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Effect of exercise on the factor VIII complex: A correlation of the von Willebrand antigen and factor VIII coagulant antigen increase
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RESEARCH 15; 61-67 Printed Press Ltd.1979.
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EFFECT OF EXERCISE ON THE FACTOR VIII COMPLEX: A CORRELATION OF THE VON WILLEBRAND ANTIGEN AND FACTOR VIII COAGULANT ANTIGEN INCREASE
James E. Brown, Robert F. Baugh, and Cecil Hougr= Department of Pathology University of California, San Diego, School of Medicine La Jolla, California 92093 USA
in revised form 9.2.1979. (Received 15.12.1978; Accepted by Editor K. M. Brinkhous)
ABSTRACT Ten volunteers ran 314 miles as fast as possible and blood was drawn prior to and immediately following the exercise. Plasma factor VIII coagulant activity rose an average of 187% while the factor VIII coagulant antigen rose only an average of 107% and correlated better with the two-stage clotting assay (102%) and the von Willebrand factor antigen (115%). It is concluded that the disproportionate rise in factor VIII procoagulant activity above the von Willebrand factor is observed only when using the one-stage factor VIII clotting assay.
INTRODUCTION Rizza (1) originally observed that factor VIII levels rose in human blood following exercise. Prentice and co-workers (2) demonstrated a similar rise in plasma factor VIII levels, but extended the study to include the factor VIII-related antigen (von Willebrand antigen) which was reported to rise proportionately following a half mile run. Stibbe (3), however, reported that factor VIII coagulant activity in ten subjects rose an average of 126% following a five minute vigorous exercise, whereas factor VIII antigen (vWf) rose an average of only 46%. In order to determine whether the disproprotionate rise of factor VIII and von Willebrand factor is real or an artifact of the one-stage clotting rest, we used a two-stage assay as well as an immunoradiometric assay (IRMA) for factor VIII procoagulant activity (4, 5) as modified from that reported by Peake and Bloom (6). MATERIALS AND METHODS Volunteers among laboratory personnel (age range 21-56), five males and five females, were asked to run as fast as possible 3/4 miles (3 laps) around a circular track. All but one of the subjects exercised routinely, but only M.K. ran regularly. Blood (9 ml) was collected into polypropylene tubes containing 1 ml of 0.1 M buffered citrate one half hour before and within 61
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five minutes after the run. Platelet-poorplasma was obtained by centrifugation for 20 minutes at 1,000 g and room temperature. The one-stage assay for factor VIII used was that previously described (7) and employed as a standard, pooled plasma from ten individuals. The two-stage technique for factor VIII was modified from that of Pool and Robinson (8). Lyophilisedbovine serum was used at 4 mg/ml (one part) as a source of factor XII, XI, IX and X and aluminum hydroxide-adsorbedhemophilic plasma at l/20 was included with the bovine serum to elevate the factor V. The plasmas to be tested for factor VIII were adsorbed with aluminum hydroxide for 20 minutes (one part aluminum hydroxide + 19 parts plasma) and incubated (1 part) at 37OC with the bovine serum (1 part) in the presence of optimal cephalin (1 part) and 25 mM calcium chloride (1 part). Aliquots of this incubationmixture, one part, and one part of .025 M calcium were added simultaneouslyfollowing 5, 10 and 15 minutes of incubation to pooled normal plasma that had been aged (as whole blood) in 0.01 M EDTA for 48 hours at 4oC. The clotting times were then determined. The blank time (without adsorbed plasma) was increased from 60 seconds to 170 by using EDTA-aged plasma compared with normal human plasma as substrate. Aging the bovine serum diluted l/20 for 48 hours at 4oC depleted 99% of the factor Xa present when originally reconstitutedas measured by a spectrophotometricassay (9) and hence also increased the blank time of the assay. The IRMA for factor VIII procoagulant antigen was performed according to the method of Reisner et al. (5) with the exception that the Fab fragment of the IgG factor VIII inhibitormolecule was used rather than the Fab'. A hemophilicwith a factor VIII inhibitor-titerof about 2,000 Bethesda units/ml was obtained and IgG was purified from the inhibitorplasma by chromatography on QAB A-50 Sephadex equilibratedwith ethylene diamine, .037 M, and