cm9-3&18/:8:0701m53 somo:o
THROMBOSISRESEARCH Vd. 13.PP.D-60. 0 perpmaa mxs Lid. 1978.Printedin chat Britain.
LOCATION OF FACTCR VIII COAGULANT ACTIVITY IN .PELATIO:YI TC FACTOR VIII RELATED ANTIGEN AFTER RAPID TWO-DIMENSIONALIMMUNOFI.FCTROPHO.P.ESIS Jennifer Voke, HaemophiliaCentre, Departmentof Haematoloa, Royal Free ilospital,London NWj, UK.
(Received 6.4.1978; in revised form 3.5.1978. Accepted by Editor A.L. Bloom)
of plasma A method for rapid two-dimensionalisununoelectrophoresis factor VIII is described and compared with the conventionalover.nightmethod. $ completing both dimensions of the immunoelectrophoresis at 8-10 C within 4 hours, a proportionof the factor VIII coagulant activity is retained and this is demonstratedby means of a haemophilic plasma/agarosegel laid over the immune gel containing the precipitinarcs. Fibrin forms over the whole area of contact with normal factor VIII precipitinarcs. It is visible earliest and most clearly over the slow-movingfactor VIII. This clotting can be completely prevented by prior immersion of the immune gel in specific human antibody to factor VIII coagulant activity. Fibrin does not form over the factor VIII precipitin arcs of plasmas from patients with severe haemophilia. This method will be valuable in demonstratingthe relationshipof clotting activity to electrophoretic mobility in the various forms of factor VIII found in von Willebrand'sdisease and under experimentalconditions.
INTRODUCTION The physiologicalrelationshipsbetween factor VIII related antigen (F VIIIR:Ag}, factor VIII von Willebrand factor (F VIII:WF) and factor VIII coagulantactivity (F VIII:C) are not-yet clearly established. There is experimental evidence for a large molecular form of factor VIII (1 - 2 million daltons) which can be dissociatedinto subunits by reduction (l,2,j)and for an associatedlow molecular weight piece which has coagulant activity but does not react with precipitatingrabbit antibody to F VIIIR:Ag or have F VIII:WF activity. There is also evidence that this low molecular weight piece dissociatesfrom the large molecular forms in plasma from von Willebrand's disease patients after F VIII infusions (4) and can also be dissociated by gel filtration of normal plasma at high ionic strength (5,6,7). 53
54
LOCdTION
OF FVTII
=1CTTVTTY
Vol.13,No.l
Agarose gel immunoelectrophoresis (IE?) suggests -hat there are multiple molecular forms of F VIiIR:Ag in normal plasma asd that these -an be separated to some extent by cryoprecipitation, agarose gel chromatography and tw.odimensional (2D) IEP (2). Many patients wi*h low F VIII:::d.~e to a variant form of von Willebrand's disease lack slow-moving F %TIIR:Ag but have an increase in fast-moving F VIIIR:Ag on 2D IE? (3,lO). Patients ;Jithother conditions have been found to have 2 raizd level of F VIiIR:Ag due mainly to an increase in intermediate and fast mobility F VIII @,ll). In spite of the evidence for different forms of F VIIIR:Ag, little is known about the coagulant activity associated with them. This is partly due to technical difficulties involved in maintaining this labile activity during the experimental procedures involved. Coagulation can be detected in 2 plasma/agarose mixture by an increase in the opacity of the gel as fibrin forms (12). After Laurel1 one-dimensional IEP of normal plasma against rabbit precipitating antibody to F VIIIR:Ag, the gel is covered with a second agarose gel containing F VIII deficient plasma (13j. Fibrin fbrms over the precipitin line thus demonstrating F VIII:C in the immunoprecipitate. This fibrin formation can be prevented by immersion of the electrophoresis plate in human antibody to F VIII:C before application of the plasma/agarose layer. This method has not previously been applied to 2D IEP as F vIII:C is lost during the time required for conventional 2D IEP. The present study was therefore undertaken with two main objectives:- first to develop 2 rapid 2D IEP method which would preserve a proportion of F vTII:C, and to compare this rapid IEP with the conventional method, and secondly to apply the coagulation gel method to the 2D IEP to correlate F VIII:C with specific fractions of F VIIIR:Ag.
