British Journal of Haematology, 1976, 32, 9.
Annotation ORAL ANTICOAGULANTS, VITAMIN K AND PROTHROMBIN COMPLEX FACTORS Coumarins and indaiiediones are antagonists of vitamin K and are widely used for their anticoagulant effect. Considerable progress has recently been made in understanding their mode of action, mainly through elucidation of the role of vitamin K in the synthcsis of protlirombin and related coagulation factors. In 1963 Hemker and colleagues showed that anticoagulant treatment did not cause simple deficiency of vitamin K dependent factors; they demonstrated the presence of an abnormal protein in the plasma of patients on oral anticoagulaiit treatment and suggested that it was an inactive precursor of prothrombin. Hemker & Muller (1968) and Hemker et al(1968) named the abnormal protein PIVKA, abbreviated from protein, induced by vitamin K absence or antagonists. Later the presence of abnormal prothrombin, factor VII, IX and X (PIVKA 11, VII, IX and X) in the plasma of all patients on oral anticoagulants was confirmed by many investigators using immunological techniques (Ganrot & NilChn, 1968; Larrieu & Meyer, 1970; Denson, 1971). Studies in vitamin K deficient rats (Bell & Matschiner, 1969; Suttie, 1967, 1970) also indicated that a precursor protein is involved in the formation of prothrombin. When vitamin K was administered to vitamin K deficient rats, about half of the normal prothrombin concentration was restored quickly within the first hour, and this increase could not be blocked by cycloheximide, an inhibitor of protein synthesis. These observations suggested that vitamin K acts at a post-ribosomal site to convert an inactive precursor into a biologically active coagulation factor. Thus, the identification of the differences between normal vitamin K dependent factors and PIVKAs was obviously a key to the understanding of thc modc of action of vitamin K. Comparison of Normal Prothrombin and PIVKA II Many groups of workers have studied PIVKA I1 (Ganrot & Nilithn, 1968; Josso et al, 1968; Cesbron et al, 1973 ; MCnacht et al, 197s). It has the same main antigenic determinants as normal prothrombin and is chemically indistinguishable from it except in its inability to bind calcium and to absorb to barium sulphate. As calcium is essential to link prothrombin to a phospholipid surface during normal activation by factor Xa (Gitel et al, 1973), PIVKA I1 can only be converted into thrombin by physiological activators very slowly and so it does not participate in normal coagulation. It can, however, give rise to normal thrombin in the presence of snake venoms and staphylocoagulase(Minacht, 1975). This indicates that PIVKA I1 contains the normal thrombin portion of the molecule and suggests that the vitamin K dependent step involves attachment of calcium binding prosthetic groups or modification of amino acid residues to form the calcium binding site. Stenflo (1973, 1974, 1975) has studied the NH,-terminal calcium binding part of both bovine prothrombin and bovine PIVKA I1 obtained from cows on long-term dicoumarol. Correspondence: Dr Milica BrozoviE, Central Middlesex Hospital, Acton Lane, London NWIO7NS.
9
Annotation
I0
1-75. Fragments from normal prothrombin and from bovine PIVKA 11 had identical amino acid composition and both lacked carbohydrate; the normal fragment could bind 10 calcium ions, whereas the precursor could bind at most one calcium ion. However, distinct differences were observed between peptide maps prepared from the tryptic digests of the completely reduced and carboxy-methylated fragments of normal prothrombin and of PIVKA 11. Stenflo has isolated on a preparative scale the peptides that are different between the two. One of these peptides (that from normal prothrombin), containing residues 4-10, had an abnormally high electrophoretic mobility for its amino acid composition, suggesting the presence of extra negativc charges. The unusual mobility of this peptide has also been reported by Magnusson (1973). The peptide contained Gly-Phe-Leu-Glu-Glu-Val-Arg. It was digested by aminopeptidase M and carboxypeptidase B, leaving a tetrapeptide : LeuGlu-Glu-Val. NMR spectropscopy and mass spectrometry indicated that the peptide obtained HOOC
COOH 1
COOH
\/ CH
I I
CHt
I
HZN-CH-COOH
HZN-CH-COOH
Glutamic acid
y-carboxyglutamic acid
FIG I
from normal prothrombin contained an extra COOH group on each of the two glutamic acid residues, turning it into a hitherto unidentified amino acid, y-carboxyglutamic acid (see Fig I). Another peptide with high calcium affinity and abnormal electrophoretic mobility was isolated from normal prothrombin by Stenflo. It appeared to contain several y-carboxyglutamic acid residues. Corresponding peptides from PIVKA I1 had electrophoretic mobility consistent with their amino acid composition and no evidence for the presence of y-carboxyglutaniic acid could be found. Thus the structural difference between normal prothrombin and the precursor protein or PIVKA I1 is confined to NH,-terminal end of the polypeptide chain. The vitamin K dependent structures are y-carboxyglutamic acid residues, and vitamin K is involved in the carboxylation of glutamic acid. In the absence of y-carboxyglutamic acid residues, the calcium binding properties of prothrombin are abolished, and the resultant PIVKA I1 cannot participate in coagulation. The amino acid sequence of the NH,-terminal part of factor IX and of the light chain factor X show considerable homology with the NH,-terminal part ofprothrombin (Fujikawa et al, 1974). As PIVKA IX and PIVKA X cannot bind calcium (Reekers et al, 1973), this suggests that they may also contain y-carboxyglutamic acid residues.
