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Affinity chromatography of coagulation factors II, VIII, IX and X on matrix-bound phospholipid vesicles
THROMBOSIS RESEARCH 23; 481-489, 1981 0049-3848/81/180481-09$02.00/O Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
AFFINITY CHROMATOGRAPHY OF COAGULATION FACTORS II, PHOSPHOLIPID VESICLES VIII, IX AND X ON MATRIX-BOUND Lars-Olov Department School x) On
Andersson x> , Le Phuc
Thuy
and James
E. Brown
of Pathology, University of California San Diego of Medicine, La Jolla, California 92093, USA.
leave from Research Stockholm, Sweden.
Department,
Biochemistry,
KABI
AB,
(Received 28.4.1981; in revised form 3.8.1981. Accepted by Editor P. Wallen)
ABSTRACT
Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine were coupled to cyanogenbromide activated agarose gels. The gels obtained showed phospholipid-related procoagulant activity in the Russell's Viper Venom clotting test. Upon column 5tromatography containing of prothrombin complex concentrate in Ca Factor IX and Factor X were bound buffer, prothrombin, to the gel and could be eluted by desorption with buffer containing citrate. When purified Factor VIII/van Wille brand Factor dissolved in buffer without Ca2+ was applied to the column the main part of Factor VIII activity Desorption with 1 M NaCl did not elute any was bound. Factor VIII but 1 M KSCN did, suggesting that the Factor VIII-phospholipid binding mainly is dependent on hydrophobic interactions.
INTRODUCTION There is considerable evidence that phospholipid surfaces play an important role in the blood clotting process. They appear to be essential both for the activation of Factor X as well as for the activation of prothrombin. In the activation of prothrombin, phospholipid structures on the surface of the released platelet probably bind activated Factor V and activated Factor X thereby forming the prothrombinase complex which then transforms prothrombin to thrombin (l-3). The situation is probably similar in the formation of the Factor X activator complex about which relatively little is known. Phospholipid vesicles have been shown to bind Factor V (4) and Factor VIII (5,6) in the absence of Ca2+ and the factors II, VII, IX and X in the presence of Ca2+ KEYWORDS:
Affinity chromatography, phospholipid, tor VIII, Factor IX, Factor X. 481
Factor
II, Fac-
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PURIFICATION OF II, VIII, IX & X
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The binding of bovine prothrombin to phospholipid vesicles (7). has been studied in some detail (4,8-10) and the dissociation constant for a certain set of conditions has been determined to be around 2 x 10e6 M. In this study phospholipid vesicles have been coupled to a gel matrix and the adsorption and elution of coagulation factors II, VIII, IX and X has been studied. MATERIALS AND METHODS Materials The phosphatidylserine (PS) and phosphatidylethanolamine (PE) preparations were obtained from Sigma Chemical Co., St. The PS was prepared from brain and more than Louis, MO., USA. The PE was from brain and more than 95% pure. It was 98% pure. delivered as a solution in CH Cl. Dipalmitoyl-l-14C-phosphatidylcholine (PC) was obtained from New England Nuclear, Boston, solution. Mass., USA, in toulene-benzene The specific activity was 100 mCI/mmole. Biogel A 15 M and Affi-gel 10 was purchased from BioRad LaPreactivated Sepharose Macroboratories, Richmond, CA., USA. beads 6 MB was from Pharmacia Fine Chemicals, Uppsala, Sweden. Cyanogen bromide was from Sigma Chemical Co., St. Louis, MO., USA. The Human Prothrombin complex concentrate used was from the American Red Cross and has been prepared by DEAE-Sephadex Purified Factor VIII/van Willebrand Facadsorption and elution. tor complex was prepared by gel filtration on Biogel A 15 M of Kabi high purity Factor VIII. The void volume fractions were collected and frozen or used directly for the adsorption experiments. The specific activity was usually around 10 unitsfmg, the quotient Factor VIII R:Ag/Factor VIII activity around 3 and the quotient Factor VIII C:Ag/Factor VIII activity 1.8. Russell's Viper Venom and Taipan Snake Venom were obtained from Sigma Chemical CO., St. Louis, MO., USA. Preparation and coupling of phospholipid vesicles To 12.5 mg of PS in a poly-propylene tube was added 12.5 mg of PE in CH3Cl solution and 15 ug of 14C-labelled PC in touleneAfter mixing, the solvents were evaporated by benzene solution. immersing the tube in a water bath and blowing nitrogen through. When the tube was completely dry, 10 ml of 0.02 M imidazole, 0.15 M NaCl buffer pH 7.3 was added and nitrogen bubbled through for Then the tube was put in an ice bath and soniabout 5 minutes. cated for 8 minutes using a Heatsystems-Ultrasonics Inc., Plainview, USA, sonicator. For coupling of the phospholipid vesicle preparation to agasolution pH rose gel, 10 ml of Biogel A 15 M in 5 M K3P04-K2HP04 12.0 was activated with 20 mg CNBr/ml for 10 minutes at +5'C. The gel was then washed with ice-cold water and 10 ml of the PE-PS vesicle preparation was added. The pH of the suspension was adjusted to 9.2 and allowed to stand at room temperature for two After that the gel was washed with hours under slow stirring. imidazole-NaCl buffer and with imidazole buffer containing 1 M NaCl. Remaining reactive groups were blocked by treatment with 0.1 M ethanolamine overnight. Determination of prothrombin Prothrombin was assayed using Taipan Snake Venom (Oxyiranos scutellantus scutellantus) to convert the prothrombin to thrombin which was then determined by measuring clotting of bovine fibrinogen (11).