acetic acid, .073 Mj pH 7.0. The purified IgG following lyophilizationhad a specific activity of 298 Bethesda units/mg. The IgG was hydrolyzedwith papain by the method of Porter (10) and the 40-80X ammonium sulfate cut used as the Fab fragment. The inhibitor titer had dropped (98%) to 7 Bethesda units/mg (assume for Fab an El% = 10). This was dialyzed vs. 0.1 M phosphate, pH 7.5, and frozen at -200C. It had a major band at 55,OOrmolecular weight upon SDS gel electrophoresisin 5% acrylamide. Aggregateswere removed by gel filtrationon Bio-Gel A 1.5 M prior to labeling and 50 ug labeled with 1 mCi of 1251 by the chloramine T method (11). The free iodine was separated from the protein incorporatediodine by chromatographyon Sephadex G-25 and the iodinated Fab molecule, 5-10 uCi/pg protein (about 0.8 ml) mixed with a preparation of bovine factor VIII (0.2 ml) containing150 units/ml of bovine factor VIII (5 mg/ml) in 0.02 M imidazole, 0.15 M NaCl, pH 6.9, for one hour at 370C. The mixture was then applied to a Bio-Gel A 1.5 M column (0.9 x 56 cm) equilibratedwith 0.02 M imidazole,0.15 M NaCl, 0.02 M imidazole,0.15 M NaCl, 0.02% sodium azide, pH 6.9. Fractions (1.6 ml each) at the exclusion volume (complexof Fab and factor VIII) ranged from 0.84 to 1.2% (average 1.02%) of the total recovered radioactivity(five preparations) from the column. These were pooled and concentratedto 0.8 ml using a 75,000 molecular weight cut-off collodion bag (Schleicherand Schuell, Inc., Keene, NH). 125I Fab from the factor VIII was achieved by The dissociationof the mixing enough 1 M glycine HCl, pH 2.57 (0.11 ml), with the complex (0.8 ml) to bring the pH down to 2.8, followed by incubationat 37OC for one hour and then addition of 0.10 ml of 20 mgfml BSA solution. Chromatographyof this complex on a 0.9 x 56 cm column of Bio-Gel A 0.5 M using 0.1 M acetate, 0.5 M NaCl, pH 4.0, as buffer yielded two peaks of radioactivityand 40-50% dissociation
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(second peak). The second peak upon a 5% acrylamide gel sliced into 2 mm segments shwed a major band at 55,000 molecular weight. The second peak from the Bio-Gel A 0.5 M column was concentrated by a collodion membrane (25,000 molecular weight cut-off) to about 1 ml and contained about 300,000-500,000 cpm. A typical assay for factor VIII coagulant antigen (VIII:CAg) was performed as follows: 110 h of plasmas diluted l/20 and l/40 into 0.02 M imidazole, 1% BSA, 0.15 M NaCl, 0.02% NaN3, pH 7.3 for assays before and after exercise, respectively, were added to a 400 ul polypropylene tube. Fifty pl of a solution of 20 mg/ml normal human IgG in pH 7.3 imidazole-saline was added as carrier and then 10 ul of 1251 Fab inhibitor was added, approximately 2,200 cpm. These were incubated for 18 hours at room temperature and then 105 ul of saturated (4oC) ammonium sulfate was added followed by centrifugation (2OC) for 20 minutes at 10,000 g. Supernatant fluid was removed and the pellet counted in an automated well type gamma counter. Assays were done in duplicate both before and after exercise and on fresh as well as frozen plasmas. A typical standard curve was as follows Plasma dilution l/5 l/10 l/20 l/40 typical hemophilic plasma l/5 bovine plasma l/5 buffer bank
% bound radioactivity 53.4 44.1 35.8 30.3 22.4 42.6 12.0
and was linear when plotted on log-log paper. The sensitivity of the IRMA for factor VIII:CAg is similar to the 3% level previously reported (4, 5). A typical hemophilic plasma (5 samples) exhibited a 5% cross reaction. It is unknown why the consistently higher percentage of Fab binding (beyond the buffer blank) with the hemophilic plasmas is observed. Perhaps the increased protein in the ammonium sulfate precipitate when testing hemophilic plasma at a l/5 dilution is responsible for non-specifically carrying with it more Fab fragment. The IRMA was further validated as being specific for factor VIII in a blind study with the University Hospital in San Diego. Twelve patient plasma samples (< 1% to 100% VIII:C) were assayed for factor VIII:CAg and compared with the factor VIII:C values as determined independently using a onestage clotting assay (7). Except for the three severe hemophilics (~1% VIII:C) which assayed at 4-6X VIII:CAg, a good correlation was obtained in all cases but one. This patient had a factor VIII inhibitor (4 Bethesda units/ml) with VIII:C less than 1X, but assayed at 33% VIII:CAg. Bovine plasma exhibited a parallel dose response curve and assayed 44% relative to human plasma for VIII:CAg. The von Willebrand factor antigen was measured by the Laurel1 immunoelectrophoresis method (12). The percentage increases of the parameters measured were calculated by subtracting the pre-exercise value from the post exercise value and then dividing by the pre-exercise value and multiplying by 100. Bovine factor VIII (3,000-fold pure) was prepared as previously described (13), as was bovine thrombin (1,300 units/mg) (14). RESULTS
