Monoclonal antibodies directed to the calcium-free conformation of human protein S

Four mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/l CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate adsorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/l CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analytical considerations for free protein S assays in protein S deficiency

Protein S is an anticoagulant protein that circulates in plasma in complex with C4b-binding protein (C4BP) or in free form. Deficiency of protein S increases the risk of venous thrombosis. Measurement of free protein S, as compared to total levels, has been shown to be superior for prediction of protein S deficiency. We studied the effects of different handling protocols for an immuno- and a ligand (C4BP)-based assay for free protein S. When the assay was performed at 37 degrees C, the levels of free protein S in plasma from protein S deficient patients were approximately twice those obtained at room temperature. The reason for this phenomenon was that plasmas from protein S deficient patients exhibited a time-, temperature-, and dilution-dependent increase in free protein S, which was more pronounced than corresponding dilution of the normal plasma that was used to create the standard curve. These findings demonstrate the importance of assay procedure and sample handling in assays for free protein S.     

Identification of different forms of human C4b-binding protein lacking beta-chain and protein S binding ability

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.     

Multicentre evaluation of IL Test Free PS: a fully automated assay to quantify free protein S

Deficiency of the anticoagulant vitamin K-dependent protein S (PS) is associated with increased risk of venous thrombosis. In human plasma, PS circulates in two forms: as free protein (free PS) and PS bound to C4b-binding protein (C4BP), a regulator of the complement system. Assays for free PS have higher sensitivity and specificity for protein S deficiency than assays for total protein S. We have extensively evaluated the analytical performance of a novel assay for free PS, the IL Test Free Protein S, which takes advantage of the affinity of C4BP for free PS, and compared its performance to existing methods. IL Test Free Protein S is a rapid, fully automated turbidimetric assay consisting of two reagents: a C4BP coated latex and an anti-PS monoclonal antibody coated latex. The test range, precision and linearity were adequate and the assay tolerated high concentrations of interfering substances of clinical significance. The reference range agreed with previously published studies. The analysis of 903 patient samples belonging to 20 different clinical categories with the new assay yielded free PS results that agreed well with those obtained using the assays established in the participating laboratories. The study demonstrated the IL Test Free Protein S to be rapid, reliable and easy to perform.     

Monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for the measurement of vitamin K-dependent protein S: the effect of antibody immunoreactivity on plasma protein S antigen determinations.

Two monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution curves obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution - plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-asparaginase treatment reduces the anticoagulant potential of the protein C system without affecting vitamin K-dependent carboxylation

The changes in plasma levels of the vitamin K-dependent natural anticoagulants protein C (PC) and protein S (PS) and procoagulant factors II, IX and X were evaluated in 8 adult patients during treatment with L-asparaginase (L-ase i.v. 120,000 U/m² over 10 days). PC anticoagulant activity and factor IX, X and II coagulant activity decreased proportionally to their half-lives to a nadir of 50–60% of pretreatment values after 2–5 L-ase infusions, suggesting that inhibition of protein synthesis rather than consumption is the main mechanism responsible for the observed changes. Free PS antigen levels declined at a rate similar to total PS antigen, reaching a nadir of 56% of pretreatment values after 3 L-ase infusions; however, due to C4b-binding protein levels higher than total PS levels (p <0.05), they were constantly lower than the corresponding total PS antigen levels (0.05 < p <0.001). This implicates that total PS antigen levels cannot be taken as an indicator of PS activity. No differences between the antigenic levels and the anticoagulant activities of PC and free PS could be observed suggesting that L-ase does not affect the mechanisms of vitamin K-dependent carboxylation of Gla-residues. The faster rate of decline of PC and PS activities relative to that of factor II may be responsible for the onset of an hypercoagulable state during the early phase of L-ase treatment.     