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Determination of Factor IX Factor IX was assayed by a one-stage method as described by Veltkamp et al. (12) using hemophilia B plasma. Determination of Factor X Factor X was determined by transforming it to activated form by incubation with Russell's Viper Venom followed by a clotting assay using Factor X deficient bovine plasma. The assay was performed essentially as described by Bachmann et al. (13). Determination of Factor VIII and related components Factor VIII activity was assayed using a one-stage method essentially according to Langdell (14) using human hemophiliac plasma as substrate. Factor VIII related antigen (F. VIII R:Ag) was determined using electroimmunoassay (15). Factor VIII coaguusing an immuno radiolant antigen (F. VIII C:Ag) was determined metric assay (IRMA) as described previously (16). Polyacrylamide gel electrophoresis SDS polyacrylamide gel electrophoresis was performed according to Laemmli (17). RESULTS Coupling of phospholipid vesicles Phosphatidylserine-phosphatidylethanolamine vesicles were prepared by sonication of suspensions containing 2.5 mg/ml of each phospholipid in 0.02 M imidazole, 0.15 M NaCl buffer pH 7.3. $4C-labelled phosphatidylcholine which had The vesicles contained been added before sonication. Coupling to Sepharose gel was accomplished by incubating the vesicles with CNBr-activated Sepharose 4B at pH 9.2 for two hours. After washing and blocking of remaining reactive groups with ethanolamine an afiquot of gel was taken out and subjected to scintillation counting after dissolving the gel in dimethylformamide. The degree of coupling as determined by the 14C-counting corresponded to 36 ng of phospholiBy amino acid analysis for serine and ethanolpid/ml of gel. amine the value obtained was 41 ug phospholipid/ml of gel. In separate experiments it was found that essentially the same degree of coupling of phospholipid was obtained when Affi-gel 10 In contrast the coupling was 60% lower when CNBr-actiwas used. vated Sepharose Macrobeads were used. Assay for phospholipid procoagulant activity using the Kussell's Viper Venom clotting test (18) showed that the phospholipid-Sepharose gel had an activity corresponding to 24 ng PS-PE phospholipid/ml of gel. Prothrombin, Factor IX and Factor X Prothrombin comnlex concentrate was dissolved in 0.02 M imibuffer pH 7.3 to give a solution dazole, 0.15 M NaC1,'5 mM CaCl containing 2.1 units prothrombfn/ml, 1.6 units Factor IX/ml and 1.7 units Factor X/ml. When 0.5 ml of this solution was applied to a mini-column containing 7 ml of PE-PS-Sepharose and equilibrated with the same buffer almost all prothrombin, Factor IX and Factor X were adsorbed to the gel as evident from Figure la. Upon desorption with 0.02 M imidazole, 0.15 M NaCl, 0.01 M citracomplex eluted. The recoveries te buffer 7.3,‘ the prothrombin were for prothrombin 75%, for Factor IX 86% and for Factor X 81%. In Figure 2 is shown the SDS polyacrylamide gel electrophoresis patterns obtained with starting material, the fraction passing through and desorption fraction.