The subjects were ranked in the order in which they finished the 314 mile run, along with values for the factor VIII parameters measured. Certain trends can be seen in examining the data. The time with which one runs was roughly indicative of the amount of exertion involved in the exercise and hence the degree of increase in factor VIII parameters. On a separate
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occasion two individuals (J.B. and C.C.) listed in Table 1 jogged at a brisk
TABLE1 Effect
of Exercise on Plasma Factor VIII Complex Units/ml Factor
314 Subject
K.P. A.M. J.B. M.K. C.J. C.C. S.L. M.M. C.H. C.L. Average
mile time
VIII:C
One-stage Pre
Two-stage
Post
Pre
VIII:CAg
Post
Pre
Post
vWf:Ag Pre
Post
4:31 1.02 3.90 1.05 2.88 1.07 2.84 0.89 2.33 4:34 0.80 2.48 1.76 2.64 0.98 2.04 1.25 3.19 4:43 0.91 3.44 0.94 2.62 1.10 2.57 0.65 1.78 4:51 1.44 3.28 1.80 3.44 0.96 2.00 1.89 3.23 5:Ol 1.08 4.06 0.87 2.88 0.81 1.94 0.85 2.35 5:05 1.52 5.59 0.92 2.00 1.48 2.63 1.93 5.25 5:51 0.80 1.64 1.56 2.20 0.86 1.71 0.77 1.68 5:58 1.26 2.84 0.94 2.90 0.98 2.64 0.83 2.05 6:32 1.20 2.22 1.33 1.76 1.15 1.70 2.13 2.53 6:32 0.92 2.16 1.36 1.92 1.64 2.72 1.25 2.33 ____________________~~____~~~~__~~~~~~~~~~~~~~~~~~~~~~__~~~~~_~~~_~_ 5:22
1.10
Increase
3.16 187%
1.25
2.52 102%
1.10
2.28
1.24
107%
pace for one half hour and one (T.L.) ran in place for ten minutes. results of these less vigorous exercises are shown in Table 2.
2.67 115%
The
TABLE 2 Effect of Jogging 30 Minutes on Factor VIII Complex
Subject J.B. C.C. T.L. Average
% increase (initial level, units/ml) One-stage vWf:Ag VIII:CAg factor VIII 35 (1.27) 9 (1.24) 34 (0.71) 27 (1.48) 20 (1.57) 25 (1.16) 43 (2.10) 41 (1.22) 38-(;Tr63) _______________-____~~~~~~~~~~~-~~~~~ 35
23
32
The serum levels of von Willebrand antigen were identical to the plasma values both before and after exercise and were, in fact, used in order to obtain an average value for the percentage increase of vWf antigen following The factor VIII clotting activity is consumed during clotting and exercise. it was noted that in five subjects so studied the VIII:CAg remained at an average value of 69% of respective plasma values with a parallel dose response curve. The same proportional increase in serum VIII:CAg was noted following In order to see if thrombin might exercise as that found for the plasmas. have an effect on the VIII:CAg levels , purified bovine factor VIII was treated
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with bovine thrombin at different concentrations and assayed following an 18 hour incubation. The thrombin has a slight effect on the VIII:CAg levels, but only at high concentration (Table 3). No increase in VIII:CAg was noted when
TABLE 3 Effect of Thrombin (18 Hrs.) on Purified Bovine Factor VIII Thrombin (U/ml) None (control) 0.03 3 30
One-stage VIII:C (U/ml) 51 125 21 1.5
VIII:CAg % initial level 100 100 89 61
thrombin acted at levels which increased the one-stage clotting assay up to three-fold following 18 hours. DISCUSSION As seen in Table 1, a larger increase of factor VIII one-stage clotting activity occurs following a five minute exercise (187%) than the factor VIII coagulant antigen (107X), two-stage factor VIII activity (102%) or the von Willebrand antigen (115%). A disproportional increase in factor VIII onestage clotting activity above von Willebrand factor activity was recently reported by Stibbe (3). She found that the factor VIII-Ag.