Acquired hypoprothrombinemia due to non-neutralizing antibodies to prothrombin: mechanism and management

A patient developed bleeding due to an acute acquired specific prothrombin deficiency. Unlike previously described patients, this patient had no evidence of an associated lupus anticoagulant. Prothrombin activity and antigen were decreased concordantly and the patient's plasma did not neutralize the activity of added prothrombin or interfere with its measurement by electroimmunoassay. Nevertheless, immunoelectrophoresis and experiments using 125I-prothrombin revealed a prothrombin-binding antibody. The residual prothrombin in the patient's plasma was in the form of a prothrombin-antibody complex. Administration of adrenal corticosteroids was associated with a rise in prothrombin activity and cessation of bleeding, but circulating prothrombin was still bound to the antibody. This suggests that non- neutralizing antibodies to prothrombin cause plasma prothrombin deficiency because of a rapid clearance of prothrombin-antibody complexes, which is slowed by adrenal corticosteroids. The antibody had a relatively low affinity for prothrombin (Kd 5 to 8 X 10(-7)) and was transient. It is possible, therefore, that the antibody arose not to prothrombin itself, but to an antigen sharing an epitope with prothrombin.     

Inhibition of platelet prothrombinase activity by a lupus anticoagulant

Lupus anticoagulants are spontaneously occurring antibodies with specificity for negatively charged phospholipids. The plasma of a patient with such a polyclonal antibody of IgM type demonstrated low levels of factor VIII coagulant activity (VIII:C) and factors IX, XI and XII when analyzed by biologic clotting assays, whereas in immunochemical assays, normal levels of VIII coagulant antigen and factor IX were obtained. After immunoadsorption of patient plasma with anti-IgM Sepharose, normal biologic activities were demonstrated in clotting assays for VIII:C, factors IX, XI, and XII. The addition of the patient's isolated IgM to normal plasma resulted in grossly abnormal results in these coagulation assays, and a pattern similar to that of the patient's plasma was obtained. The inhibitory effect of the patient's lupus anticoagulant on blood coagulation was demonstrated also in platelet-rich plasma. The results of the clotting assays indicated that the anticoagulant inhibited several of the reactions in the blood coagulation cascade. The availability of purified components made it possible to demonstrate an inhibiting effect on the activation of prothrombin by factor Xa in the presence of isolated platelets, as well as in a system where purified factor V and well defined phospholipid vesicles were substituted for the platelets.     

Anti-prothrombin antibodies and the lupus anticoagulant

The investigators have evaluated the frequency and manifestations of anti-prothrombin antibodies in patients with the lupus anticoagulant. Thirty-one of 42 patients with lupus anticoagulants associated with a variety of underlying conditions (74%) had evidence on crossed immunoelectrophoresis of anti-prothrombin antibodies. Twenty-four of 25 patients with an activated partial thromboplastin time exceeding 50 seconds and 14 of 15 patients with a prothrombin time exceeding control by more than two seconds had demonstrable anti-prothrombin antibodies. Three of the 31 patients with anti-prothrombin antibodies had essentially no measurable plasma prothrombin, a presumed result of accelerated clearance of prothrombin/prothrombin antibody complexes. Each of these patients had bled abnormally. The remaining patients with anti-prothrombin antibodies had neither substantial hypoprothrombinemia nor hemorrhagic manifestations, which confirms the non-neutralizing property of anti-prothrombin antibodies associated with the lupus anticoagulant. Since lupus anticoagulant immunoglobulins are known to react with phospholipids, the high prevalence of antibodies binding prothrombin led us to test the hypothesis of antibody polyreactivity. Adsorption of three lupus anticoagulant plasmas with insolubilized prothrombin markedly diminished evidence of both prothrombin/prothrombin antibody complexes and anticoagulant activity. Eluates of the insolubilized prothrombin contained IgG that not only bound prothrombin but possessed lupus anticoagulant activity.     

An IgG antiprothrombin antibody enhances prothrombin binding to damaged endothelial cells and shortens plasma coagulation times.

OBJECTIVE: To test the hypothesis that some lupus anticoagulants are antiprothrombin antibodies, and that such antibodies enhance prothrombin binding to endothelial cells (EC) and thus promote clotting on the cell surface. METHODS: We generated a monoclonal antiprothrombin antibody (designated IS6) from a patient with primary antiphospholipid syndrome (APS). The antibody was analyzed for its binding properties, lupus anticoagulant activity, and pathophysiologic activity, using an EC-based plasma coagulation assay. RESULTS: IS6 is the first patient-derived monoclonal IgG antiprothrombin antibody. It bound to prothrombin with low affinity, reacted with 3 phospholipids (cardiolipin, phosphatidylethanolamine, and phosphatidylserine), and showed lupus anticoagulant activity. Moreover, IS6 enhanced the binding of prothrombin to damaged EC and shortened the EC-based plasma coagulation times. CONCLUSION: These findings suggest that IS6 may promote coagulation in areas of damaged EC in the host, and thus contribute to thrombosis in patients with APS.     