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FIG. la Affinity cnromatography of 0.5 ml prothrombin complex concentrate (2.1 units/ml prothrombin, 1.6 units/ml Factor IX and 1.7 units/ml Factor X) on PE-PS-agarose gel. Column dimensions 1 x 9 The column was equilibrated with 0.02 M imidazole, 0.15 M cm. The elution buffer was 0.02 M NaCl, 5 mM CaCl2 buffer pH 7.3. 0.15 M NaCl 0.01 M citrate pH 7.3. w UV-adsorption imidazole, prothrombin activity AA Factor IX activity and at 280 nm, 0-0 Factor X activity. 0-0
FIG. lb Affinity chromatography of 2.0 ml prothrombin complex concentrate (3.6 units/ml prothrombin and 2.8 units/ml Factor IXj on PEColumn dimensions 1 x 9 cm. PS-agarose gel. The column was equilibrated with 0.02 M imidazole, 0.15 M NaCl, 5 mM CaC12 buffer pH The elution buffer was G.02 M imidazole, G.15 M NaCl, 0.01 7.3. prothromM citrate pH 7.3. u UV-adsorption at 280 nm, 0 -0 A Factor IX activity. bin activity and ATo test the binding capacity of the PE-PS Sepharose gel, 2.0 ml of prothrombin complex solution containing 3.6 units/ml pro-
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thrombin and 2.8 units/ml Factor IX was applied to the column. In Figure lb the results are shown. In this case about half of the prothrombin is bound and the other half passes through. In contrast more than 95% of the Factor IX is bound indicating that Factor IX is more strongly bound to phospholipid than prothrombin. Calculation of the amount of prothrombin bound gives the value 47 pg/ml of gel. After repeated use for prothrombin complex adsorption and elution part of the gel was tested for phospholipid procoagulant activity by the Russell's Viper Venom clotting test. The activivariation as with the newly ty was the same within experimental prepared gel.
FIG. 2 Polyacrylamide gel electroPhoresis of materials from the affinity chromatography shown in Figure la. A - starting prothrombin complex material, B - break through fraction, C - desorption fraction.
-’
ABC Factor VIII/van Willebrand Factor When a solution of Factor VIII/van Willebrand Factor, prepared by gel filtration of a high purity Factor VIII concentrate, was applied to a mini-column containing PE-PS-Sepharose part of Factor VIII activity was adsorbed and part passed through as seen in Figure 3. Desorption by 1 M NaCl did not result in any elution of Factor VIII activity. Subsequent elution with 1.0 M KSCN did however result in elution of considerable amounts of Factor VIII activity. KSCN was removed by gel filtration using Sephadex Total recovery G25 before determination of Factor VIII activity. of Factor VIII activity was 25% where 11% is unbound and 14% in The fairly low recovery of Factor VIII actithe desorption peak. vity is probably in part related to the observed lability of the eluted material in 1 M KSCN. Determination of Factor VIII R:Ag showed that the break through peak contained most of this materiThe al but there was also some present in the desorption peak. ratio Factor VIII R:Ag/Factor VIII activity was 13.4 for the break Assay for through fraction and 1.6 for the desorption fraction. Factor VIII C:Ag by IRMA showed a similar pattern as for the Factor VIII activity but the ratios Factor VIII C:Ag/Factor VIII activity were different in the two peaks being 2.4 in the break through fraction and 0.55 in the desorption fraction.
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PLIRIFICATIONOF II, VIII, IX & X
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FIG. 3 Affinity chromatography of Factor VIII/van Willebrand Factor on PE-PS-agarose gel. Column dimensions 1 x 3 cm. The column was equilibrated with 0.02 M imidazole, 0.1 M NaCl and 0.02% The column was eluted with 1 M NaN3 pH 6.8. NaCl and then 1 M KSCN in the equilibrating bufFactor VIII activity, Afer. 0-@ A Factor VIII C:Ag, M Factor VIII R:Ag. DISCUSSION Phospholipid vesicles can be coupled to agarose gels and their activities as regards procoagulant activity and binding of coagulation factors is preserved. This indicated that the structures of the vesicles does not change very much during the coupling and subsequent washing procedures. The stability of the linkage between phospholipid vesicles and the agarose gels appears adequate as only trace amounts of phospholipid leaks from the gel during the various desorption and washing steps. It is possible that the matrix-bound vesicles are fixed to the gel by multipoint attachment; several PS and PE molecules in the same vesicle might be covalently linked to the agarose. The fairly low degree of coupling obtained could be due to the necessity for multipoint attachment in order to get a stable matrix-bound vesicle that is not removed in the washing steps. However, one can not exclude the fact that other effects like steric hinderance are in part responsible for the low coupling yields obtained. As expected, prothrombin, Factor IX and Factor X are adsorbed to the matrix-bound vesicles when calcium ions are present. Very good recoveries are obtained upon desorption with citratecontaining buffer. The degree of purification is also good judging from the specific activities obtained and the SDS polyacrylamide gel electrophoresis pattern. It is likely that Factor VII and protein C also are present in the desorption material. The capacity of the gel to adsorb prothrombin is surprisingly high,