(vWf antigen) and the ristocetin co-factor rose proportionately but significantly less than the factor VIII one-stage clotting activity. We have extended the study to include the factor VIII coagulant antigen and a modified two-stage assay. The results obtained with these two tests on purified bovine factor VIII showed no increase in values when trace amounts of thrombin were added in amounts sufficient to increase the one-stage assay many-fold. Rizza and Eipe (15) found a reasonably good correlation between the one- and two-stage factor VIII assays following exercise, but the mean values for the one-stage assays were slightly higher. In our study the one-stage assay gave consistently higher values following exercise than the two-stage method. The one-stage assay also gives consistently higher values relative to the two-stage factor VIII assay when comparing bovine and human plasmas for factor VIII:C. Bovine plasma is eight to twelve times as active as human plasma in the one-stage assay, but only 1.9 times as active as human plasma in the two-stage factor VIII:C assay and only 44% as active relative to human plasma in the VIII:CAg assay. The factor VIII increase is real and not attributable to an increase in factor XII activity affecting the assay system as suggested by Iatridis and Ferguson (16). We found no increase in factor XII in the three individuals in whom the factor VIII one-stage assay increased the greatest degree following exercise. The activated partial thromboplastin times (APTT's) shortened an average of 10.3 seconds in these three individuals (45.2-34.9 seconds) but the ox brain prothrombin times were unaffected. We found that purified bovine factor VIII will also shorten the AI?TT's as much as 15 seconds when added in ten-fold unit excess (unpublished observations). The finding that factor VIII prepared from exercised donors disappears more rapidly from the circulation than non-exercised factor VIII when infused
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into hemophilics (17) suggests a modified form of factor VIII is released following exercise. The studies reported here indicate that the factor VIII released is modified (possiblyby thrombin) or activated as it is more active in the one-stage assay than in the other three parameters. The data suggest that factor VIII and the von Willebrand factor are released from the same storage pool following exercise. ACKNOWLEDGEMENTS This work was supported by USPHS Research Grant HL 15230 and a grant from the Hemophilia Foundation of Southern California. We thank ChristineJacoby and Cheryl Carton for technicalassistance. REFERENCES 1. RIZZA, C.R. Effect of exercise on the level of antihaemophilicglobulin in human blood. J. Physiol. (London)156, 128, 1961. 2. PRENTICE, C.R.M., FORBES, C.D., and SMITH, S.M. Rise of factor VIII after exercise and adrenalin infusion,measured by immunologicaland biological techniques. Thromb. Res. 1, 493, 1972. 3. STIBBE, J. Effect of exercise on F VIII-complex:Proportionalincrease of ristocetin cofactor (von Willebrand Factor) and f-VIII-AGN,but disproportionalincrease of F-VIII-AHF. Thromb. Res. 10, 163, 1977. 4. LAZARCHICK,J. and HOYER, L.W. Immunoradiometricmeasurementof the factor VIII procoagulantantigen. J. Clin. Invest. 62, 1048, 1978. 5. REISNER, H. M., BARROW, E.S., and GRAHAM, J.B. Radioimmunoassayfor coagulant factor VIII-relatedantigen (VIII:CAg). Thromb. Res., In press. 6. PEAKE, I.R. and BLOOM, A.L. Immunoradiometricassay of procoagulantfactor VIII antigen in plasma and serum and its reduction in haemophilia. Lancet 1, 473, 1978. 7. LANGDELL, R.D., WAGNER, R.H., and BRINKHOUS, K.M. Effect of antihemophilic factor on one-stage clotting tests. J. Lab. Clin. Med. 41, 637, 1953. 8. POOL, J.B. and ROBINSON, J. Assay of plasma antihaemophilicglobulin (AHG). Brit. J. Haemat. 5, 17, 1959. 9. BROWN, J.E., BAUGH, R.F., and HOUGIE, C. Substrate inhibitionof the intrinsic generation of activated factor X (Stuart factor). Thromb. Res. 13,.893, 1978. 10. PORTER, R. P. The hydrolysis of rabbit y-globulin and antibodieswith crystallinepapain. Biochem. J. 73, 119, 1959. 11. HUNTER, W.M. and GREENWOOD, F.C. Preparation of iodine-labeledhuman growth hormone of high specific activity. Nature 194, 495, 1962. 12. BAUGH, R.F., BROWN, J.E., SARGEANT,R.B., and HOUGIE, C. Separationof human factor VIII activity from von Willebrand'santigen and ristocetin platelet aggregating activity. Biochim. Biophys. Acta 371, 360, 1974.
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13. BROWN, J.E., BAUGH, R.F., SARGEANT, R.B., and HOUGIE, C. Separation of bovine factor VIII-related antigen from bovine antihemophilic factor. Proc. Sot. Exp. Biol. Med. 147, 608, 1974. 14. LIJNDBLAD,R.L. A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethyl-sulfonyl fluoride. Biochemistry 10, 2501, 1971. 15. RIZZA, C.R. and EIPE, J. Exercise, factor VIII and the spleen. Brit. J. Haemat. 20, 269, 1971. 16. IATRIDIS, S.G. and FERGUSON, J.H. Effect of exercise on blood clotting and fibrinolysis. J. Appl. Physiology 18, 337, 1963. 17. van GASTEL, C., SIXMA, J.J., BORST-EILERS, E., et al. Preparation and infusion of cryoprecipitate from exercised donors. Brit. J. Haemat. 25. 461, 1973.