Prothrombin Houston: a dysprothrombin identifiable by crossed immunoelectrofocusing and abnormal Echis carinatus venom activation

A 72-yr-old male with a lifelong history of easy bruisability and posttraumatic bleeding had a prolonged prothrombin time and activated partial thromboplastin time. His plasma Stypven, Taipan, and Echis carinatus venom clotting times were prolonged. The presence of a dysprothrombin was confirmed by the discrepancy between plasma prothrombin coagulant activity and prothrombin antigen levels. His plasma prothrombin was capable of being completely absorbed onto and then eluted from barium sulfate. Crossed immunoelectrophoresis of his plasma prothrombin, and normal plasma prothrombin, into agarose containing rabbit anti-human factor II antibody were similar. Crossed immunoelectrofocusing, a procedure combining isoelectric focusing in disc gels with electroimmunoassay in the second dimension, demonstrated that the patient's prothrombin antigen was more basic than normal. The eluate from barium sulfate absorbtion of patient plasma, when reacted with Echis carinatus venom (which directly cleaves prothrombin to thrombin) clotted purified fibrinogen at a rate slower than normal plasma eluate. SDS-slab gel electrophoresis revealed that the prothrombin present in the patient's eluate was cleaved by Echis carinatus venom. These studies suggest that the coagulopathy of prothrombin Houston results from the generation of a dysfunctional thrombin.     

Diagnosis of lupus anticoagulant in the lupus anticoagulant-hypoprothrombinemia syndrome: report of two cases and review of the literature

We report a severe hemorrhagic disorder in two pediatric patients with lupus anticoagulant (LA) associated to acquired factor II (prothrombin) deficiency. In both patients, hemorrhagic symptoms resolved after corticosteroid therapy. Serial coagulation studies showed that Staclot LA assay was more sensitive than DVVconfirm and Staclot PNP tests to confirm the presence of LA when associated with severe factor II deficiency. Both patients had non-neutralizing anti-prothrombin antibodies and their titers inversely correlated with factor II activity (r = -1.0, P < 0.0001). Associated findings in these patients included positive immunologic tests for systemic lupus erythematosus, a positive anti-cardiolipin antibody, and anti-beta(2) GPI antibodies in one case. Our findings point out the difficulty in diagnosing LA associated with acquired factor II deficiency and suggest that, in confirmation of its phospholipid dependency, the inclusion of a source of normal human plasma in the test sequence to correct for any factor deficiency and a confirmatory step utilizing hexagonal (II) phase phospholipids may be crucial to the diagnosis of LA in some patients with LA-hypoprothrombinemia syndrome.     

Radioimmunoassay of human prothrombin—The quantitation of plasma factor II antigen

Human factor II (prothrombin) was isolated by chromatographic techniques and showed a single band on electrophoresis. ¹²⁵I-tagged factor II was prepared by standard chloramine-T oxidation and sodium metabisulfite reduction. The radioimmunoassay (RIA) method used was a specific double-antibody radioimmunoassay similar to that described for the pituitary trophic hormones. The method showed high precision and sensitivity. A dose-response curve was generated by adding known amounts of normal human plasma to human barium sulfate-absorbed oxalated plasma. A straight-line relationship existed between 15 to 140% coagulant activity and 7 to 50 μg/ml antigen content. Plasma factor II antigen in 20 normals was 86.1 ± 8.4 μg/ml. Eight patients with induced vitamin K deficiency revealed normal antigen levels and reduced coagulant activity, whereas three patients with severe hepatocellular disease were found to have both antigen and coagulant activity reduced. Although coagulant activity decreased with prolonged storage, it was found that antigen content did not change with up to three months' storage at ? 20°C. The use of the RIA for measuring prothrombin protein in conjunction with coagulation factor assays may have clinical application in studying the mechanism of action of vitamin K, the developmental aspects of factor II production and activation, and the understanding of acquired coagulopathies.     