Kinetics of PIVKAs PIVKA I1 appears in plasma fiithin hours of the first dose of an oral anticoagulant (Brozovil & Gurd, 1971). The levels of PIVKA I1 gradually increase to reach a plateau after 3-5
days.
Annotation
I1
The plasma half-life of PIVKA 11 is only 18 h compared to 72 h for prothrombin (Lavergne &Josso, 1975). Although the sum of PIVKA I1 and prothrombin in the plasma of patients on oral anticoagulants is always slightly lower than the normal prothrombin level, the very short half life of PIVKA I1 implies that the synthetic rate of PIVKA is much higher during oral anticoagulant treatment than in the normal state. This may explain the observations of Karpatkin & Karpatkin (1973) who injected plasma of coumarin-treated rabbits into normal animals and found that it caused a rise in the plasma prothrombin level (as well in the levels of factors VII, IX and X). They suggested that a hormone-like mediator was induced by low plasma levels of the vitamin K dependent factors. According to Lavergne & Josso (1975) prothrombin synthesis is accelerated by the mediator induced by low prothrombin concentration in plasma, and is repressed by the increasing intracellular levels of the precursor. In patients and animals on oral anticoagulants, inactive molecules of factor IX (Larrieu & Meyer, 1970; Denson, 1971 ; Veltkamp et al, 1971; Lechner, 197z), factor X (Denson, 1971; Lindhout & Kop-Klaassen, 1975) and factor VII (Goodnight et a/, 1971; Denson, 1971; Levanon et al, 1972; Howarth et al, 1974) have been demonstrated in plasma. The amounts of PIVKA IX, X and VII in plasma are much smaller and more variable than the amount of PIVKA 11 in the same patient, and it is tempting to postulate that this is due to the very short half-life of all the precursor proteins. The relevance of PIVKA to anticoagulant treatment and its management is at present difficult to assess. However, it is known that the sensitivity of different thromboplastins to the coumarin-induced defect depends on the capacity of the thromboplastin to recognize the inhibitory effect of PIVKA X (Hemker et a!, 1968) or to react with PIVKA I1 (Brozovii et al, 1973). This has important consequences for the standardization of the control of anticoagulant treatment. It is also possible, although not proven, that thc presence of PIVKAs in plasma enhances the anticoagulant effect of couinarin treatment. MILICABROZOVIC Dcpurtment of Haematology,
Northwick Park Hospital, Harrow, Middlcscx REFERENCES
J.T. (1969) Synthesis and BELL,R.G. & MATSCHINER, destruction of prothrombin in thc rat. Arrhives .f Biochemistry and Biophysics, 135, I 52. BROZOVIC, M. & GURD,L. (1971) Rate of appearance of abnormal prothrombin following initiation of oral-anticoagulant treatment. (Letter). Lancet, ii, 427. D.J. (1973) BROZOVIC, M., GURU,L. & HOWARTH, Consumption of abnornial prothrombin in the prcscnce of different types of thromboplastin. Thrombosis Researcli, 2, 557. CESBRON, N., BOYER, C., GUILLIN, M.-C. & MBNACHB, D. (1973) Human coumarin prothrombin. Chromatographic, coagulation and immunologic studies. Thrombosis et Diathesis Haemorrhagica, 30, 43 7.