RESEARCH 15; 61-67 Printed Press Ltd.1979.
in Great Britain oo4g-3848/7g/o6ol-oo61
$02.00/O
EFFECT OF EXERCISE ON THE FACTOR VIII COMPLEX: A CORRELATION OF THE VON WILLEBRAND ANTIGEN AND FACTOR VIII COAGULANT ANTIGEN INCREASE
James E. Brown, Robert F. Baugh, and Cecil Hougr= Department of Pathology University of California, San Diego, School of Medicine La Jolla, California 92093 USA
in revised form 9.2.1979. (Received 15.12.1978; Accepted by Editor K. M. Brinkhous)
ABSTRACT Ten volunteers ran 314 miles as fast as possible and blood was drawn prior to and immediately following the exercise. Plasma factor VIII coagulant activity rose an average of 187% while the factor VIII coagulant antigen rose only an average of 107% and correlated better with the two-stage clotting assay (102%) and the von Willebrand factor antigen (115%). It is concluded that the disproportionate rise in factor VIII procoagulant activity above the von Willebrand factor is observed only when using the one-stage factor VIII clotting assay.
INTRODUCTION Rizza (1) originally observed that factor VIII levels rose in human blood following exercise. Prentice and co-workers (2) demonstrated a similar rise in plasma factor VIII levels, but extended the study to include the factor VIII-related antigen (von Willebrand antigen) which was reported to rise proportionately following a half mile run. Stibbe (3), however, reported that factor VIII coagulant activity in ten subjects rose an average of 126% following a five minute vigorous exercise, whereas factor VIII antigen (vWf) rose an average of only 46%. In order to determine whether the disproprotionate rise of factor VIII and von Willebrand factor is real or an artifact of the one-stage clotting rest, we used a two-stage assay as well as an immunoradiometric assay (IRMA) for factor VIII procoagulant activity (4, 5) as modified from that reported by Peake and Bloom (6). MATERIALS AND METHODS Volunteers among laboratory personnel (age range 21-56), five males and five females, were asked to run as fast as possible 3/4 miles (3 laps) around a circular track. All but one of the subjects exercised routinely, but only M.K. ran regularly. Blood (9 ml) was collected into polypropylene tubes containing 1 ml of 0.1 M buffered citrate one half hour before and within 61
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five minutes after the run. Platelet-poorplasma was obtained by centrifugation for 20 minutes at 1,000 g and room temperature. The one-stage assay for factor VIII used was that previously described (7) and employed as a standard, pooled plasma from ten individuals. The two-stage technique for factor VIII was modified from that of Pool and Robinson (8). Lyophilisedbovine serum was used at 4 mg/ml (one part) as a source of factor XII, XI, IX and X and aluminum hydroxide-adsorbedhemophilic plasma at l/20 was included with the bovine serum to elevate the factor V. The plasmas to be tested for factor VIII were adsorbed with aluminum hydroxide for 20 minutes (one part aluminum hydroxide + 19 parts plasma) and incubated (1 part) at 37OC with the bovine serum (1 part) in the presence of optimal cephalin (1 part) and 25 mM calcium chloride (1 part). Aliquots of this incubationmixture, one part, and one part of .025 M calcium were added simultaneouslyfollowing 5, 10 and 15 minutes of incubation to pooled normal plasma that had been aged (as whole blood) in 0.01 M EDTA for 48 hours at 4oC. The clotting times were then determined. The blank time (without adsorbed plasma) was increased from 60 seconds to 170 by using EDTA-aged plasma compared with normal human plasma as substrate. Aging the bovine serum diluted l/20 for 48 hours at 4oC depleted 99% of the factor Xa present when originally reconstitutedas measured by a spectrophotometricassay (9) and hence also increased the blank time of the assay. The IRMA for factor VIII procoagulant antigen was performed according to the method of Reisner et al. (5) with the exception that the Fab fragment of the IgG factor VIII inhibitormolecule was used rather than the Fab'. A hemophilicwith a factor VIII inhibitor-titerof about 2,000 Bethesda units/ml was obtained and IgG was purified from the inhibitorplasma by chromatography on QAB A-50 Sephadex equilibratedwith ethylene diamine, .037 M, and acetic acid, .073 Mj pH 7.0. The purified IgG following lyophilizationhad a specific activity of 298 Bethesda units/mg. The IgG was hydrolyzedwith papain by the method of Porter (10) and