Complexes of anti-prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces

We investigated the mechanism by which anti-prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity-purified anti-prothrombin antibodies from LAC-positive plasma samples (alpha-FII-LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified alpha-FII-LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when alpha-FII-LAC+ were added to prothrombin-deficient plasma or when purified anti-prothrombin antibodies from LAC-negative plasma samples (alpha-FII-LAC-) were added. When a combination of prothrombin and alpha-FII-LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The alpha-FII-LAC+ alone or a combination of prothrombin and alpha-FII-LAC- did not show inhibition. Ellipsometry studies showed that, in the presence of alpha-FII-LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with alpha-FII-LAC-. Our results show that complexes of prothrombin and anti-prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti-beta2-glycoprotein I antibodies.     

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

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.     

Direct method for detection and characterization of cell surface receptors for insulin by means of 125I-labeled autoantibodies against the insulin receptor.

Autoantibodies directed against the cell surface receptors for insulin are found in some patients with extreme insulin resistance. These antibodies specifically inhibit the binding of insulin to its receptor. A purified IgG fraction from one patient's plasma was labeled with 125I. The 125I-labeled antireceptor antibody, which initially represented about 0.3% of the total 125I-IgG, was enriched by selective adsorption and subsequent elution from cells rich in insulin receptors. The 125I-antireceptor antibody bound to cells and the binding was inhibited by whole plasma and purified IgG from this patient, as well as whole plasma from another patient with autoantibodies to the insulin receptor. Insulins that differed 300-fold in biological potency and affinity inhibited binding of 125I-antireceptor antibody in direct proportion to their ability to bind to the insulin receptor. The binding of 125I-antireceptor antibody was closely correlated with the binding of 125I-insulin over a wide range of receptor concentrations on different cell types. Experimentally induced reduction of the insulin receptor concentration was associated with parallel decreases in the binding of 125I-antireceptor antibody and 125I-insulin. The preparation of 125I-antireceptor antibody with a high specific activity by cytoadsorption and elution has provided a sensitive method for the detection of receptors and autoantibodies to cell surface components.     

Antiphospholipid antibody: functional specificity for inhibition of prothrombin activation by the prothrombinase complex

The antiphospholipid syndrome (APS) is associated with production of autoantibodies with lupus anticoagulant (LA) activity. These antibodies cause prolongation of in vitro clotting tests by inhibition of the conversion of prothrombin to thrombin in the presence of anionic phospholipid (PL). The extent to which this inhibition reflects antibody binding to, or functional inhibition of, phospholipids alone, prothrombin alone, or a prothrombin–phospholipid complex is pertinent to our understanding of the pathophysiology of this syndrome. Immunoglobulin fractions (IgG) from 18 patients with LA activity were tested for inhibitory activity against prothrombin activation by either factor Xa, in a purified prothrombinase system, or by purified fractions of snake venoms ( E. carinatus , E. multisquamatus ) which cleave prothrombin at the same initial site as the prothrombinase complex but do not require anionic phospholipid as a cofactor. Parallel testing of the same IgG samples for prothrombin binding by immunoassay was performed. Although all IgG samples inhibited the prothrombinase reaction, only three exhibited any inhibition of venom protease prothrombin activation in either the presence or absence of PL. Only one sample exhibited prothrombin binding by Western blot. These results suggest that lupus anticoagulant antibodies are heterogenous and that many, if not most, of the autoantibody populations responsible for LA activity impair prothrombin activation by interaction either with phospholipid alone or with a restricted range of prothrombin–phospholipid epitopes expressed by prothrombin only as part of the intact prothrombin–prothrombinase complex.     

Factor XI antigen and activity in human platelets

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.     

Understanding the antiphospholipid syndrome and its treatment

The antiphospholipid syndrome (APS) is a clinicopathologic disorder characterized by the persistent presence of anticardiolipin antibodies, lupus anticoagulants, or both in the plasma of patients with arterial or venous thrombosis or obstetric complications. The antiphospholipid antibodies involved in this syndrome have a relatively weak affinity for phospholipid-binding proteins such as b(2) glycoprotein I and prothrombin. In certain conditions they form bivalent antibody-protein complexes that bind with a relatively high affinity to negatively charged phospholipid surfaces. Evidence is accumulating for a pathogenetic role of antiphospholipid antibodies via interference with surface-mediated anticoagulant and procoagulant processes. The syndrome is characterized by the recurrence of thrombotic and obstetric complications. Retrospective studies have suggested that patients with APS and thrombosis need a high-intensity anticoagulant treatment. A few small prospective studies support treatment with a targeted INR of 2 to 3.     

Disturbed platelet aggregation to collagen associated with an antibody against an 85- to 90-Kd platelet glycoprotein in a patient with prolonged bleeding time.