Protein S activity in patients with heredofamilial protein S deficiency and in patients with juvenile venous thrombosis. Results of a functional method

Using an ACL 300R coagulometer (Instrumentation Laboratory) we assessed the clinical usefulness of a new method to measure PS activity (PS:Act), based on the prolongation of prothrombin time of a mixture of diluted plasma sample, PS depleted plasma previously incubated with Protac for protein C activation, bovine thromboplastin and calcium ions. The results were compared with those from immunological assays. PS:Act was measured in 42 apparently healthy subjects, in 12 patients with hereditary PS deficiency (HPSD group) diagnosed on the basis of immunologic tests and in 48 patients with episodes of juvenile venous thromboembolism at least three months prior to testing (JVTE group). All the HPSD patients had PS:Act below the normal range (< 62%). In JVTE group 9 patients (18.7%) showed abnormal results for PS:Act, 4 (8.3%) had low levels of free PS:Ag; all patients had normal total PS:Ag levels. Levels of antiphospholipid antibodies (immunologic test) were normal in the 9 JVTE patients with low PS:Act. When all the results were considered together (n=102), the correlation coefficient between PS:Act and free PS:Ag was 0.78 (p<0.01).     

Behavior of protein S during long-term oral anticoagulant therapy

It has recently been reported that a natural anticoagulant, protein S (PS), is depressed during oral anticoagulation. Since more detailed information is required from the clinical standpoint, we measured plasma levels of PS [both total and free (not complexed) PS antigen], C4b-binding protein (C4bp) and other vitamin K-dependent proteins (factors II, VII, IX, X and protein C) in 60 plasma samples from patients on long-term oral anticoagulant therapy with warfarin. Together with the reduction of other vitamin K-dependent plasma proteins, PS decreased during warfarin treatment, being dependent on the intensity of the therapy. A considerable variation in plasma PS levels was also observed among individuals with a similar intensity of anticoagulation. Plasma concentration of C4bp was closely correlated with total PS level, and free PS/total PS ratio was independent of thrombotest values. These findings indicate that long-term oral anticoagulant therapy results in the suppression of the synthesis of PS, and that its reduction is on the whole balanced with C4bp and vitamin K-dependent coagulation factors. It was suggested that the metabolism of C4bp might be regulated by the plasma PS level, although this hypothesis needs further exploration.     

The Ser460Pro mutation in recombinant protein S Heerlen does not affect its APC-cofactor and APC-independent anticoagulant activities

Protein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen. To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasma-derived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.     

Impairment of the protein C anticoagulant pathway in a patient with systemic lupus erythematosus, anticardiolipin antibodies and thrombosis

We have identified an inhibitor of the protein C anticoagulant pathway in the plasma of a patient with systemic lupus erythematosus and a history of recurrent deep vein thrombosis, fetal wastage, and seizures. The patient's plasma contained anticardiolipin antibodies as well as a weak lupus anticoagulant. Examination of this patient's plasma revealed normal levels of protein C and protein S antigen, normal levels of functional protein C, as well as essentially normal levels of every blood coagulation factor. In a modified prothrombin time assay, the activated protein C-mediated prolongation of the clotting time observed in normal plasma was not observed in this patient's plasma. Gel permeation chromatography of the patient's plasma revealed that the inhibitory material was a high molecular weight protein that coeluted with the IgM peak. The inhibitor did not appear to circulate as a complex with protein C, since the inhibitor could easily be separated from protein C during fractionation procedures, and did not interfere with the activation of protein C in plasma as assessed by a functional amidolytic assay. Our findings suggest that the recurrent thrombotic episodes observed in this patient may have occurred as a result of the patient's antiphospholipid antibody neutralizing specific phospholipids essential for the full expression of the anticoagulant activity of activated protein C.     

Normal reference ranges of antithrombin,protein C and protein S: Effect of sex,age and hormonal status

Introduction

Antithrombin (AT), protein C (PC) and protein S (PS) deficiencies are risk factors for venous thromboembolism. Overlapping values between heterozygous carriers and normal individuals often make a correct classification of a deficiency difficult. The aim of this study was to investigate the effect of sex, age, menopause and hormone therapy on natural anticoagulant plasma levels in a large group of healthy individuals, and to evaluate the need of separate reference ranges.