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the weight ratio between bound prothrombin and matrix-bound phospholipid is 1.3 which can be compared with the value 1.2 obtained from studies of prothrombin binding to phospholipid vesicles in solution (9). The observed stronger binding of Factor IX as compared to prothrombin may have a functional significance as the concentration of Factor IX in plasma is-considerably lower-than that of prothrombin and thus stronger binding of Factor IX is necessary in order to get a suitable concentration of Factor IX on phospholipid. The data obtained in the studies of Factor VIII/van Willebrand Factor are not as clearcut as those obtained with the vitamin K dependent coagulation factors. This probably depends on the fact that the Factor VIII/van Willebrand Factor complex applied to the column is not a single entity but a series of large Different parts of the commolecular weight protein complexes. plexes may react differently and give rather complicated binding and dissociation reactions. However, the observation that almost all of the Factor VIII related antigen passes through the column and only a very small amount is desorbed, suggests that the von Willebrand Factor part of the complex is not adsorbed to the phos This is in agreement with the results of a recent stupholipid. dy on binding of Factor VIII/van Willebrand Factor to phospholiFactor VIII on the other hand is pid vesicles in solution (6). This can be explained by combound although not quantitatively. petitive effects of binding to von Willebrand Factor resulting in part of Factor VIII eluting as Factor VIII/van Willebrand Factor The observation complex and part being bound to phospholipid. that desorption with buffer containing 1 M NaCl does not elute any Factor VIII activity whereas desorption with 1 M KSCN does, suggests that the binding of Factor VIII to phospholipid is not primarily dependent on electrostatic interactions but rather on hydrophobic interactions. A somewhat surprising finding is that Factor VIII activity is higher than Factor VIII C:Ag in the desorption fraction. A possible explanation would be that the Factor VIII C:Ag structure detected by our antibody preparation is more labile to KSCN solution than is the structures necessary for Factor VIII activity. This is supparted by the recent finding (19) that Factor VIII C: Ag as measured by the same antibody preparation is more labile to heat than is the Factor VIII activity. The results obtained in this study show that a phospholipid vesicle-agarose gel can be used for affinity chromatography of a number of coagulation factors. Further it can probably also be casused as a model system for the two steps in the coagulation cade that require a phospholipid surface, that is the activation The observation of Factor X and the activation of prothrombin. that the gel particles do have high procoagulant activity in the Russell's Viper Venom test support that concept. ACKNOWLEDGEMENTS for her secretarial asWe are grateful to Ms. Mona Stendahl This work was supported by USPHS Research Grant HLsistance. 15230 and HL-06132-01.
488
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REFERENCES 1.
MILETICH, J.P., JACKSON, C.M. of the Factor X, binding site 1978. Chem, 253, 6908-6919,
and MAJERUS, P.W. on human platelets.
2.
NESHEIM, M.E., TASWELL, J.B. and MANN, K.G. The contribution of Bovine Factor Va to the activity of prothrombinase. J Biol Chem, 254, 10952-10962, 1979.
3.
DAHLBACK, B. and STENFLO, J. by platelet-bound Factor Xa. 1980.
4.
BLOOM, J.W., NESHEIM, M.E. and MANN, K.G. binding properties of bovine factor V and chemistry, Is, 4419-4425, 1979.
5.
HEMKER, H.C., KAHN, M.J.P. and DEVILEE, P.P. The adsorption of coagulation factors onto phospholipids. Thromb Diath Haemorrh, 24, 206-223, 1970.
6.
ANDERSSON, L.-O. and BROWN, J.E. Factor VIII/van Willebrand Factor sicles. Biochem J (in press).
7.
PAPAHADJOPOULOS, D. and HANAHAN, D.J. Observations on the interaction of phospholipids and certain clotting factors in prothrombin activator formation. Biochim Biophys Acta, 3, 436-439, 1964.
8.
NELSESTUEN, G.L. and LIM, T.K. Equilibria involved in prothrombinand blood-clotting Factor X membrane binding. 4164-4171, 1977. 16 -Biochemistry, -'
9.
NELSESTUEN, G.L. and BRODERIUS, M. Interaction of prothrombin and blood-clotting Factor X with membranes of varying Biochemistry, 16, 4172-4177, 1977. composition.
The Eur
activation J Biochem,
Properties J Biol
of prothrombin 104, 549-557,
Phospholipidfactor V Bioa' -
On the interactions of with phospholipid ve-
10.