We studied a 5-year-old girl presenting with a markedly prolonged bleeding time. Her platelets were refractory to collagen stimulation, but the response to other agonists was normal. There were no coagulation abnormalities as measured by standard tests. Two-dimensional electrophoresis showed no abnormalities of the patient's platelet membrane glycoproteins. When the patient's plasma or purified plasma IgG was mixed with normal platelets, collagen-induced platelet aggregation was blocked. Western blotting showed the presence of an antibody in the patient's plasma directed against a protein of molecular weight 85 to 90 Kd under both reducing and nonreducing conditions. This protein comigrated with glycoprotein (GP) IV immunoprecipitated by OKM5 from 125I-labeled platelets. Immunoprecipitation of 125I-labeled normal platelet glycoproteins with the patient's IgGs also yielded an 85- to 90-Kd protein that migrated on the diagonal following nonreduced/reduced two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite similarities in electrophoretic behavior, the antigen was not demonstrated to be GPIV, since purified GPIV was not recognized by the antibody.     

Circulating inhibitors of blood coagulation associated with procainamide-induced lupus erythematosus

We studied a patient being treated with procainamide in whom we observed a high antinuclear antibody titer and prolonged activated partial thromboplastin (PTT), prothrombin (PT), and Stypven times (ST). Serum antibody concentrations against single-stranded DNA were elevated while those against native DNA were not elevated, suggesting the procainamide-induced lupus syndrome. Dilution of the patient's plasma with normal plasma failed to correct the PTT and PT, indicating the presence of an inhibitor(s) to blood coagulation. The anticoagulant activity was associated with the IgG fraction of the patient's serum. Addition of purified or partially purified human factors IX, X, VIII, VII, XIa, prekallikrein, high molecular weight kininogen, or phospholipids to the patient's plasma failed to correct the PTT, PT, or ST; however, purified human factor XII and prothrombin corrected the PTT and ST, respectively. These results indicate that production of antibodies directed against antigenic determinants on coagulation proteins can be a manifestation of procainamide-induced lupus erythematosus.     

ANTI-TSH ANTIBODY WITH HIGH SPECIFICITY TO HUMAN TSH IN SERA FROM A PATIENT WITH GRAVES'' DISEASE: ITS ISOLATION FROM, AND INTERACTION WITH, TSH RECEPTOR ANTIBODIES

A patient with thyrotoxic Graves' disease had an apparent measurable level of serum TSH (2.5 microU/ml) by double-antibody radioimmunoassay (RIA). The serum IgG bound with both [125I]human(h)TSH and [125I]bovine(b)TSH. The [125I]hTSH binding was more effectively displaced by human than bovine TSH, whereas [125I]bTSH binding was displaced exclusively by bTSH. Scatchard analyses revealed that [125I]hTSH binding showed two components, whereas [125I]bTSH binding had only one component. Serum TSH determined by RIA became undetectable 21 months after antithyroid drug treatment with a parallel decrease of [125I]hTSH binding IgG activity. Four thyrotrophin binding inhibitory immunoglobulins (TBII) from other patients did not interfere with the binding of the patient's serum to [125I]h- or bTSH. Furthermore, the in-vitro thyroid stimulating activities of three thyroid stimulating antibodies (TSAb) were not affected by the addition of this patient's IgG. On the other hand, this patient's Ig (3 mg/ml) abolished the in-vitro thyroid stimulation by bTSH (100 microU/ml), but did not affect that by hTSH (100 microU/ml). The anti-hTSH antibody, TSH receptor antibody and anti-bTSH antibody in the serum, which contains TSAb as well as anti-TSH antibodies, could be partially purified by hTSH-agarose and subsequently by guinea pig fat cell membrane affinity absorptions. However, the anti-hTSH antibody fraction obtained had both hTSH binding activity and thyroid stimulating activity, and this fraction did not show any inhibitory effect on the in-vitro thyroid stimulation of autologous TSH receptor antibody or hTSH. The possible significance of anti-TSH antibodies is discussed.     