MAT!SUALS AND METHODS
Venous blood was anticoagulated with citrate (9 parts blood to 1 part O.ljM trisodium citrate) and the plasma separated by centrifugation at 4'C at 15OOg for 20 minutes. Polystyrene tubes were used. Plasma samples tested both by 2D IEP and the coagulation gel method were kept at 4'C and tested within a few hours of collection. Samples used only for 2D IEP were either tested on the day of collection or after l-3 weeks at -4OOC. Only samples with F'VIII:C levels within the normal range were used as normal controls. Haemophilic plasmas were obtained from patients who had not been treated for at least 2 weeks and who had undetectable levels of F VIII:C. Precipitating rabbit antibody to F VlIIR:Ag (Behringwerke) was used at a dilution of 1 in 100 in 1% agarose for the second dimension of the IEP. 'Ihis antibody had no neutralizing effect on F VIII:C. Human antibody to F VIII:C was obtained from a patient with severe haemophilia and high-titre antibody to F VIII:C (4000 u/ml) as assayed after 1 hour's incubation (14). lml of plasma was diluted with 9 ml 0.9% saline for use. The electrophoresis buffer, pH 8.6, contained 10.31 g/l sodium 'barbitone and 1.325 g/l barbituric acid in distilled water. 5 litres of buffer were needed in one electrophoresis tank for 2 gels. Double layers of surgical lint were used as wicks.
LOClTTOS
vol.l~,so.1
OF FVTTT
ACTIVITY
55
1% agarose (piles-Seravao' in barbirove el+c-ro.A_ buffer was !‘sed A.. for zhe ._ _ aeis and 2;%$ in 0.9% saline for the clotting gels. Aliquots of s-orad _..sterile universais until need+ gel :-1?re " _ at i 3 '1 -_ .-.,.
phoro3is
Gels were prepared 'between glass plates lrrxn thick using 1,mnplastic Y-shaped farmers he1.d in place with bulldog clips. Coated glass plates were prepared by dipping the plates ir,warm 0.1-0.2s agarose and sllowing shem zo drain. 31s prevented movement of the gels between the firs; and second s-ages of the IE?. The ag.aroae .&as pipetxed into the space oetiieen 2 veriizal $;a115 *EI ir: .diametarwere 2';: glates. T”cs se13 c;esp_:rxi lc‘i? :c 3 x hl. in 2~ gels at Lntervaia of 2cm as shown in i"iqJre 1.
FIG. 1 Diagram of the immunoelectrophoresis plate prior to pouring tne second dimension gel. ~~u~m~bered arrows show the directions of the electrophorstiz runs.
?? Glass
plate.
First dimension gel left in place for second dimension run. The rest of the first dimension gel has been removed. a
U-shaped former ssndwiched between two glass plaxs.
??i:iellpunched
in agarose.
Samples of neat plasma were applied to the gels using a Ziptrol capillary pipette. Test and cop+ .Acrol plasmas were applied alternately so that 4 test and iicontrol plasmas were run on each plate. Slectrophoresia was carried out at 3-100, ,_. on a zooling platten. The first dimension xas run for 1 hour at 60 volts/gel as measured across the
56
LOCATION
OF FVTII
ACTIVITY
Vol. 13,Xo.l
gel from edge to edge in zhe direction of tne current, (a‘bout 0.25 mA/m wid-lnof gel). After 'he first stage (gel without antibody) gel strips were removed as shown in figure 1 and, using the U-shaped formers again, gel containingrabbit antibody was poured around the first dimension strips containingthe separatedplasma proteins. The second electrophoresis,run at right angles to the first, was at 8-10°C for 2$ hours at 6Q volts/gel measured across the gel in the direction of the current (about 0.2 mA/rmn width of gel). To compare the rapid 2D IEP with the conventionalslow method, 20 samples were also run at 15 volts/gel for 16 hours in the second dimension. 25 normal subjects, 3j haemophiliacsand 10 patients with von Willebrands disease were tested by 2D IEP. 10 of the haomophiliacshad antibody to F vIII:C and two of these patients were tested on four differentoccasions when antibody titres were at different levels. Plasma samples were diluted in barbitone buffer when necessary, to give approximatelynormal levels of F VIIIR:Ag.