the 40-80X ammonium sulfate cut used as the Fab fragment. The inhibitor titer had dropped (98%) to 7 Bethesda units/mg (assume for Fab an El% = 10). This was dialyzed vs. 0.1 M phosphate, pH 7.5, and frozen at -200C. It had a major band at 55,OOrmolecular weight upon SDS gel electrophoresisin 5% acrylamide. Aggregateswere removed by gel filtrationon Bio-Gel A 1.5 M prior to labeling and 50 ug labeled with 1 mCi of 1251 by the chloramine T method (11). The free iodine was separated from the protein incorporatediodine by chromatographyon Sephadex G-25 and the iodinated Fab molecule, 5-10 uCi/pg protein (about 0.8 ml) mixed with a preparation of bovine factor VIII (0.2 ml) containing150 units/ml of bovine factor VIII (5 mg/ml) in 0.02 M imidazole, 0.15 M NaCl, pH 6.9, for one hour at 370C. The mixture was then applied to a Bio-Gel A 1.5 M column (0.9 x 56 cm) equilibratedwith 0.02 M imidazole,0.15 M NaCl, 0.02 M imidazole,0.15 M NaCl, 0.02% sodium azide, pH 6.9. Fractions (1.6 ml each) at the exclusion volume (complexof Fab and factor VIII) ranged from 0.84 to 1.2% (average 1.02%) of the total recovered radioactivity(five preparations) from the column. These were pooled and concentratedto 0.8 ml using a 75,000 molecular weight cut-off collodion bag (Schleicherand Schuell, Inc., Keene, NH). 125I Fab from the factor VIII was achieved by The dissociationof the mixing enough 1 M glycine HCl, pH 2.57 (0.11 ml), with the complex (0.8 ml) to bring the pH down to 2.8, followed by incubationat 37OC for one hour and then addition of 0.10 ml of 20 mgfml BSA solution. Chromatographyof this complex on a 0.9 x 56 cm column of Bio-Gel A 0.5 M using 0.1 M acetate, 0.5 M NaCl, pH 4.0, as buffer yielded two peaks of radioactivityand 40-50% dissociation
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(second peak). The second peak upon a 5% acrylamide gel sliced into 2 mm segments shwed a major band at 55,000 molecular weight. The second peak from the Bio-Gel A 0.5 M column was concentrated by a collodion membrane (25,000 molecular weight cut-off) to about 1 ml and contained about 300,000-500,000 cpm. A typical assay for factor VIII coagulant antigen (VIII:CAg) was performed as follows: 110 h of plasmas diluted l/20 and l/40 into 0.02 M imidazole, 1% BSA, 0.15 M NaCl, 0.02% NaN3, pH 7.3 for assays before and after exercise, respectively, were added to a 400 ul polypropylene tube. Fifty pl of a solution of 20 mg/ml normal human IgG in pH 7.3 imidazole-saline was added as carrier and then 10 ul of 1251 Fab inhibitor was added, approximately 2,200 cpm. These were incubated for 18 hours at room temperature and then 105 ul of saturated (4oC) ammonium sulfate was added followed by centrifugation (2OC) for 20 minutes at 10,000 g. Supernatant fluid was removed and the pellet counted in an automated well type gamma counter. Assays were done in duplicate both before and after exercise and on fresh as well as frozen plasmas. A typical standard curve was as follows Plasma dilution l/5 l/10 l/20 l/40 typical hemophilic plasma l/5 bovine plasma l/5 buffer bank
% bound radioactivity 53.4 44.1 35.8 30.3 22.4 42.6 12.0
and was linear when plotted on log-log paper. The sensitivity of the IRMA for factor VIII:CAg is similar to the 3% level previously reported (4, 5). A typical hemophilic plasma (5 samples) exhibited a 5% cross reaction. It is unknown why the consistently higher percentage of Fab binding (beyond the buffer blank) with the hemophilic plasmas is observed. Perhaps the increased protein in the ammonium sulfate precipitate when testing hemophilic plasma at a l/5 dilution is responsible for non-specifically carrying with it more Fab fragment. The IRMA was further validated as being specific for factor VIII in a blind study with the University Hospital in San Diego. Twelve patient plasma samples (< 1% to 100% VIII:C) were assayed for factor VIII:CAg and compared with the factor VIII:C values as determined independently using a onestage clotting assay (7). Except for the three severe hemophilics (~1% VIII:C) which assayed at 4-6X VIII:CAg, a good correlation was obtained in all cases but one. This patient had a factor VIII inhibitor (4 Bethesda units/ml) with VIII:C less than 1X, but assayed at 33% VIII:CAg. Bovine plasma exhibited a parallel dose response curve and assayed 44% relative to human plasma for VIII:CAg. The von Willebrand factor antigen was measured by the Laurel1 immunoelectrophoresis method (12). The percentage increases of the parameters measured were calculated by subtracting the pre-exercise value from the post exercise value and then dividing by the pre-exercise value and multiplying by 100. Bovine factor VIII (3,000-fold pure) was prepared as previously described (13), as was bovine thrombin (1,300 units/mg) (14). RESULTS
