Dedicated removal of immunoglobulin (Ig)A, IgM, and Factor (F)XI/activated FXI from human plasma IgG.

BACKGROUND: Adverse events can be associated with treating critically ill patients with immunoglobulin (Ig)G. Some adverse events are due to contaminants like IgA and activated Factor (F)XI. Therefore, new purification strategies are needed for dedicated removal of these contaminants without impairing IgG recovery. STUDY DESIGN AND METHODS: An immunoglobulin fraction containing IgG, IgM, and IgA was prepared by caprylic acid precipitation of cryoprecipitate-poor plasma. The capacities of the cation exchangers (S HyperCel and CM Ceramic HyperD F) and anion exchangers (HyperCel STAR AX and Q HyperCel) to remove IgA, IgM, and spiked FXI were tested following a design of experiment approach using microplates and chromatographic column scale-up. FXI removal was also evaluated using Mustang S chromatographic membranes. IgG/IgG subclasses, IgA, IgM, and FXI were assessed by enzyme-linked immunosorbent assay, and caprylic acid, by gas chromatography. RESULTS: Extensive removal of IgA and IgM, but not FXI, was achieved by a two-step chromatographic process combining S HyperCel used in the IgG binding and elution mode and HyperCel STAR AX used in the IgG flow-through mode, providing high IgG and IgG subclass recovery (>85%), high purity (>99.5%), and efficient removal of IgA (<0.5%) and IgM (undetectable). Twenty-six-fold FXI removal was achieved by processing the resulting purified IgG fraction through Mustang S cation-exchanger membranes at pH 6.0 and 12.7 mS/cm. Caprylic acid was removed by S HyperCel. CONCLUSIONS: Combining S HyperCel and HyperCel STAR AX extensively removed IgA and IgM, with good IgG recovery. Mustang® S membranes can be used for dedicated removal of FXI.

Part of purified human plasma kallikrein removed together with a remaining IgG fraction--immunoblot experiments and functional tests.

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.

Defective vaccinia virus as a biologically safe tool for the overproduction of recombinant human secretory proteins.

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.

A binding site for thrombin in the apple 1 domain of factor XI.

Previously we defined a binding site for high molecular weight kininogen (HK) in the A1 domain of factor XI (FXI). Since thrombin can activate FXI and HK inhibits the activation of FXI by thrombin, we have identified a thrombin binding site in FXI. Both the recombinant A1 domain (Glu1-Ser90) and a synthetic peptide (Phe56-Ser86) containing the HK binding site inhibited FXI activation by thrombin. Both a monoclonal antibody, 5F7, recognizing the A1 domain, and the rA1 domain were shown to be competitive inhibitors of thrombin-catalyzed FXI activation. The peptides Ala45-Arg54 and Val59-Arg70 acted synergistically to inhibit FXI activation by thrombin. Mutant rA1 domain constructs (Val64 --> Ala and Ile77 --> Ala), which do not inhibit FXI binding to HK, retain full capacity to inhibit FXI activation by thrombin. The peptide Ala45-Arg54 inhibited thrombin-catalyzed FXI activation, whereas it had no effect on FXI binding to HK. In contrast, the peptide Asn72-Leu83 (which inhibited FXI binding to HK) did not inhibit FXI activation by thrombin. Thus, a thrombin binding site exists in the A1 domain of FXI spanning residues Ala45-Arg70 that is contiguous with but separate and distinct from the HK binding site. These sites may regulate which ligand is bound to FXI and through which pathway FXI is activated.

Factor XI: purification from porcine plasma by affinity chromatography and some properties of factor XI and activated factor XI.

Porcine factor XI and activated factor XI were purified by the introduction of affinity chromatography on high molecular mass kininogen. On the affinity chromatography, it was observed that high affinity exists between porcine factor XI and high molecular mass kininogen. In the preparation, however, factor XII, plasma prekallikrein and high molecular mass kininogen were not detected. The factor XI forms a dimer, and is a heterogeneous molecule, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity of activated factor XI and inhibition profile of activated factor XI against proteinase inhibitors were investigated by comparison with those of bovine and human activated factor XI. From these results, the properties of porcine activated factor XI show great similarities with those of bovine and human activated factor XI.

Nanofiltration, a new specific virus elimination method applied to high-purity factor IX and factor XI concentrates.

We have validated the use of two new regenerated multilayered structured cellulose membranes (BMM), Planova 15 N and Planova 35 N, with respective mean pore sizes of 15 and 35 nm, as a new filtration system to eliminate viruses in highly purified factor IX and factor XI concentrates. Virus spiking experiments indicated that single dead-end filtration on the membranes could remove more than 5.7-7.8 log10 of human immunodeficiency virus, bovine viral diarrhoea virus, porcine pseudorabies virus, reovirus type 3, and simian virus 40, as well as the small non-enveloped viruses, poliovirus Sabin type 1 and bovine parvovirus. In vitro control tests and animal studies (Wessler stasis model, rat hypotension model) of the two concentrates did not reveal any significant differences with the non-nanofiltered material. Viral filtration of plasma derivatives on porous polymeric membranes might be an essential step in the improvement of their viral safety.