The mobility of the F VIIIR:Ag was determined by measuring the distance of the peak of the precipitinarc from the first dimension baseline, drawn through the centres of the wells. The peak distance of each patient's sample was compared with that of a normal donor pool (29) and the mobility expressed as a ratio of patient/normal. Known fast mobility von Willebrand plasmas were used as abnormal controls. All samples were tested on at least two occasions. Any abnormality in the shape of the precipitinarc was noted. Each clotting gel was prepared by mixing equal volumes (5ml) of haemophilicplasma warmed briefly to 37'C, with 0.6% molten agarose in saline at 56OC. Gels were poured using the same U-shaped farmers and left to stand at 4'C to set. The electrophoresisgel was immersed in a dish of 0.025 M calcium chloride for 3 minutes at 37OC and was then overlaid with the clotting gel. Both gels were kept in a moist chamber at 4'C and examined after 1 hour. As soon as fibrin formation was apparent in the clotting gel it was fixed by immersion in 4% formaldehydesolution prior to photographyover dark ground illumination. To check that the coagulantactivity was due to F vIII:C, one of two identical electrophoresisgels was immersed in diluted haemophilicplasma containinga high titre antibody to F VIII:C for 30 minutes at 37'C after completionof the second dimension of the electrophoresisand before recalcification. The second gel was immersed in the same dilution of a haemophilic plasma without antibody for the same length of time.
RESULTS
Clear, reproducibleF VIII precipitinarcs were obtained using the four-hour 2D electrophoresismethod. The mobility ratios measured by the rapid method were no different from those obtained by the conventionalslow electrophoresis. Differencesin F VIIIR:Ag levels (obtainedby serially diluting a plasma with a high level of F VIIIR:Ag in barbitone buffer! did not alter the mobility compared with an undiluted normal pool. However, precipitinarcs were difficult to see at levels of F VIIIR:Ag less than 10% of normal.
Vol.13,No.l
57
L.OCXTIO?; OF F\?KII ACTIVITY
Xo significans difference was found between +,neF VIII mobility ratios of 25 normal donors and 4C patients with severe haemophilia (Table 1). 5 of zhe 10 von Willebrand plasmas gave consistently fast mobility ratios, varying F VIII from 1.3 - 1.73. There was a significant difference between r;i=e mo'oility ratios of the normal donors and the 6 von Uillebrand pa:ienzs with mobility ratios greater than 1.3 (p =
THROMBOSISRESEARCH Vd. 13.PP.D-60. 0 perpmaa mxs Lid. 1978.Printedin chat Britain.
LOCATION OF FACTCR VIII COAGULANT ACTIVITY IN .PELATIO:YI TC FACTOR VIII RELATED ANTIGEN AFTER RAPID TWO-DIMENSIONALIMMUNOFI.FCTROPHO.P.ESIS Jennifer Voke, HaemophiliaCentre, Departmentof Haematoloa, Royal Free ilospital,London NWj, UK.
(Received 6.4.1978; in revised form 3.5.1978. Accepted by Editor A.L. Bloom)
of plasma A method for rapid two-dimensionalisununoelectrophoresis factor VIII is described and compared with the conventionalover.nightmethod. $ completing both dimensions of the immunoelectrophoresis at 8-10 C within 4 hours, a proportionof the factor VIII coagulant activity is retained and this is demonstratedby means of a haemophilic plasma/agarosegel laid over the immune gel containing the precipitinarcs. Fibrin forms over the whole area of contact with normal factor VIII precipitinarcs. It is visible earliest and most clearly over the slow-movingfactor VIII. This clotting can be completely prevented by prior immersion of the immune gel in specific human antibody to factor VIII coagulant activity. Fibrin does not form over the factor VIII precipitin arcs of plasmas from patients with severe haemophilia. This method will be valuable in demonstratingthe relationshipof clotting activity to electrophoretic mobility in the various forms of factor VIII found in von Willebrand'sdisease and under experimentalconditions.