The subjects were ranked in the order in which they finished the 314 mile run, along with values for the factor VIII parameters measured. Certain trends can be seen in examining the data. The time with which one runs was roughly indicative of the amount of exertion involved in the exercise and hence the degree of increase in factor VIII parameters. On a separate
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occasion two individuals (J.B. and C.C.) listed in Table 1 jogged at a brisk
TABLE1 Effect
of Exercise on Plasma Factor VIII Complex Units/ml Factor
314 Subject
K.P. A.M. J.B. M.K. C.J. C.C. S.L. M.M. C.H. C.L. Average
mile time
VIII:C
One-stage Pre
Two-stage
Post
Pre
VIII:CAg
Post
Pre
Post
vWf:Ag Pre
Post
4:31 1.02 3.90 1.05 2.88 1.07 2.84 0.89 2.33 4:34 0.80 2.48 1.76 2.64 0.98 2.04 1.25 3.19 4:43 0.91 3.44 0.94 2.62 1.10 2.57 0.65 1.78 4:51 1.44 3.28 1.80 3.44 0.96 2.00 1.89 3.23 5:Ol 1.08 4.06 0.87 2.88 0.81 1.94 0.85 2.35 5:05 1.52 5.59 0.92 2.00 1.48 2.63 1.93 5.25 5:51 0.80 1.64 1.56 2.20 0.86 1.71 0.77 1.68 5:58 1.26 2.84 0.94 2.90 0.98 2.64 0.83 2.05 6:32 1.20 2.22 1.33 1.76 1.15 1.70 2.13 2.53 6:32 0.92 2.16 1.36 1.92 1.64 2.72 1.25 2.33 ____________________~~____~~~~__~~~~~~~~~~~~~~~~~~~~~~__~~~~~_~~~_~_ 5:22
1.10
Increase
3.16 187%
1.25
2.52 102%
1.10
2.28
1.24
107%
pace for one half hour and one (T.L.) ran in place for ten minutes. results of these less vigorous exercises are shown in Table 2.
2.67 115%
The
TABLE 2 Effect of Jogging 30 Minutes on Factor VIII Complex
Subject J.B. C.C. T.L. Average
% increase (initial level, units/ml) One-stage vWf:Ag VIII:CAg factor VIII 35 (1.27) 9 (1.24) 34 (0.71) 27 (1.48) 20 (1.57) 25 (1.16) 43 (2.10) 41 (1.22) 38-(;Tr63) _______________-____~~~~~~~~~~~-~~~~~ 35
23
32
The serum levels of von Willebrand antigen were identical to the plasma values both before and after exercise and were, in fact, used in order to obtain an average value for the percentage increase of vWf antigen following The factor VIII clotting activity is consumed during clotting and exercise. it was noted that in five subjects so studied the VIII:CAg remained at an average value of 69% of respective plasma values with a parallel dose response curve. The same proportional increase in serum VIII:CAg was noted following In order to see if thrombin might exercise as that found for the plasmas. have an effect on the VIII:CAg levels , purified bovine factor VIII was treated
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with bovine thrombin at different concentrations and assayed following an 18 hour incubation. The thrombin has a slight effect on the VIII:CAg levels, but only at high concentration (Table 3). No increase in VIII:CAg was noted when
TABLE 3 Effect of Thrombin (18 Hrs.) on Purified Bovine Factor VIII Thrombin (U/ml) None (control) 0.03 3 30
One-stage VIII:C (U/ml) 51 125 21 1.5
VIII:CAg % initial level 100 100 89 61
thrombin acted at levels which increased the one-stage clotting assay up to three-fold following 18 hours. DISCUSSION As seen in Table 1, a larger increase of factor VIII one-stage clotting activity occurs following a five minute exercise (187%) than the factor VIII coagulant antigen (107X), two-stage factor VIII activity (102%) or the von Willebrand antigen (115%). A disproportional increase in factor VIII onestage clotting activity above von Willebrand factor activity was recently reported by Stibbe (3). She found that the factor VIII-Ag.(vWf antigen) and the ristocetin co-factor rose proportionately but significantly less than the factor VIII one-stage clotting activity. We have extended the study to include the factor VIII coagulant antigen and a modified two-stage assay. The results obtained with these two tests on purified bovine factor VIII showed no increase in values when trace amounts of thrombin were added in amounts sufficient to increase the one-stage assay many-fold. Rizza and Eipe (15) found a reasonably good correlation between the one- and two-stage factor VIII assays following exercise, but the mean values for the one-stage assays were slightly higher. In our study the one-stage assay gave consistently higher values following exercise than the two-stage