Materials and Methods

AT and PC were measured with a chromogenic assay, antigenic free PS with an ELISA test. To evaluate the effect of sex, age, oral contraception, hormonal status (and their interaction) on AT, PC and PS levels, linear regression models were used. Biological relevance and the value of the normal deviate z were chosen as rules to decide for separate reference ranges.

Results

The study population consisted of 1837 healthy adult individuals (741 men, 1096 women), aged 18-85 years (median age: 44 years). In men AT levels decreased after the age of 50 years. Men had higher levels of PS than women, particularly at young ages. In women, after correction for menopause, only PC levels increased with age. Menopause affected AT and PS, but not PC levels. Oral contraceptive intake was associated with a decrease of AT and PS, and an increase of PC levels.

Conclusions

For AT, PC and PS, sex- and age-specific normal reference ranges can be useful, in order to better discriminate true carriers of a natural anticoagulant deficiency.     

Functional and immunologic protein S in normal pregnant women and in full-term newborns

Total and free protein S antigen and C4b-binding protein (C4bp) were determined by rocket immuno-electrophoresis, and functional protein S was assayed by a coagulation method, throughout pregnancy and normal puerperium and in a group of normal full-term newborns (FTN). The functional protein S assay is based on a modification of the APTT, using a mixture of test sample, protein S deficient plasma, activated protein C, phospholipids and calcium. This protein S functional assay is specific for protein S since the APTT prolongation by normal plasma was abolished by incubation of plasma with monospecific, rabbit anti-protein S IgG. The ratios of functional protein S/free protein S antigen in healthy men (n = 13) and women (n = 14) were 1.0 +/- 0.13 (mean +/- SD) and 1.03 +/- 0.20, respectively. During pregnancy there is a decrease in functional protein S and a progressive decrease in total and free protein S antigen, with a functional/free protein S ratio of 0.75 +/- 0.28 in the third trimester of pregnancy (n = 16). In early puerperium the functional protein S level was lower than the free protein S antigen level (ratio about 0.5). In the FTN group, the free protein S level was 39% and protein S activity was about 70% that of adults, with a functional/free protein S ratio of 1.84 +/- 0.31. C4bp values were 23.5 +/- 10.3% in the FTN group, and crossed immunoelectrophoresis showed that in this group the major protein S peak corresponded to free protein S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein C and protein S levels in uremic patients before and after dialysis

The inhibitory capacity of the natural protein C (PC)/protein S (PS) system was evaluated measuring both the functional activity and the antigen level of both these inhibitors in 30 uremic patients before and after a dialytic treatment and in 30 healthy normal volunteers. PC functional activity was determined by two methods, one clotting and one chromogenic. PS antigen level was measured both as free protein and as total content. Unlike previous authors, we found that PC functional activity and the antigen level were normal in patients before dialysis, with a significant increase after. PS functional activity and free and total antigen levels were all normal before dialysis, and all except free antigen showed a significant post-treatment rise.     

Membrane binding and anticoagulant properties of protein S natural variants

Introduction

Protein S (PS) is a vitamin K-dependent plasma glycoprotein with a key role in the control of coagulation pathway on phospholipid membranes. We compared anticoagulant and membrane binding properties of PS altered by natural mutations (N217S, DelI203D204) affecting the epidermal growth factor like-domain 4 (EGF4) and causing PS deficiency.

Materials and methods

Binding of recombinant, immunopurified PS (rPS) to several conformation-specific antibodies, to C4BP and to phospholipid liposomes was investigated by ELISA. PS binding to cells was analysed by flow cytometry. PS inhibitory activities were studied in plasma and purified systems.