LIM, T.K., BLOOMFIELD, V.A. and NELSESTUEN, G.L. Structure of the prothrombinand blood clotting Factor X-membrane Biochemistry, 2, 4177-4181, 1977. complexes.
11.
AUSTIN, D.E.G. and RHYMES, I.L. Factor II assay (Taipan method) in "A laboratory manual of blood coagulation". Blackwell Scientific Publications, pp 46, 1975.
12.
VELTKAMP, J.K., DRION, E.F. and LOELIGER, E.A. Detection the carrier state in hereditary coagulation disorders I. Thromb Diath Haemorrh, 19-20, 279-303, 1968.
13.
BACHMANN, F., DUCKERT, F. and KOLLER, F. The Stuart-Prower factor assay and its clinical significance. Thromb Diath Haemorrh,L, 24-38, 1958.
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PURIFICATION OF II, VIII, IX & X
14.
LANGDELL, R.D., WAGNER, R.H. and BRINKHOLJS, antihemophilic factor on one-stage clotting Clin Med, 5, 637-647, 1953.
K.M. Effect of tests. J Lab
15.
Quantitative LAURELL, C.B. trophoresis in agarose gel them, 15, 45-52, 1966.
16.
BROWN, J.E., BAUGH, R.F. and HOUGIE,_C. Effect of exercise on the Factor VIII complex: a correction of the von Willebrand antigen and Factor VIII coagulant antigen increase. Thromb Res, 15, 61-67, 1979.
17.
LAEMMLI, U.K. Cleavage of structural assembly of the head of bacteriophage 685, 1970.
18.
SANDBERG, H. and ANDERSSON, of platelet factor 3 using K, 12, 113-124, 1979.
19.
Evidence for a second molecular BROWN, J.E. and HOLJGIE, C. Thrombos Haemostas, 46, Abstracts defect in hemophilia A. and Haemztasis, pp of VIII Intern. Congr. on Thrombosis 188, 1981.
estimation of proteins containing antibodies.
by elecAnal Bio-
proteins during the T4. Nature -227, 680-
L.-O. A highly sensitive assay a chromogenic substrate. Thromb
AFFINITY CHROMATOGRAPHY OF COAGULATION FACTORS II, PHOSPHOLIPID VESICLES VIII, IX AND X ON MATRIX-BOUND Lars-Olov Department School x) On
Andersson x> , Le Phuc
Thuy
and James
E. Brown
of Pathology, University of California San Diego of Medicine, La Jolla, California 92093, USA.
leave from Research Stockholm, Sweden.
Department,
Biochemistry,
KABI
AB,
(Received 28.4.1981; in revised form 3.8.1981. Accepted by Editor P. Wallen)
ABSTRACT
Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine were coupled to cyanogenbromide activated agarose gels. The gels obtained showed phospholipid-related procoagulant activity in the Russell's Viper Venom clotting test. Upon column 5tromatography containing of prothrombin complex concentrate in Ca Factor IX and Factor X were bound buffer, prothrombin, to the gel and could be eluted by desorption with buffer containing citrate. When purified Factor VIII/van Wille brand Factor dissolved in buffer without Ca2+ was applied to the column the main part of Factor VIII activity Desorption with 1 M NaCl did not elute any was bound. Factor VIII but 1 M KSCN did, suggesting that the Factor VIII-phospholipid binding mainly is dependent on hydrophobic interactions.