DENSON, K.W.E. (1971) Thc lcvels of factors 11, VII, IX and X by antibody neutralization techniques in the plasma of patients recciving phenindionc therapy. British Jormal qf Haernatology, 20, 643. FUJIXAWA, K., COAN,M.H., ENFIELD, D.L., TITANI, K., ERICSSON,L.H. 81 DAVIE,W.E. (1974) A comparison of bovine prothrombin, factor IX (Christmas factor), and factor X (Stuart factor). Proceedings Offhe National AcadPmy qf Sciences of the United Stafes ofAmerica, 71,427. P.O. & NILBHN,J.-E. (1968) Plasma proGANROT, thrombin during treatment with dicoumarol. 11. Demonstration of an abnormal prothrombin fraction. Scandinavian Journal of Clinical and Laboratory Investigation, 22, 23. GITEL,S.N., OWEN,W.G., ESMON, C.T. FC JACKSON,
I2
Annotation
C.M.(1973) A polypeptide region of bovine prothrombin specific for binding to phospholipids. Proceedings of the National Academy of Sciences of the United States of America, 70, 1344. GOODNIGHT, S.H., JR, FEINSTEIN, D.I., aSTERUD, B. & RAPAPORT, S.I. (1971) Factor VII antibody-neutralizing material in hereditary and acquired factor VII deficiency. Blood, 38, I. HEMKER, H.C. & MULLER, A.D. (1968) Kinetic aspects of the interaction of blood-clotting enzymes. VI. Localization of the site of blood-coagulation inhibition by the protein induced by vitamin K absence (PIVKA). Thrombosis et Diafhesis Haemorrhagica, 20, 78. HEMKER, H.C., VELTKAMP, J.J., HENSEN,A. & LOELIGER,E.A. (1963) Nature of prothrombin biosynthesis: preprothrombinaemia in vitamin Kdeficiency. Nature, 200, 589. HEMKER,H.C., VELTKAMP, J.J. & LOELIGER, E.A. (1968) Kinetic aspects of the interaction of blood clotting enzymes. 111. Demonstration of an inhibitor of prothrombin conversion in vitamin K deficiency. Thrombosis et Diathesis Haemorrhagica, 19, 346. HOWARTH, D.J., BROZOVIC,M., STIRLING,Y. & REED,M. (1974) Factor VII during warfarin treatment. Scandinavian Journal of Haematology, 12, 346. Josso, F., LAVERGNE, J.M., GOUAULT,M., PROUWARTELLE, 0. & SOULIER, J.P. (1968) Diffgrents Ctats mol6culaires du facteur I1 (prothrombine). Leur Ctude h l’aide de la staphylocoagulase et d’anticorps antifacteur 11. I. Le facteur I1 chez les sujets trait& par les antagonistes de la vitamine K. Thrombosis et Diathesis Haemorrhagica, 20, 88. KARPATKIN, M. & KARPATKIN, S. (1973) Evidence for a humoral agent capable of raising vitamin-Kdependant coagulation factors in rabbits. British Journal of Haematology, 24, 5 53. LARRIEU,M.J. & MEYER,D. (1970) Abnormal factor IX during anticoagulant treatment. (Letter). Lancet, ii, 1085. LAVERGNE, J.M. & Josso, F. (197s) Metabolism of PIVKA I1 in man. Prothrombin and Related Clotting Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 183. Leiden University Press. LECHNER, K. (1972) Immune reactive factor IX in acquired factor IX deficiency. Thrombosis et Diathesis Haemorrhagica, 27, 19. LEVANON, M., RIMON,S., SHANI,M., RAMOT,B. & GOLDBERG, E. (1972) Active and inactive factor factor VII in Dubin-Johnson syndrome with factorVII; deficiency, hereditary factor-VII deficiency and
on coumadine administration. British Journal of Haematology, 23, 669. LINDHOUT,M.J. & KOP-KLAASSEN, B.H.M. (1975) Proteins induced by vitamin K antagonists (PIVKA’s). Prothrombin Related Coagulation Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 274. Leiden University Press. MAGNUSSON, S. (1973) Primary structure studies on thrombin and prothrombin. Thrombosis et Diathesis Haemorrhagica, Suppl. 54, 31. MBNACHB, D., GUILLIN, M.C., BOYER, C. & CESBRON, N. (1975) Preliminary studies on human coumarin prothrombin. Prothrombin and Related Clotting Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 159. Leiden University Press. REEKERS, P.P.M., LINDHOUT,M.J., KOP-KLAASSEN, B.H.M. & HEMKER, H.C. (1973) Demonstration of three anomalous plasma proteins induced by a vitamin K antagonist. Biochimica et Biophysica Acta, 317, 559. SHAH,D.V. & SUTTIE,J.W. (1972) The effect of vitamin K and warfarin on rat liver prothrombin concentrations. Archives of Biochemistry, 150,91. STENFLO, J. (1973) Vitamin K and the biosynthesis of prothrombin. 