependent coagulation test raises the possibility that lupus anticoagulant (LA) might interfere with this test, thus complicating the control of anticoagulant treatment. The effect of 6 affinity-purified preparations of anti- (a)β2-glycoprotein I (GPI) antibodies with LA activity on the PT was tested. Instead of prolonging PT as expected, the aβ2-GPI antibodies reduced the PT of both normal plasma and anticoagulated plasma by a mean of 2.4 seconds and 5.6 seconds, respectively. This effect was also observed using other 5 commercially available preparations of thromboplastin. The aβ2-GPI–mediated reduction in PT was dose-dependent and was lost upon removal of β2-GPI. The failure of aβ2-GPI antibodies to express LA activity in PT was found to depend on the fact that calcium ions were added together with PL at the beginning of the assay. In fact, modification of the standard diluted Russell viper venom time (dRVVT) test by adding calcium ions together with PL resulted in a loss of aβ2-GPI anticoagulant activity. The procoagulant effect was not as evident in an assay that used stimulated monocytes as a source of thromboplastin. These results show that aβ2-GPI antibodies exhibit an ‘in vitro’ procoagulant effect in PT and an anticoagulant effect in dRVVT only when the interaction with their antigen and PL occurs in the absence of calcium ions.

Synchronized Inhibition of the Phospholipid Mediated Autoactivation of Factor XII in Plasma by β2-glycoprotein I and Anti-β2-glycoprotein I

1995 ◽  
Vol 73 (05) ◽  
pp. 798-804 ◽  
Author(s):  
Inger Schousboe ◽  
Margit Søe Rasmussen
Keyword(s):  
Lupus Erythematosus ◽  
Lupus Anticoagulant ◽  
Response Curve ◽  
Inhibitory Effect ◽  
Factor Xii ◽  
High Antibody ◽  
Contact Activation ◽  
Maximal Inhibition ◽  
Lupus Anticoagulants ◽  
Glycoprotein I

SummaryLupus anticoagulants are a group of antibodies commonly found in patients with autoimmune diseases such as systemic lupus erythematosus. Lupus anticoagulants inhibit phospholipid dependent coagulation and may bind to negatively charged phospholipids. Recent studies have suggested an association between anti-β2-glycoprotein I and a lupus anticoagulant, whose activity is frequently dependent on the presence of β2-glycoprotein I. Based on these observations, the effect of anti-β2-glycoprotein I on the autoactivation of factor XII in plasma was investigated. Autoactivation initiated by the presence of negatively charged phospholipids, but not by sulfatide, was strongly inhibited by immunoaffinity purified anti-β2-glycoprotein I. The dose-response curve of anti-β2-gly coprotein I was identical with that of a precipitating antibody, showing no inhibition at low and high antibody dilutions and maximal inhibition at an intermediate dilution. At high antibody concentrations, an increased rate of factor Xlla activation was observed. This increase was of the same magnitude as the decreased rate observed in plasma supplemented with the same amount of β2-glycoprotein I as in the plasma itself. This confirms the inhibitory effect of β2-GP-I on the contact activation and shows that inhibition is effective on the autoactivation of factor XII in plasma. The inhibitory action of β2-glycoprotein I was independent of the inhibition caused by the anti- β2-glycoprotein I/β2-glycoprotein I complex suggesting a synchronized inhibition of factor XII autoactivation by β2-glycoprotein I and anti-β2-gly coprotein I. The inhibition caused by the antibody is suggested to be caused by a reduced availability of negatively charged phospholipids due to the binding of the anti-β2- GP-I/β2-GP-I complex. This complex may be a lupus anticoagulant.

Factor X Activation by washed human platelets

1979 ◽  
Author(s):  
E van Wijk ◽  
L Kahlé ◽  
J ten Cate
Keyword(s):  
Human Factors ◽  
Trypsin Inhibitor ◽  
Calcium Ions ◽  
Factor Xa ◽  
Human Platelets ◽  
Soybean Trypsin Inhibitor ◽  
Thrombin Inhibitor ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Factor X Activation

In a system of washed human platelets, Ca2+and purified human factors X anc II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX alone for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.

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