Results and conclusions

Conformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800nM concentration reduced inhibition of coagulation (rPS217S, 10.2-17.3%; rPSDelI203D204, 5.8-8.9% of rPSwt) in FXa 1-stage clotting assay with APC. In thrombin generation assays the inhibition of ETP was reduced to 51.6% (rPS217S) and 24.1% (rPSDelI203D204) of rPSwt. A slightly shortened lag time (minutes) was also observed (rPS217S, 2.58; rPSDelI203D204, 2.33; rPSwt, 3.17; PS deficient plasma, 2.17).In flow cytometry analysis both mutants efficiently bound apoptotic cells in adhesion or in suspension. The affinity for phosphatidylserine-rich vesicles (apparent Kd: rPSwt 27.7 ± 1.6 nM, rPS217S 146.0 ± 16.1 nM and rPSDelI203D204 234.1 ± 28.1 nM) was substantially increased by membrane oxidation (10.9 ± 0.6, 38.2 ± 3.5 and 81.4 ± 6.0 nM), which resulted in a virtually normal binding capacity of mutants at physiological PS concentration.These properties help to define the molecular bases of PS deficiency, and provide further elements for PS-mediated bridging of coagulation and inflammation.     

Determination of total, free and complexed protein s in plasma by ELISA, and comparison with a standard electroimmunoassay

An enzyme-linked immunosorbent assay (ELISA) for measuring total, free and complexed protein S in plasma was developed. To assay free protein S, C4b-binding protein-bound protein S (C4b-BP-PS) was extracted by addition of polyethyleneglycol (PEG) 6000 (5%, final concentration) to plasma samples. Microtiter plates were coated with rabbit anti-human protein S, and bound protein S was detected with labelled anti-protein S antibody. Diluted plasma samples were incubated in the plates overnight at 22 °C to permit C4b-BP-PS complexes to dissociate. Mean variation coefficients of 2.1 and 3.2% (intra-assay) and 4.3 and 7.9% (inter-assay) were found for total and free protein S assays, respectively. The ELISA measures free and complexed protein S with equal efficiency as is demonstrated by the fact that the sum of free protein S and C4b-BP-PS complex levels in normal individuals, women in their third trimester of gestation and patients with acute deep vein thrombosis (DVT), equaled the level of total protein S present in the corresponding plasma. Total protein S values obtained with the ELISA, in all groups studied, correlated well with those obtained with a standard electroimmunoassay (EIA) (r=0.93; N=40). However, total protein S levels measured by EIA were lower than those assayed by ELISA in pregnant women and in DVT patients. Furthermore, addition of several amounts of purified C4b-BP to NHP, which reduced the recovery of free protein S, did not influence the total protein S values measured by ELISA but slightly decreased the recovery of total protein S measured by EIA. These results indicate the necessity of using assays which accurately and reliably measure the total amount of protein S antigen. After addition of C4b-BP to NHP, the residual functional protein S level was lower than the residual level of free protein S antigen; this lends support to the idea that C4b-BP-PS complex inhibits the activated protein C cofactor activity of protein S.     

Regulation of endothelial cell protein c activation and fibrinolysis by procoagulant albumin

Endothelial cell regulation of protein C activation and fibrinolysis are important components of the hemostatic response to vascular injury or perturbation. Procoagulant albumin (P-A1), a constituent of normal human plasma has been purified and identified as an inducer of endothelial cell tissue factor activity. The purpose of the studies reported herein was to investigate the effects of P-A1 on human endothelial cell protein C activation and fibrinolysis. P-A1 suppressed protein C activation, enhanced release of plasminogen activator inhibitor-1, but had no effect on tissue-plasminogen activator release. Plasminogen activator inhibitor-1 released by P-A1 was functional as evidenced by the capacity to form a covalent complex with ¹²⁵I-urokinase. Inactive albumin (isolated during the same purification procedure as P-A1, but without tissue factor-inducing activity) did not suppress protein C activation or increase plasminogen activator inhibitor-1 release. These results indicate that P-A1, a component of human plasma, can modulate multiple vascular hemostatic properties, and support the hypothesis that P-A1 is involved in normal or pathologic hemostasis.     