111. Structural comparison of an NH2-terminal fragment from normal and dicoumarol-induced bovine prothrombin. Journal of Biological Chemistry, 248, 6325. STENFLO, J. (1974) Vitamin K and the biosynthesis of prothrombin. IV. Isolation of peptides containing prosthetic groups from normal prothrombin and the corresponding peptides from dicoumarolinduced prothrombin. Journal ofBiologica1 Chemistry, 249, 5527. J. (1975) Structural comparisons of normal STENFLO, and dicoumarol induced prothrombin. Prothrombin and Related Clotting Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 152. Leiden University Press. SUTTIE,J.W. (1967) Control of prothrombin and factor VII biosynthesis by vitamin K. Archives of Biochemistry andBiophysics, 118,166. SUTTIE,J.W. (1970) The effect of cycloheximide administration on vitamin K-stimulated prothrombin formation. Archives of Biochemistry and Biophysics, 141, 571. J.J., MUIS,H., MULLER, A.D., HEMKER, VELTKAMP, H.C. & LOELIGER, E.A. (1971) Additional evidence for the existence of a precursor molecule of the prothrombin complex in oral anticoagulation. Thrombosis et Diathesis Haemorrhagica, 25, 3 12.
Annotation ORAL ANTICOAGULANTS, VITAMIN K AND PROTHROMBIN COMPLEX FACTORS Coumarins and indaiiediones are antagonists of vitamin K and are widely used for their anticoagulant effect. Considerable progress has recently been made in understanding their mode of action, mainly through elucidation of the role of vitamin K in the synthcsis of protlirombin and related coagulation factors. In 1963 Hemker and colleagues showed that anticoagulant treatment did not cause simple deficiency of vitamin K dependent factors; they demonstrated the presence of an abnormal protein in the plasma of patients on oral anticoagulaiit treatment and suggested that it was an inactive precursor of prothrombin. Hemker & Muller (1968) and Hemker et al(1968) named the abnormal protein PIVKA, abbreviated from protein, induced by vitamin K absence or antagonists. Later the presence of abnormal prothrombin, factor VII, IX and X (PIVKA 11, VII, IX and X) in the plasma of all patients on oral anticoagulants was confirmed by many investigators using immunological techniques (Ganrot & NilChn, 1968; Larrieu & Meyer, 1970; Denson, 1971). Studies in vitamin K deficient rats (Bell & Matschiner, 1969; Suttie, 1967, 1970) also indicated that a precursor protein is involved in the formation of prothrombin. When vitamin K was administered to vitamin K deficient rats, about half of the normal prothrombin concentration was restored quickly within the first hour, and this increase could not be blocked by cycloheximide, an inhibitor of protein synthesis. These observations suggested that vitamin K acts at a post-ribosomal site to convert an inactive precursor into a biologically active coagulation factor. Thus, the identification of the differences between normal vitamin K dependent factors and PIVKAs was obviously a key to the understanding of thc modc of action of vitamin K. Comparison of Normal Prothrombin and PIVKA II Many groups of workers have studied PIVKA I1 (Ganrot & Nilithn, 1968; Josso et al, 1968; Cesbron et al, 1973 ; MCnacht et al, 197s). It has the same main antigenic determinants as normal prothrombin and is chemically indistinguishable from it except in its inability to bind calcium and to absorb to barium sulphate. As calcium is essential to link prothrombin to a phospholipid surface during normal activation by factor Xa (Gitel et al, 1973), PIVKA I1 can only be converted into thrombin by physiological activators very slowly and so it does not participate in normal coagulation. It can, however, give rise to normal thrombin in the presence of snake venoms and staphylocoagulase(Minacht, 1975). This indicates that PIVKA I1 contains the normal thrombin portion of the molecule and suggests that the vitamin K dependent step involves attachment of calcium binding prosthetic groups or modification of amino acid residues to form the calcium binding site. Stenflo (1973, 1974, 1975) has studied the NH,-terminal calcium binding part of both bovine prothrombin and bovine PIVKA I1 obtained from cows on long-term dicoumarol. Correspondence: Dr Milica BrozoviE, Central Middlesex Hospital, Acton Lane, London NWIO7NS.