INTRODUCTION The physiologicalrelationshipsbetween factor VIII related antigen (F VIIIR:Ag}, factor VIII von Willebrand factor (F VIII:WF) and factor VIII coagulantactivity (F VIII:C) are not-yet clearly established. There is experimental evidence for a large molecular form of factor VIII (1 - 2 million daltons) which can be dissociatedinto subunits by reduction (l,2,j)and for an associatedlow molecular weight piece which has coagulant activity but does not react with precipitatingrabbit antibody to F VIIIR:Ag or have F VIII:WF activity. There is also evidence that this low molecular weight piece dissociatesfrom the large molecular forms in plasma from von Willebrand's disease patients after F VIII infusions (4) and can also be dissociated by gel filtration of normal plasma at high ionic strength (5,6,7). 53
54
LOCdTION
OF FVTII
=1CTTVTTY
Vol.13,No.l
Agarose gel immunoelectrophoresis (IE?) suggests -hat there are multiple molecular forms of F VIiIR:Ag in normal plasma asd that these -an be separated to some extent by cryoprecipitation, agarose gel chromatography and tw.odimensional (2D) IEP (2). Many patients wi*h low F VIII:::d.~e to a variant form of von Willebrand's disease lack slow-moving F %TIIR:Ag but have an increase in fast-moving F VIIIR:Ag on 2D IE? (3,lO). Patients ;Jithother conditions have been found to have 2 raizd level of F VIiIR:Ag due mainly to an increase in intermediate and fast mobility F VIII @,ll). In spite of the evidence for different forms of F VIIIR:Ag, little is known about the coagulant activity associated with them. This is partly due to technical difficulties involved in maintaining this labile activity during the experimental procedures involved. Coagulation can be detected in 2 plasma/agarose mixture by an increase in the opacity of the gel as fibrin forms (12). After Laurel1 one-dimensional IEP of normal plasma against rabbit precipitating antibody to F VIIIR:Ag, the gel is covered with a second agarose gel containing F VIII deficient plasma (13j. Fibrin fbrms over the precipitin line thus demonstrating F VIII:C in the immunoprecipitate. This fibrin formation can be prevented by immersion of the electrophoresis plate in human antibody to F VIII:C before application of the plasma/agarose layer. This method has not previously been applied to 2D IEP as F vIII:C is lost during the time required for conventional 2D IEP. The present study was therefore undertaken with two main objectives:- first to develop 2 rapid 2D IEP method which would preserve a proportion of F vTII:C, and to compare this rapid IEP with the conventional method, and secondly to apply the coagulation gel method to the 2D IEP to correlate F VIII:C with specific fractions of F VIIIR:Ag.
MAT!SUALS AND METHODS
Venous blood was anticoagulated with citrate (9 parts blood to 1 part O.ljM trisodium citrate) and the plasma separated by centrifugation at 4'C at 15OOg for 20 minutes. Polystyrene tubes were used. Plasma samples tested both by 2D IEP and the coagulation gel method were kept at 4'C and tested within a few hours of collection. Samples used only for 2D IEP were either tested on the day of collection or after l-3 weeks at -4OOC. Only samples with F'VIII:C levels within the normal range were used as normal controls. Haemophilic plasmas were obtained from patients who had not been treated for at least 2 weeks and who had undetectable levels of F VIII:C. Precipitating rabbit antibody to F VlIIR:Ag (Behringwerke) was used at a dilution of 1 in 100 in 1% agarose for the second dimension of the IEP. 'Ihis antibody had no neutralizing effect on F VIII:C. Human antibody to F VIII:C was obtained from a patient with severe haemophilia and high-titre antibody to F VIII:C (4000 u/ml) as assayed after 1 hour's incubation (14). lml of plasma was diluted with 9 ml 0.9% saline for use. The electrophoresis buffer, pH 8.6, contained 10.31 g/l sodium 'barbitone and 1.325 g/l barbituric acid in distilled water. 5 litres of buffer were needed in one electrophoresis tank for 2 gels. Double layers of surgical lint were used as wicks.
LOClTTOS
vol.l~,so.1
OF FVTTT
ACTIVITY
55
1% agarose (piles-Seravao' in barbirove el+c-ro.A_ buffer was !‘sed A.. for zhe ._ _ aeis and 2;%$ in 0.9% saline for the clotting gels. Aliquots of s-orad _..sterile universais until need+ gel :-1?re " _ at i 3 '1 -_ .-.,.
phoro3is
Gels were prepared 'between glass plates lrrxn thick using 1,mnplastic Y-shaped farmers he1.d in place with bulldog clips. Coated glass plates were prepared by dipping the plates ir,warm 0.1-0.2s agarose and sllowing shem zo drain. 31s prevented movement of the gels between the firs; and second s-ages of the IE?. The ag.aroae .&as pipetxed into the space oetiieen 2 veriizal $;a115 *EI ir: .diametarwere 2';: glates. T”cs se13 c;esp_:rxi lc‘i? :c 3 x hl. in 2~ gels at Lntervaia of 2cm as shown in i"iqJre 1.