Activation of factor XI in plasma is dependent on factor XII.

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.

A therapeutic, highly purified factor XI concentrate from human plasma.

A highly purified factor XI (FXI) concentrate was prepared from human plasma by a process comprising a filter adsorption step and chromatography on a cation exchange resin. The freeze-dried FXI, which solubilized quickly, had high specific activity (130-150 U/mg protein), high potency (approx. 100 U/mL), and excellent stability for at least 24 hours at room temperature in the liquid state. The overall recovery was about 220 U of FXI per liter of plasma. Minor protein contaminants (C1-inhibitor, fibronectin, IgG, and alpha-2-macroglobulin) were found to be between 0.13 and 0.46 mg per 1000 U of FXI. Fibrinogen and relevant coagulation factors (factors II, V, VII, IX, X, XII, XIII, and VIII/von Willebrand factor) were undetectable, as evidenced by immunologic and immunoelectrophoretic data. Components of the kinin system were present in trace amounts or were undetectable. No evidence of activated factors such as factors Xa and IXa was found. Proteolytic activity, as assessed by S-2288 chromogenic substrate, was negligible and thrombin was undetectable. A solvent-detergent treatment was included prior to chromatographic purification to enhance viral safety against lipid-enveloped viruses. In vitro and in vivo animal studies demonstrated the absence of thrombogenic, hypotensive, or toxic effects. No thrombogenic activity was found in the Wessler model in rabbits at doses of 900 to 1100 U of FXI per kg of body weight. This FXI preparation could be beneficial in substitution therapy of congenital or acquired FXI deficiency, especially as a way to avoid the use of fresh-frozen plasma.

Production and therapeutic use of a factor XI concentrate from plasma.

Factor XI deficiency is an uncommon bleeding disorder usually manifested by excessive bleeding after surgery or trauma. Until recently the only effective therapy has been fresh-frozen plasma (FFP) infusion. We describe the efficacy and safety of a new factor XI concentrate produced from human donor plasma by a modification of the method used for antithrombin III concentrate. The mean recovery of factor XI in the circulation measured on 62 occasions was approximately 91% of the injected dose, and the mean half-disappearance-time was 52 h. The concentrate was used for 31 invasive procedures in 30 patients, including 16 patients who had a definite bleeding tendency on previous occasions, with normal haemostasis being achieved in all but 1. Only 1 patient (previously experiencing allergy to FFP) experienced adverse effects during infusion. Monitoring of liver function tests and viral antibody status in suitable patients has shown no evidence of transmission of hepatitis viruses, HIV-1 or parvovirus B19. We conclude that this concentrate provides effective treatment for patients with factor XI deficiency. Preliminary results suggest safety from virus transmission, but this needs to be established in further studies of previously untreated patients.

Purification of factor XI and some properties of activated factor XI from porcine plasma.

Porcine Factor (F.) XI was purified by following three successive chromatographies. By this procedure, about 5.5 mg of F. XI was obtained from 500 ml of the plasma. The F. XI forms dimer, and is heterogeneous molecule, judging from SDS-polyacrylamide gel electrophoresis. The properties of isolated porcine F. XIa are great similar with those of bovine F. XIa.

Activation of the contact system in ascites from patients with gastrointestinal cancer.

Our observations indicates that the plasma contact system is activated in ascites from patients with gastrointestinal cancer: Factor XII is activated, plasma kallikrein is present in complex with the protease inhibitor alpha 2-macroglobulin, and the plasma kallikrein substrate high molecular weight kininogen, is highly degraded. Contact activation seems to take place in spite of a high level of inhibition. Activation of the contact system generates mediators, which may play a role in the accumulation of ascites.

Location of the disulfide bonds in human coagulation factor XI: the presence of tandem apple domains.

Factor XI is a plasma glycoprotein that participates in the blood coagulation cascade. Of the 19 disulfide bonds present in each of the subunits of the human protein, 16 were determined by amino acid sequence analysis of peptide fragments produced by chemical and enzymatic digestion. Four apple domains of 90 or 91 amino acids were identified in the tandem repeats present in the amino-terminal portion of each subunit of factor XI. The disulfide bonds in the carboxyl-terminal portion of the molecule were similar to those in the catalytic region of other serine proteases. The two identical subunits of factor XI were connected by a single disulfide bond at Cys321 linking each of the fourth apple domains while each of the Cys residues at position 11 in the first apple domains forms a disulfide bond with another Cys residue.

Purification and characterization of platelet factor XI.