9
Annotation
I0
1-75. Fragments from normal prothrombin and from bovine PIVKA 11 had identical amino acid composition and both lacked carbohydrate; the normal fragment could bind 10 calcium ions, whereas the precursor could bind at most one calcium ion. However, distinct differences were observed between peptide maps prepared from the tryptic digests of the completely reduced and carboxy-methylated fragments of normal prothrombin and of PIVKA 11. Stenflo has isolated on a preparative scale the peptides that are different between the two. One of these peptides (that from normal prothrombin), containing residues 4-10, had an abnormally high electrophoretic mobility for its amino acid composition, suggesting the presence of extra negativc charges. The unusual mobility of this peptide has also been reported by Magnusson (1973). The peptide contained Gly-Phe-Leu-Glu-Glu-Val-Arg. It was digested by aminopeptidase M and carboxypeptidase B, leaving a tetrapeptide : LeuGlu-Glu-Val. NMR spectropscopy and mass spectrometry indicated that the peptide obtained HOOC
COOH 1
COOH
\/ CH
I I
CHt
I
HZN-CH-COOH
HZN-CH-COOH
Glutamic acid
y-carboxyglutamic acid
FIG I
from normal prothrombin contained an extra COOH group on each of the two glutamic acid residues, turning it into a hitherto unidentified amino acid, y-carboxyglutamic acid (see Fig I). Another peptide with high calcium affinity and abnormal electrophoretic mobility was isolated from normal prothrombin by Stenflo. It appeared to contain several y-carboxyglutamic acid residues. Corresponding peptides from PIVKA I1 had electrophoretic mobility consistent with their amino acid composition and no evidence for the presence of y-carboxyglutaniic acid could be found. Thus the structural difference between normal prothrombin and the precursor protein or PIVKA I1 is confined to NH,-terminal end of the polypeptide chain. The vitamin K dependent structures are y-carboxyglutamic acid residues, and vitamin K is involved in the carboxylation of glutamic acid. In the absence of y-carboxyglutamic acid residues, the calcium binding properties of prothrombin are abolished, and the resultant PIVKA I1 cannot participate in coagulation. The amino acid sequence of the NH,-terminal part of factor IX and of the light chain factor X show considerable homology with the NH,-terminal part ofprothrombin (Fujikawa et al, 1974). As PIVKA IX and PIVKA X cannot bind calcium (Reekers et al, 1973), this suggests that they may also contain y-carboxyglutamic acid residues.
Kinetics of PIVKAs PIVKA I1 appears in plasma fiithin hours of the first dose of an oral anticoagulant (Brozovil & Gurd, 1971). The levels of PIVKA I1 gradually increase to reach a plateau after 3-5
days.