method. The one-stage assay also gives consistently higher values relative to the two-stage factor VIII assay when comparing bovine and human plasmas for factor VIII:C. Bovine plasma is eight to twelve times as active as human plasma in the one-stage assay, but only 1.9 times as active as human plasma in the two-stage factor VIII:C assay and only 44% as active relative to human plasma in the VIII:CAg assay. The factor VIII increase is real and not attributable to an increase in factor XII activity affecting the assay system as suggested by Iatridis and Ferguson (16). We found no increase in factor XII in the three individuals in whom the factor VIII one-stage assay increased the greatest degree following exercise. The activated partial thromboplastin times (APTT's) shortened an average of 10.3 seconds in these three individuals (45.2-34.9 seconds) but the ox brain prothrombin times were unaffected. We found that purified bovine factor VIII will also shorten the AI?TT's as much as 15 seconds when added in ten-fold unit excess (unpublished observations). The finding that factor VIII prepared from exercised donors disappears more rapidly from the circulation than non-exercised factor VIII when infused
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into hemophilics (17) suggests a modified form of factor VIII is released following exercise. The studies reported here indicate that the factor VIII released is modified (possiblyby thrombin) or activated as it is more active in the one-stage assay than in the other three parameters. The data suggest that factor VIII and the von Willebrand factor are released from the same storage pool following exercise. ACKNOWLEDGEMENTS This work was supported by USPHS Research Grant HL 15230 and a grant from the Hemophilia Foundation of Southern California. We thank ChristineJacoby and Cheryl Carton for technicalassistance. REFERENCES 1. RIZZA, C.R. Effect of exercise on the level of antihaemophilicglobulin in human blood. J. Physiol. (London)156, 128, 1961. 2. PRENTICE, C.R.M., FORBES, C.D., and SMITH, S.M. Rise of factor VIII after exercise and adrenalin infusion,measured by immunologicaland biological techniques. Thromb. Res. 1, 493, 1972. 3. STIBBE, J. Effect of exercise on F VIII-complex:Proportionalincrease of ristocetin cofactor (von Willebrand Factor) and f-VIII-AGN,but disproportionalincrease of F-VIII-AHF. Thromb. Res. 10, 163, 1977. 4. LAZARCHICK,J. and HOYER, L.W. Immunoradiometricmeasurementof the factor VIII procoagulantantigen. J. Clin. Invest. 62, 1048, 1978. 5. REISNER, H. M., BARROW, E.S., and GRAHAM, J.B. Radioimmunoassayfor coagulant factor VIII-relatedantigen (VIII:CAg). Thromb. Res., In press. 6. PEAKE, I.R. and BLOOM, A.L. Immunoradiometricassay of procoagulantfactor VIII antigen in plasma and serum and its reduction in haemophilia. Lancet 1, 473, 1978. 7. LANGDELL, R.D., WAGNER, R.H., and BRINKHOUS, K.M. Effect of antihemophilic factor on one-stage clotting tests. J. Lab. Clin. Med. 41, 637, 1953. 8. POOL, J.B. and ROBINSON, J. Assay of plasma antihaemophilicglobulin (AHG). Brit. J. Haemat. 5, 17, 1959. 9. BROWN, J.E., BAUGH, R.F., and HOUGIE, C. Substrate inhibitionof the intrinsic generation of activated factor X (Stuart factor). Thromb. Res. 13,.893, 1978. 10. PORTER, R. P. The hydrolysis of rabbit y-globulin and antibodieswith crystallinepapain. Biochem. J. 73, 119, 1959. 11. HUNTER, W.M. and GREENWOOD, F.C. Preparation of iodine-labeledhuman growth hormone of high specific activity. Nature 194, 495, 1962. 12. BAUGH, R.F., BROWN, J.E., SARGEANT,R.B., and HOUGIE, C. Separationof human factor VIII activity from von Willebrand'santigen and ristocetin platelet aggregating activity. Biochim. Biophys. Acta 371, 360, 1974.
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13. BROWN, J.E., BAUGH, R.F., SARGEANT, R.B., and HOUGIE, C. Separation of bovine factor VIII-related antigen from bovine antihemophilic factor. Proc. Sot. Exp. Biol. Med. 147, 608, 1974. 14. LIJNDBLAD,R.L. A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethyl-sulfonyl fluoride. Biochemistry 10, 2501, 1971. 15. RIZZA, C.R. and EIPE, J. Exercise, factor VIII and the spleen. Brit. J. Haemat. 20, 269, 1971. 16. IATRIDIS, S.G. and FERGUSON, J.H. Effect of exercise on blood clotting and fibrinolysis. J. Appl. Physiology 18, 337, 1963. 17. van GASTEL, C., SIXMA, J.J., BORST-EILERS, E., et al. Preparation and infusion of cryoprecipitate from exercised donors. Brit. J. Haemat. 25. 461, 1973.