INTRODUCTION There is considerable evidence that phospholipid surfaces play an important role in the blood clotting process. They appear to be essential both for the activation of Factor X as well as for the activation of prothrombin. In the activation of prothrombin, phospholipid structures on the surface of the released platelet probably bind activated Factor V and activated Factor X thereby forming the prothrombinase complex which then transforms prothrombin to thrombin (l-3). The situation is probably similar in the formation of the Factor X activator complex about which relatively little is known. Phospholipid vesicles have been shown to bind Factor V (4) and Factor VIII (5,6) in the absence of Ca2+ and the factors II, VII, IX and X in the presence of Ca2+ KEYWORDS:
Affinity chromatography, phospholipid, tor VIII, Factor IX, Factor X. 481
Factor
II, Fac-
482
PURIFICATION OF II, VIII, IX & X
vo1.23, No.6
The binding of bovine prothrombin to phospholipid vesicles (7). has been studied in some detail (4,8-10) and the dissociation constant for a certain set of conditions has been determined to be around 2 x 10e6 M. In this study phospholipid vesicles have been coupled to a gel matrix and the adsorption and elution of coagulation factors II, VIII, IX and X has been studied. MATERIALS AND METHODS Materials The phosphatidylserine (PS) and phosphatidylethanolamine (PE) preparations were obtained from Sigma Chemical Co., St. The PS was prepared from brain and more than Louis, MO., USA. The PE was from brain and more than 95% pure. It was 98% pure. delivered as a solution in CH Cl. Dipalmitoyl-l-14C-phosphatidylcholine (PC) was obtained from New England Nuclear, Boston, solution. Mass., USA, in toulene-benzene The specific activity was 100 mCI/mmole. Biogel A 15 M and Affi-gel 10 was purchased from BioRad LaPreactivated Sepharose Macroboratories, Richmond, CA., USA. beads 6 MB was from Pharmacia Fine Chemicals, Uppsala, Sweden. Cyanogen bromide was from Sigma Chemical Co., St. Louis, MO., USA. The Human Prothrombin complex concentrate used was from the American Red Cross and has been prepared by DEAE-Sephadex Purified Factor VIII/van Willebrand Facadsorption and elution. tor complex was prepared by gel filtration on Biogel A 15 M of Kabi high purity Factor VIII. The void volume fractions were collected and frozen or used directly for the adsorption experiments. The specific activity was usually around 10 unitsfmg, the quotient Factor VIII R:Ag/Factor VIII activity around 3 and the quotient Factor VIII C:Ag/Factor VIII activity 1.8. Russell's Viper Venom and Taipan Snake Venom were obtained from Sigma Chemical CO., St. Louis, MO., USA. Preparation and coupling of phospholipid vesicles To 12.5 mg of PS in a poly-propylene tube was added 12.5 mg of PE in CH3Cl solution and 15 ug of 14C-labelled PC in touleneAfter mixing, the solvents were evaporated by benzene solution. immersing the tube in a water bath and blowing nitrogen through. When the tube was completely dry, 10 ml of 0.02 M imidazole, 0.15 M NaCl buffer pH 7.3 was added and nitrogen bubbled through for Then the tube was put in an ice bath and soniabout 5 minutes. cated for 8 minutes using a Heatsystems-Ultrasonics Inc., Plainview, USA, sonicator. For coupling of the phospholipid vesicle preparation to agasolution pH rose gel, 10 ml of Biogel A 15 M in 5 M K3P04-K2HP04 12.0 was activated with 20 mg CNBr/ml for 10 minutes at +5'C. The gel was then washed with ice-cold water and 10 ml of the PE-PS vesicle preparation was added. The pH of the suspension was adjusted to 9.2 and allowed to stand at room temperature for two After that the gel was washed with hours under slow stirring. imidazole-NaCl buffer and with imidazole buffer containing 1 M NaCl. Remaining reactive groups were blocked by treatment with 0.1 M ethanolamine overnight. Determination of prothrombin Prothrombin was assayed using Taipan Snake Venom (Oxyiranos scutellantus scutellantus) to convert the prothrombin to thrombin which was then determined by measuring clotting of bovine fibrinogen (11).
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Determination of Factor IX Factor IX was assayed by a one-stage method as described by Veltkamp et al. (12) using hemophilia B plasma. Determination of Factor X Factor X was determined by transforming it to activated form by incubation with Russell's Viper Venom followed by a clotting assay using Factor X deficient bovine plasma. The assay was performed essentially as described by Bachmann et al. (13). Determination of Factor VIII and related components Factor VIII activity was assayed using a one-stage method essentially according to Langdell (14) using human hemophiliac plasma as substrate. Factor VIII related antigen (F. VIII R:Ag) was determined using electroimmunoassay (15). Factor VIII coaguusing an immuno radiolant antigen (F. VIII C:Ag) was determined metric assay (IRMA) as described previously (16). Polyacrylamide gel electrophoresis SDS polyacrylamide gel electrophoresis was performed according to Laemmli (17). RESULTS Coupling of phospholipid vesicles Phosphatidylserine-phosphatidylethanolamine vesicles were prepared by sonication of suspensions containing 2.5 mg/ml of each phospholipid in 0.02 M imidazole, 0.15 