Annotation
I1
The plasma half-life of PIVKA 11 is only 18 h compared to 72 h for prothrombin (Lavergne &Josso, 1975). Although the sum of PIVKA I1 and prothrombin in the plasma of patients on oral anticoagulants is always slightly lower than the normal prothrombin level, the very short half life of PIVKA I1 implies that the synthetic rate of PIVKA is much higher during oral anticoagulant treatment than in the normal state. This may explain the observations of Karpatkin & Karpatkin (1973) who injected plasma of coumarin-treated rabbits into normal animals and found that it caused a rise in the plasma prothrombin level (as well in the levels of factors VII, IX and X). They suggested that a hormone-like mediator was induced by low plasma levels of the vitamin K dependent factors. According to Lavergne & Josso (1975) prothrombin synthesis is accelerated by the mediator induced by low prothrombin concentration in plasma, and is repressed by the increasing intracellular levels of the precursor. In patients and animals on oral anticoagulants, inactive molecules of factor IX (Larrieu & Meyer, 1970; Denson, 1971 ; Veltkamp et al, 1971; Lechner, 197z), factor X (Denson, 1971; Lindhout & Kop-Klaassen, 1975) and factor VII (Goodnight et a/, 1971; Denson, 1971; Levanon et al, 1972; Howarth et al, 1974) have been demonstrated in plasma. The amounts of PIVKA IX, X and VII in plasma are much smaller and more variable than the amount of PIVKA 11 in the same patient, and it is tempting to postulate that this is due to the very short half-life of all the precursor proteins. The relevance of PIVKA to anticoagulant treatment and its management is at present difficult to assess. However, it is known that the sensitivity of different thromboplastins to the coumarin-induced defect depends on the capacity of the thromboplastin to recognize the inhibitory effect of PIVKA X (Hemker et a!, 1968) or to react with PIVKA I1 (Brozovii et al, 1973). This has important consequences for the standardization of the control of anticoagulant treatment. It is also possible, although not proven, that thc presence of PIVKAs in plasma enhances the anticoagulant effect of couinarin treatment. MILICABROZOVIC Dcpurtment of Haematology,
Northwick Park Hospital, Harrow, Middlcscx REFERENCES
J.T. (1969) Synthesis and BELL,R.G. & MATSCHINER, destruction of prothrombin in thc rat. Arrhives .f Biochemistry and Biophysics, 135, I 52. BROZOVIC, M. & GURD,L. (1971) Rate of appearance of abnormal prothrombin following initiation of oral-anticoagulant treatment. (Letter). Lancet, ii, 427. D.J. (1973) BROZOVIC, M., GURU,L. & HOWARTH, Consumption of abnornial prothrombin in the prcscnce of different types of thromboplastin. Thrombosis Researcli, 2, 557. CESBRON, N., BOYER, C., GUILLIN, M.-C. & MBNACHB, D. (1973) Human coumarin prothrombin. Chromatographic, coagulation and immunologic studies. Thrombosis et Diathesis Haemorrhagica, 30, 43 7.
DENSON, K.W.E. (1971) Thc lcvels of factors 11, VII, IX and X by antibody neutralization techniques in the plasma of patients recciving phenindionc therapy. British Jormal qf Haernatology, 20, 643. FUJIXAWA, K., COAN,M.H., ENFIELD, D.L., TITANI, K., ERICSSON,L.H. 81 DAVIE,W.E. (1974) A comparison of bovine prothrombin, factor IX (Christmas factor), and factor X (Stuart factor). Proceedings Offhe National AcadPmy qf Sciences of the United Stafes ofAmerica, 71,427. P.O. & NILBHN,J.-E. (1968) Plasma proGANROT, thrombin during treatment with dicoumarol. 11. Demonstration of an abnormal prothrombin fraction. Scandinavian Journal of Clinical and Laboratory Investigation, 22, 23. GITEL,S.N., OWEN,W.G., ESMON, C.T. FC JACKSON,
I2
Annotation