FIG. 1 Diagram of the immunoelectrophoresis plate prior to pouring tne second dimension gel. ~~u~m~bered arrows show the directions of the electrophorstiz runs.
?? Glass
plate.
First dimension gel left in place for second dimension run. The rest of the first dimension gel has been removed. a
U-shaped former ssndwiched between two glass plaxs.
??i:iellpunched
in agarose.
Samples of neat plasma were applied to the gels using a Ziptrol capillary pipette. Test and cop+ .Acrol plasmas were applied alternately so that 4 test and iicontrol plasmas were run on each plate. Slectrophoresia was carried out at 3-100, ,_. on a zooling platten. The first dimension xas run for 1 hour at 60 volts/gel as measured across the
56
LOCATION
OF FVTII
ACTIVITY
Vol. 13,Xo.l
gel from edge to edge in zhe direction of tne current, (a‘bout 0.25 mA/m wid-lnof gel). After 'he first stage (gel without antibody) gel strips were removed as shown in figure 1 and, using the U-shaped formers again, gel containingrabbit antibody was poured around the first dimension strips containingthe separatedplasma proteins. The second electrophoresis,run at right angles to the first, was at 8-10°C for 2$ hours at 6Q volts/gel measured across the gel in the direction of the current (about 0.2 mA/rmn width of gel). To compare the rapid 2D IEP with the conventionalslow method, 20 samples were also run at 15 volts/gel for 16 hours in the second dimension. 25 normal subjects, 3j haemophiliacsand 10 patients with von Willebrands disease were tested by 2D IEP. 10 of the haomophiliacshad antibody to F vIII:C and two of these patients were tested on four differentoccasions when antibody titres were at different levels. Plasma samples were diluted in barbitone buffer when necessary, to give approximatelynormal levels of F VIIIR:Ag.
The mobility of the F VIIIR:Ag was determined by measuring the distance of the peak of the precipitinarc from the first dimension baseline, drawn through the centres of the wells. The peak distance of each patient's sample was compared with that of a normal donor pool (29) and the mobility expressed as a ratio of patient/normal. Known fast mobility von Willebrand plasmas were used as abnormal controls. All samples were tested on at least two occasions. Any abnormality in the shape of the precipitinarc was noted. Each clotting gel was prepared by mixing equal volumes (5ml) of haemophilicplasma warmed briefly to 37'C, with 0.6% molten agarose in saline at 56OC. Gels were poured using the same U-shaped farmers and left to stand at 4'C to set. The electrophoresisgel was immersed in a dish of 0.025 M calcium chloride for 3 minutes at 37OC and was then overlaid with the clotting gel. Both gels were kept in a moist chamber at 4'C and examined after 1 hour. As soon as fibrin formation was apparent in the clotting gel it was fixed by immersion in 4% formaldehydesolution prior to photographyover dark ground illumination. To check that the coagulantactivity was due to F vIII:C, one of two identical electrophoresisgels was immersed in diluted haemophilicplasma containinga high titre antibody to F VIII:C for 30 minutes at 37'C after completionof the second dimension of the electrophoresisand before recalcification. The second gel was immersed in the same dilution of a haemophilic plasma without antibody for the same length of time.
RESULTS
Clear, reproducibleF VIII precipitinarcs were obtained using the four-hour 2D electrophoresismethod. The mobility ratios measured by the rapid method were no different from those obtained by the conventionalslow electrophoresis. Differencesin F VIIIR:Ag levels (obtainedby serially diluting a plasma with a high level of F VIIIR:Ag in barbitone buffer! did not alter the mobility compared with an undiluted normal pool. However, precipitinarcs were difficult to see at levels of F VIIIR:Ag less than 10% of normal.
Vol.13,No.l
57
L.OCXTIO?; OF F\?KII ACTIVITY
Xo significans difference was found between +,neF VIII mobility ratios of 25 normal donors and 4C patients with severe haemophilia (Table 1). 5 of zhe 10 von Willebrand plasmas gave consistently fast mobility ratios, varying F VIII from 1.3 - 1.73. There was a significant difference between r;i=e mo'oility ratios of the normal donors and the 6 von Uillebrand pa:ienzs with mobility ratios greater than 1.3 (p =