Familial dysfunction of protein S

We describe a previously unreported defect of protein S characterized by low levels of cofactor activity for activated protein C contrasting with low normal levels of total and free protein S antigen. The distribution of protein S between the free form and the form complexed with the complement component C4b-binding protein was normal on two-dimensional immunoelectrophoresis. The proband developed juvenile deep-vein thrombosis while taking oral contraceptives. Her defect was transmitted in an autosomal dominant fashion from her asymptomatic mother. Other relatives carrying the same laboratory abnormality (mother, maternal uncle, two sisters and one brother) were also asymptomatic. We postulate that the defect is due to a dysfunctional protein S present in plasma in normal amounts and with normal proportions of the free and complexed forms of the protein.     

Decreased maternal protein S activity is associated with fetal growth restriction

Introduction

Protein S (PS) activity has been shown to decrease during normal pregnancy. The aim of this study was to determine any correlation between decreased maternal PS activity and fetal growth restriction (FGR).

Methods

We carried out a retrospective study of maternal PS activity and complement 4b-binding protein (C4BP) concentration in 102 patients with FGR and 58 patients with fetuses that had normal growth. Among pregnancies affected by FGR, 14 diagnoses were made in the second trimester and 88 in the third trimester. Patients whose fetuses had normal growth were matched with FGR subjects for maternal age and gestational age at sampling (29 cases each in the second and third trimester).

Results

Mean PS activity of the control group in the third trimester was significantly lower than in the second trimester (56.5 ± 16.5% vs 35.8 ± 13.8%). PS activity in women with FGR was significantly decreased in both the second trimester (36.6 ± 13.2%) and third trimester (30.2 ± 12.2%) compared with control group levels. Plasma concentrations of C4BP for the control group were significantly higher in the third trimester than in the second trimester (90.5 ± 17.5% vs 81.1 ± 13.6%). However, in women with FGR, plasma C4BP concentrations in both the second trimester (84.0 ± 14.8%) and the third trimester (86.0 ± 17.7%) were comparable with concentrations of the control group.

Conclusions

Maternal PS activity decreased as normal pregnancies progressed but decreased over time in cases with FGR. Excessive decreases in PS activity during pregnancy could contribute to development of FGR.     

A collaborative study on the measurement of protein S antigen in plasma.

A collaborative study on the measurement of protein S (PS) antigen (total and free) in a freeze-dried ampouled test plasma by assay against local house standard plasmas was carried out in eleven laboratories. Potency estimates of total PS showed good agreement between laboratories with a geometric coefficient of variation (gcv) of 5.9% and an overall combined potency of 0.84 units per ml. Potency estimates of free PS antigen in the test sample were associated with increased variability between laboratories resulting from the polyethylene glycol (PEG) precipitation step which is used to separate free PS from PS bound to the C4b binding protein. Free PS in the test could be expressed relative to either total PS in the house standards (e.g. 0.28 units per ml) or relative to free PS in the house standards following PEG precipitation (e.g. 0.71 units free PS per ml).     

Chromatography of vWF on dextran sulphate sepharose

A relatively simple and reproducible chromatographic separation using Dextran Sulphate (DS) Agarose is described for the purification of vWf:Ag from cryoprecipitate or plasma source material. The elution profiles suggest high affinity of vWf for the matrix permitting resolution from Fibrinogen, IgG and the unbound Albumin. The major contaminant fibronectin can be removed prior to the chromatography step by Gelatin-Sepharose adsorption. Chromatography of ¹²⁵I-vWf, added to cryoprecipitate as a marker, gives two distinct peaks one of which elutes with the column wash through fractions and expresses negligible biological activity. The re-eluted bound material expresses normal vWf:RCo activity with full multimer integrity as assessed by SDS Agarose electrophoresis. DS sepharose chromatography offers an excellent method for the purification of ¹²⁵I-vWf since all the viable label is resolved from excess free radiolabel and denatured protein. Low recoveries of VIII:C were demonstrated at both R.T. or 4°C possibly due to the presence of Tri-Sodium citrate and the absence of sufficient free CaCl ₂ in the buffers.