M NaCl buffer pH 7.3. $4C-labelled phosphatidylcholine which had The vesicles contained been added before sonication. Coupling to Sepharose gel was accomplished by incubating the vesicles with CNBr-activated Sepharose 4B at pH 9.2 for two hours. After washing and blocking of remaining reactive groups with ethanolamine an afiquot of gel was taken out and subjected to scintillation counting after dissolving the gel in dimethylformamide. The degree of coupling as determined by the 14C-counting corresponded to 36 ng of phospholiBy amino acid analysis for serine and ethanolpid/ml of gel. amine the value obtained was 41 ug phospholipid/ml of gel. In separate experiments it was found that essentially the same degree of coupling of phospholipid was obtained when Affi-gel 10 In contrast the coupling was 60% lower when CNBr-actiwas used. vated Sepharose Macrobeads were used. Assay for phospholipid procoagulant activity using the Kussell's Viper Venom clotting test (18) showed that the phospholipid-Sepharose gel had an activity corresponding to 24 ng PS-PE phospholipid/ml of gel. Prothrombin, Factor IX and Factor X Prothrombin comnlex concentrate was dissolved in 0.02 M imibuffer pH 7.3 to give a solution dazole, 0.15 M NaC1,'5 mM CaCl containing 2.1 units prothrombfn/ml, 1.6 units Factor IX/ml and 1.7 units Factor X/ml. When 0.5 ml of this solution was applied to a mini-column containing 7 ml of PE-PS-Sepharose and equilibrated with the same buffer almost all prothrombin, Factor IX and Factor X were adsorbed to the gel as evident from Figure la. Upon desorption with 0.02 M imidazole, 0.15 M NaCl, 0.01 M citracomplex eluted. The recoveries te buffer 7.3,‘ the prothrombin were for prothrombin 75%, for Factor IX 86% and for Factor X 81%. In Figure 2 is shown the SDS polyacrylamide gel electrophoresis patterns obtained with starting material, the fraction passing through and desorption fraction.
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FIG. la Affinity cnromatography of 0.5 ml prothrombin complex concentrate (2.1 units/ml prothrombin, 1.6 units/ml Factor IX and 1.7 units/ml Factor X) on PE-PS-agarose gel. Column dimensions 1 x 9 The column was equilibrated with 0.02 M imidazole, 0.15 M cm. The elution buffer was 0.02 M NaCl, 5 mM CaCl2 buffer pH 7.3. 0.15 M NaCl 0.01 M citrate pH 7.3. w UV-adsorption imidazole, prothrombin activity AA Factor IX activity and at 280 nm, 0-0 Factor X activity. 0-0
FIG. lb Affinity chromatography of 2.0 ml prothrombin complex concentrate (3.6 units/ml prothrombin and 2.8 units/ml Factor IXj on PEColumn dimensions 1 x 9 cm. PS-agarose gel. The column was equilibrated with 0.02 M imidazole, 0.15 M NaCl, 5 mM CaC12 buffer pH The elution buffer was G.02 M imidazole, G.15 M NaCl, 0.01 7.3. prothromM citrate pH 7.3. u UV-adsorption at 280 nm, 0 -0 A Factor IX activity. bin activity and ATo test the binding capacity of the PE-PS Sepharose gel, 2.0 ml of prothrombin complex solution containing 3.6 units/ml pro-
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thrombin and 2.8 units/ml Factor IX was applied to the column. In Figure lb the results are shown. In this case about half of the prothrombin is bound and the other half passes through. In contrast more than 95% of the Factor IX is bound indicating that Factor IX is more strongly bound to phospholipid than prothrombin. Calculation of the amount of prothrombin bound gives the value 47 pg/ml of gel. After repeated use for prothrombin complex adsorption and elution part of the gel was tested for phospholipid procoagulant activity by the Russell's Viper Venom clotting test. The activivariation as with the newly ty was the same within experimental prepared gel.
FIG. 2 Polyacrylamide gel electroPhoresis of materials from the affinity chromatography shown in Figure la. A - starting prothrombin complex material, B - break through fraction, C - desorption fraction.
-’
ABC Factor VIII/van Willebrand Factor When a solution of Factor VIII/van Willebrand Factor, prepared by gel filtration of a high purity Factor VIII concentrate, was applied to a mini-column containing PE-PS-Sepharose part of Factor VIII activity was adsorbed and part passed through as seen in Figure 3. Desorption by 1 M NaCl did not result in any elution of Factor VIII activity. Subsequent elution with 1.0 M KSCN did however result in elution of considerable amounts of Factor VIII activity. KSCN was removed by gel filtration using Sephadex Total recovery G25 before determination of Factor VIII activity. of Factor VIII activity was 25% where 11% is unbound and 14% in The fairly low recovery of Factor VIII actithe desorption peak. vity is probably in part related to the observed lability of the eluted material in 1 M KSCN. Determination of Factor VIII R:Ag showed that the break through peak contained most of this materiThe al but there was also some present in the desorption peak. ratio Factor VIII R:Ag/Factor VIII activity was 13.4 for the break Assay for through fraction and 1.6 for the desorption fraction. Factor VIII C:Ag by IRMA showed a similar pattern as for the Factor VIII activity but the ratios Factor VIII C:Ag/Factor VIII activity were different in the two peaks being 2.4 in the break through fraction and 0.55 in the desorption fraction.