Transient hemorrhagic diathesis associated with an inhibitor of prothrombin with lupus anticoagulant in a 1½-year-old girl: Report of a case and review of the literature

Acquired inhibitors of coagulation causing bleeding manifestations are rare in children, particularly without an associated underlying disorder such as autoimmune disease. We describe an otherwise healthy 1½-year-old girl who had extensive spontaneous bruising and prolonged bleeding from venipuncture sites. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged, with evidence of an immediate-acting inhibitor. Thrombin clotting time, fibrinogen, and platelets were normal. Biologic assay of factors II, V, VII, and X were all low, with increasing values at higher dilutions. However, by immunoassay and/or chromogenic assays, only factor II was reduced. An antibody which failed to neutralize prothrombin activity in vitro was detected against radiolabeled prothrombin. Coagulation studies normalized in parallel with clinical recovery and disappearance of the antibody. This case demonstrates acute hypoprothrombinemia-lupus anticoagulant syndrome as a rare presentation of bleeding diathesis in a healthy young child. © 1996 Wiley-Liss, Inc.     

Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor

A murine monoclonal antibody directed at or near a platelet membrane receptor for the von Willebrand factor was produced by the hybridoma technique. Purified F(ab')2 fragments and/or intact antibody completely blocked the agglutination of platelets induced by both ristocetin and bovine von Willebrand factor and the binding of von Willebrand factor antigen to platelets. The antibody also decreased platelet retention, prevented the reduction in platelet electrophoretic mobility caused by bovine von Willebrand factor, and decreased the serum prothrombin time. Radiolabeled F(ab')2 fragments bound to or approximately 2.5 X 10(4) sites on normal platelets with high affinity (KD or approximately 1.5 X 10(-8) M); there was no binding to platelets from 2 patients with the Bernard-Soulier syndrome. Immunoprecipitation and affinity chromatography studies indicated that the antibody binds to glycoprotein lb at a site contained on the externally oriented portion of the GPIb alpha chain (glycocalicin). An unidentified mol wt or approximately 20,000 molecule labeled by periodate/NaB3H4 coprecipitated and copurified with GPIb.     

Inhibition of APC anticoagulant activity on oxidized phospholipid by anti-{beta}2-glycoprotein I monoclonal antibodies

Activated protein C (APC) anticoagulant activity and the ability to be inhibited by auto-antibodies associated with thrombosis are strongly augmented by the presence of phosphatidylethanolamine (PE) and phospholipid oxidation. beta(2)-glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies present in patients with the antiphospholipid syndrome. We therefore investigated whether anti-beta(2)-GPI monoclonal antibodies (mAbs) could inhibit APC with similar membrane specificity. Five mouse mAbs that reacted with different epitopes on beta(2)-GPI were examined. Each inhibited the PE-, phospholipid oxidation-dependent enhancement of APC anticoagulant activity and required antibody divalency. A chimeric APC that retains anticoagulant activity but is relatively unaffected by protein S, PE, or oxidation was not inhibited by the antibodies. In purified systems, anti-beta(2)-GPI mAb inhibition of factor Va inactivation was greater in the presence of protein S and required beta(2)-GPI. Surprisingly, although the mAbs did increase beta(2)-GPI affinity for membranes, PE and oxidation had little influence on the affinity of the beta(2)-GPI antibody complex for the membrane vesicles. We conclude that antibodies to beta(2)-GPI inhibit APC function specifically and contribute to a hypercoaguable state by disrupting specific protein-protein interactions induced by oxidation of PE-containing membranes.     

Isolation and characterization of an antifactor V antibody causing activated protein C resistance from a patient with severe thrombotic manifestations

A 44-year-old woman with a history of severe thrombotic manifestations presented with a markedly reduced activated protein C-sensitivity ratio (APC-SR). DNA sequencing of and around the regions encoding the APC cleavage sites in the factor Va molecule excluded the presence of the factor VLeiden mutation and of other known genetic mutations. No antiphospholipid antibodies were present in the patient's plasma and both prothrombin time and activated partial thromboplastin time were normal. The total immunoglobulin fraction was isolated from the patient's plasma and found to induce severe APC resistance when added to normal plasma and to factor V-deficient plasma supplemented with increasing concentrations of factor V. Immunoblotting and immunoprecipitation experiments with the total immunoglobulin fraction purified from the patient's plasma demonstrated that the antibody recognizes factor V, is polyclonal, and has conformational epitopes on the entire factor V molecule (heavy and light chains, and B region). Thus, the immunoglobulin fraction interferes with the anticoagulant pathway involving factor V. The inhibitor was isolated by sequential affinity chromatography on protein G-Sepharose and factor V-Sepharose. The isolated immunoglobulin fraction inhibited factor Va inactivation by APC because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the cofactor. The isolated immunoglobulin fraction was also found to inhibit the cofactor effect of factor V for the inactivation of factor VIII by the APC/protein S complex. Our data provide for the first time the demonstration of an antifactor V antibody not related to the presence of antiphospholipid antibodies, which is responsible for thrombotic rather than hemorrhagic symptoms.