Factor XI activity and antigen was purified about 300 fold from human platelets through chromatography on Con-A Sepharose, SP-Sephadex C-50, immobilized goat anti-factor XI, and SP-Sephadex. The partially purified platelet factor XI (Pt-XI) could be activated by activated factor XII generated in situ from single chain factor XI in a reaction requiring high molecular weight kininogen (HMWK) and a surface. Native Pt-XI migrated as a molecule of Mr = 245,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as identified by Western blotting. On reduction, Pt-XI appeared to have a Mr = 52,000. Neither form was affected by exposure to trypsin. Incubation of Pt-XI with purified factor XII, HMWK, and kaolin produced activated platelet factor XI clotting activity and, concomitantly, the generation over time of a new chain on reduced SDS-PAGE of Mr = 44,500. The coagulant activity of the activated form could be neutralized by diisopropyl flurophosphate (DFP). Incubation of the activated mixture with 3H-DFP followed by reduced SDS-PAGE showed the active site to be associated with a unit of Mr = 44,500. The adsorption domain as defined by adsorption to kaolin was localized to the Mr = 44,500 chain containing the active site. Hence, both active site and adsorption functions, properties of separate chains in plasma factor XI, reside in the same chain of Mr = 44,500 of platelet factor XI.

Localization of the high molecular weight kininogen binding site in the heavy chain of human factor XI to amino acids phenylalanine 56 through serine 86.

We have previously demonstrated that a monoclonal antibody (5F7) directed against the heavy chain region of factor XI inhibits the binding of factor XI to high molecular weight kininogen (high Mr kininogen) and the surface-mediated proteolytic activation of factor XI by factor XIIa in the presence of high Mr kininogen. In order to identify the structural domain of factor XI that binds high Mr kininogen, CNBr-digested factor XI was passed over a 5F7 antibody affinity column. One of two CNBr peptides that bound to this 5F7 affinity column inhibited binding of 125I-factor XI to high Mr kininogen, as did intact factor XI. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of an inhibitory peptide purified by high performance liquid chromatography revealed an Mr of 10,000-15,000. Gas-phase sequencing of this peptide revealed the following amino-terminal sequence: X-X-Val-Thr-Gln-Leu-Leu-Lys-Asp-Thr. These data together with the amino acid composition of the isolated peptide indicate that both the epitope recognized by antibody 5F7 and at least a portion of the high Mr kininogen binding site are contained within the amino-terminal portion of factor XI comprising residues Glu-1 through Met-102. Further cleavage of this peptide with o-iodosobenzoic acid at a tryptophanyl peptide bond revealed that an Mr 5,000 peptide (with the amino-terminal sequence Trp-Phe-Thr-Cys-Val-Leu) bound to a high Mr kininogen affinity column and inhibited binding of 125I-factor XI to high Mr kininogen. Finally, a synthetic peptide comprising residues Phe-56 through Ser-86 inhibited 125I-factor XI binding to high Mr kininogen. These experiments strongly suggest that the high Mr kininogen binding site is contained within the domain in the heavy chain region of factor XI comprising residues Phe-56 through Ser-86.

Identification of a defective factor XI cross-reacting material in a factor XI-deficient patient.

A homozygous factor XI-deficient girl, who appeared to be positive for cross-reacting material (CRM+) was studied for clarification. Factor XI antigen (F XI:Ag) was measured by radial immunodiffusion using monospecific, heterologous anti-factor XI antibodies. Factor XI coagulant activity (F XI:C) was determined in a modified activated partial thromboplastin time (APTT) test. The ratio of F XI:C to F XI:Ag was 0.04 for the proposita, as compared with 0.7 to 0.74 in the other family members. In contrast, 12 normal individuals had ratios of F XI:C to F XI:Ag of 1.04 +/- 0.15. F XI esterolytic activity was clearly higher than F XI:C in the proband, but not in her relatives. Immunoblotting studies demonstrated F XI CRM in the patient's plasma. Chromatography on diethylaminoethanol (DEAE)-Sephadex at pH 8.4 led to an almost complete removal of F XI from the plasma. The defective F XI was not bound to a negatively charged kaolin surface due to an abnormal interaction with high-mol-wt kininogen (HMWK).

Factor XIa-alpha 1 antitrypsin complex--elevation in the patients with DIC.

We developed an assay for the factor XIa-alpha 1 antitrypsin complex (F.XIa-alpha 1AT complex) in plasma. The purified factor XI (F.XI) activated with beta-XIIa and treated with alpha 1 antitrypsin (alpha 1 AT) served as the standard complex. The assay is an enzyme-linked differential antibody immunosorbent assay. The complex level of tested plasma was measured with peroxidase-labeled anti-alpha 1AT Fab' after the addition of 10-fold diluted test plasma (200 microliter) to the anti-F.XI monoclonal antibody beads. To eliminate the effects of plasma, the standard F.XIa-alpha 1AT complex was diluted with F.XI-deficient plasma (10-fold diluted) which did not contain the complex. Purified F.XI (0.08 micrograms/assay, i.e. 100%) was added to the standard F.XIa-alpha 1AT complex, because the absorbance of the standard complex containing F.XI (0.016-0.12 micrograms/assay, i.e. 20-150%) was a little lower than that of the complex alone. The recovery of the F.XIa-alpha 1AT complex added was over 90%. Neither F.XI nor alpha 1AT alone had the color development. The complex level of 25 normal individuals was below the detectable limit (less than 0.18 ng/assay), whereas the 30 patients with disseminated intravascular coagulation (DIC) had a high level complex (0.18-4.2 ng/assay). This assay may be helpful for the diagnosis of DIC.