C.M.(1973) A polypeptide region of bovine prothrombin specific for binding to phospholipids. Proceedings of the National Academy of Sciences of the United States of America, 70, 1344. GOODNIGHT, S.H., JR, FEINSTEIN, D.I., aSTERUD, B. & RAPAPORT, S.I. (1971) Factor VII antibody-neutralizing material in hereditary and acquired factor VII deficiency. Blood, 38, I. HEMKER, H.C. & MULLER, A.D. (1968) Kinetic aspects of the interaction of blood-clotting enzymes. VI. Localization of the site of blood-coagulation inhibition by the protein induced by vitamin K absence (PIVKA). Thrombosis et Diafhesis Haemorrhagica, 20, 78. HEMKER, H.C., VELTKAMP, J.J., HENSEN,A. & LOELIGER,E.A. (1963) Nature of prothrombin biosynthesis: preprothrombinaemia in vitamin Kdeficiency. Nature, 200, 589. HEMKER,H.C., VELTKAMP, J.J. & LOELIGER, E.A. (1968) Kinetic aspects of the interaction of blood clotting enzymes. 111. Demonstration of an inhibitor of prothrombin conversion in vitamin K deficiency. Thrombosis et Diathesis Haemorrhagica, 19, 346. HOWARTH, D.J., BROZOVIC,M., STIRLING,Y. & REED,M. (1974) Factor VII during warfarin treatment. Scandinavian Journal of Haematology, 12, 346. Josso, F., LAVERGNE, J.M., GOUAULT,M., PROUWARTELLE, 0. & SOULIER, J.P. (1968) Diffgrents Ctats mol6culaires du facteur I1 (prothrombine). Leur Ctude h l’aide de la staphylocoagulase et d’anticorps antifacteur 11. I. Le facteur I1 chez les sujets trait& par les antagonistes de la vitamine K. Thrombosis et Diathesis Haemorrhagica, 20, 88. KARPATKIN, M. & KARPATKIN, S. (1973) Evidence for a humoral agent capable of raising vitamin-Kdependant coagulation factors in rabbits. British Journal of Haematology, 24, 5 53. LARRIEU,M.J. & MEYER,D. (1970) Abnormal factor IX during anticoagulant treatment. (Letter). Lancet, ii, 1085. LAVERGNE, J.M. & Josso, F. (197s) Metabolism of PIVKA I1 in man. Prothrombin and Related Clotting Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 183. Leiden University Press. LECHNER, K. (1972) Immune reactive factor IX in acquired factor IX deficiency. Thrombosis et Diathesis Haemorrhagica, 27, 19. LEVANON, M., RIMON,S., SHANI,M., RAMOT,B. & GOLDBERG, E. (1972) Active and inactive factor factor VII in Dubin-Johnson syndrome with factorVII; deficiency, hereditary factor-VII deficiency and
on coumadine administration. British Journal of Haematology, 23, 669. LINDHOUT,M.J. & KOP-KLAASSEN, B.H.M. (1975) Proteins induced by vitamin K antagonists (PIVKA’s). Prothrombin Related Coagulation Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 274. Leiden University Press. MAGNUSSON, S. (1973) Primary structure studies on thrombin and prothrombin. Thrombosis et Diathesis Haemorrhagica, Suppl. 54, 31. MBNACHB, D., GUILLIN, M.C., BOYER, C. & CESBRON, N. (1975) Preliminary studies on human coumarin prothrombin. Prothrombin and Related Clotting Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 159. Leiden University Press. REEKERS, P.P.M., LINDHOUT,M.J., KOP-KLAASSEN, B.H.M. & HEMKER, H.C. (1973) Demonstration of three anomalous plasma proteins induced by a vitamin K antagonist. Biochimica et Biophysica Acta, 317, 559. SHAH,D.V. & SUTTIE,J.W. (1972) The effect of vitamin K and warfarin on rat liver prothrombin concentrations. Archives of Biochemistry, 150,91. STENFLO, J. (1973) Vitamin K and the biosynthesis of prothrombin. 111. Structural comparison of an NH2-terminal fragment from normal and dicoumarol-induced bovine prothrombin. Journal of Biological Chemistry, 248, 6325. STENFLO, J. (1974) Vitamin K and the biosynthesis of prothrombin. IV. Isolation of peptides containing prosthetic groups from normal prothrombin and the corresponding peptides from dicoumarolinduced prothrombin. Journal ofBiologica1 Chemistry, 249, 5527. J. (1975) Structural comparisons of normal STENFLO, and dicoumarol induced prothrombin. Prothrombin and Related Clotting Factors (ed. by H. C. Hemker and J. J. Veltkamp), p 152. Leiden University Press. SUTTIE,J.W. (1967) Control of prothrombin and factor VII biosynthesis by vitamin K. Archives of Biochemistry andBiophysics, 118,166. SUTTIE,J.W. (1970) The effect of cycloheximide administration on vitamin K-stimulated prothrombin formation. Archives of Biochemistry and Biophysics, 141, 571. J.J., MUIS,H., MULLER, A.D., HEMKER, VELTKAMP, H.C. & LOELIGER, E.A. (1971) Additional evidence for the existence of a precursor molecule of the prothrombin complex in oral anticoagulation. Thrombosis et Diathesis Haemorrhagica, 25, 3 12.