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PLIRIFICATIONOF II, VIII, IX & X
~01.23,
FIG. 3 Affinity chromatography of Factor VIII/van Willebrand Factor on PE-PS-agarose gel. Column dimensions 1 x 3 cm. The column was equilibrated with 0.02 M imidazole, 0.1 M NaCl and 0.02% The column was eluted with 1 M NaN3 pH 6.8. NaCl and then 1 M KSCN in the equilibrating bufFactor VIII activity, Afer. 0-@ A Factor VIII C:Ag, M Factor VIII R:Ag. DISCUSSION Phospholipid vesicles can be coupled to agarose gels and their activities as regards procoagulant activity and binding of coagulation factors is preserved. This indicated that the structures of the vesicles does not change very much during the coupling and subsequent washing procedures. The stability of the linkage between phospholipid vesicles and the agarose gels appears adequate as only trace amounts of phospholipid leaks from the gel during the various desorption and washing steps. It is possible that the matrix-bound vesicles are fixed to the gel by multipoint attachment; several PS and PE molecules in the same vesicle might be covalently linked to the agarose. The fairly low degree of coupling obtained could be due to the necessity for multipoint attachment in order to get a stable matrix-bound vesicle that is not removed in the washing steps. However, one can not exclude the fact that other effects like steric hinderance are in part responsible for the low coupling yields obtained. As expected, prothrombin, Factor IX and Factor X are adsorbed to the matrix-bound vesicles when calcium ions are present. Very good recoveries are obtained upon desorption with citratecontaining buffer. The degree of purification is also good judging from the specific activities obtained and the SDS polyacrylamide gel electrophoresis pattern. It is likely that Factor VII and protein C also are present in the desorption material. The capacity of the gel to adsorb prothrombin is surprisingly high,
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the weight ratio between bound prothrombin and matrix-bound phospholipid is 1.3 which can be compared with the value 1.2 obtained from studies of prothrombin binding to phospholipid vesicles in solution (9). The observed stronger binding of Factor IX as compared to prothrombin may have a functional significance as the concentration of Factor IX in plasma is-considerably lower-than that of prothrombin and thus stronger binding of Factor IX is necessary in order to get a suitable concentration of Factor IX on phospholipid. The data obtained in the studies of Factor VIII/van Willebrand Factor are not as clearcut as those obtained with the vitamin K dependent coagulation factors. This probably depends on the fact that the Factor VIII/van Willebrand Factor complex applied to the column is not a single entity but a series of large Different parts of the commolecular weight protein complexes. plexes may react differently and give rather complicated binding and dissociation reactions. However, the observation that almost all of the Factor VIII related antigen passes through the column and only a very small amount is desorbed, suggests that the von Willebrand Factor part of the complex is not adsorbed to the phos This is in agreement with the results of a recent stupholipid. dy on binding of Factor VIII/van Willebrand Factor to phospholiFactor VIII on the other hand is pid vesicles in solution (6). This can be explained by combound although not quantitatively. petitive effects of binding to von Willebrand Factor resulting in part of Factor VIII eluting as Factor VIII/van Willebrand Factor The observation complex and part being bound to phospholipid. that desorption with buffer containing 1 M NaCl does not elute any Factor VIII activity whereas desorption with 1 M KSCN does, suggests that the binding of Factor VIII to phospholipid is not primarily dependent on electrostatic interactions but rather on hydrophobic interactions. A somewhat surprising finding is that Factor VIII activity is higher than Factor VIII C:Ag in the desorption fraction. A possible explanation would be that the Factor VIII C:Ag structure detected by our antibody preparation is more labile to KSCN solution than is the structures necessary for Factor VIII activity. This is supparted by the recent finding (19) that Factor VIII C: Ag as measured by the same antibody preparation is more labile to heat than is the Factor VIII activity. The results obtained in this study show that a phospholipid vesicle-agarose gel can be used for affinity chromatography of a number of coagulation factors. Further it can probably also be casused as a model system for the two steps in the coagulation cade that require a phospholipid surface, that is the activation The observation of Factor X and the activation of prothrombin. that the gel particles do have high procoagulant activity in the Russell's Viper Venom test support that concept. ACKNOWLEDGEMENTS for her secretarial asWe are grateful to Ms. Mona Stendahl This work was supported by USPHS Research Grant HLsistance. 15230 and HL-06132-01.
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