Molekularbiologische Grundlagen und Diagnostik der hereditären Defekte von Antithrombin III, Protein C und Protein S

Zusammenfassung

Die enormen Fortschritte in der Molekularbiologie in den letzten Jahren ermöglichten sowohl die Aufklärung der Nukleotidsequenzen der Gene für Antithrombin III (AT III), Protein C (PROC) und Protein S (PROS) als auch die Identifizierung zahlreicher Mutationen bei hereditären Defekten dieser wichtigen Inhibitoren des plasmatischen Gerinnungssystems. Da die Gene für AT III (13,8 kb) und PROC (11,2 kb) nicht groß und relativ leicht zu analysieren sind, gibt es bereits umfangreiche »databases« der Mutationen (50, 73). Für AT III sind 79 und für PROC 160 unterschiedliche Mutationen beschrieben.

Sowohl beim AT-III-Mangel als auch beim Protein-C-Mangel hat die Mutationsaufklärung neue Erkenntnisse über die Struktur-Funktions-Beziehung der Proteine gebracht. Beim Protein-C-Mangel steht die klinische Relevanz der DNA-Analyse im Vordergrund, da die Diagnostik des Protein-C-Mangels auf der Proteinebene nicht immer zuverlässig möglich ist.

Das Protein-S-Gen ist für die Analytik schwer zugänglich, da es groß ist (80 kb) und außerdem ein Pseudogen existiert. Es sind schon zahlreiche Mutationen bei Patienten mit Protein-S-Mangel identifiziert worden. Eine Database ist bisher nicht publiziert. Die klinische Notwendigkeit zur Mutationsaufklärung besteht ebenso wie beim Protein-C-Mangel. Es ist zu erwarten, dass zukünftig die Identifizierung von Mutationen auch beim Protein-S-Mangel beschleunigt vorangeht.

Summary

Recent progress in molecular biology enabled the elucidation of the nucleotide sequences of the genes for antithrombin III (AT III), protein C (PROC) and protein S (PROS). Furthermore numerous mutations were identified causing genetic defects of the important inhibitors of blood coagulation. As the genes for AT III (13.8 kb) and PROC (11.2 kb) are small and easy to analyze a great number of molecular defects already are described in extensive databases (50, 73): 79 different mutations for AT III and 160 for PROC are included.

The identification of mutations leading to AT III and PROC deficiency has given important information on the structure-function relationships of the proteins. In case of protein C deficiency the clinical relevance of DNA analyses is most important because the diagnosis at the protein level is often uncertain. The gene for PROS is not so easy to analyze like the other two genes. The PROS gene is large and also a PROS pseudogene exists. Although a number of mutations have been identified, there has not been published a database until now. The clinical relevance to identify gene defects in PROS deficiency is as important as for PROC deficiency. Presumably the elucidation of PROS gene defects will advance in the near future.

Schlüsselwörter

Antithrombin-III-Mangel - Protein-C-Mangel - Protein-S-Mangel - molekulare Defekte - DNA-Analytik

Keywords

Antithrombin III deficiency - protein C deficiency - protein S deficiency - molecular defects - DNA analysis

• Literatur

• 1 Alhenc-Gelas M, Emmerich J, Gandrille S. et al. Protein C infusion in a patient with inherited protein C deficiency caused by two missense mutations: Arg 178 to Gln and Arg -1 to His. Blood Coagul Fibrinolysis 1995; 6: 35-41.
• 2 Allaart CF, Aronson DO, Ruys T. et al. Hereditary protein S deficiency in young adults with arterial occlusive disease. Thromb Haemost 1990; 22: 206-10.
• 3 Allaart CF, Poort SR, Rosendaal FR. et al. Increased risk of venous thrombosis in carriers of hereditary protein C deficiency defect. Lancet 1993; 341: 134-8.
• 4 Atha DH, Stephens AW, Rosenberg RD. Evaluation of critical groups required for binding of heparin to antithrombin. Proc Natl Acad Sci USA 1984; 81: 1030-4.
• 5 Baker M, French FS, Joseph DR. Vitamin K-dependent protein S is similar to rat androgen-binding protein. Biochem J 1987; 243: 293-6.
• 6 Bertina RM. ISTH Subcommittee Meeting. Munich: 1991
• 7 Bertina RM, Koeleman BPC, Koster T. et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.
• 8 Bjork I, Olson ST, Shore JD. Molecular mechanisms of the accelerating effect of heparin on the reactions between antithrombin and the clotting proteinases. In: Lane DA, Lindahl U. (eds). Heparin: Chemical and biological properties, clinical applications. London: Edward Arnold; 1989: 229-55.
• 9 Bock SC, Harris JF, Balazs I, Trent JM. Assignment of the human antithrombin III structural gene to chromosome 1q23-25. Cyt Cell Genet 1985; 39: 67-9.
• 10 Borg JY, Owen MC, Soria C. et al. Arginine 47 is a prime heparin binding site in antithrombin. A new variant Rouen II, 47 Arg to Ser. J Clin Invest 1988; 81: 1292-6.
• 11 Borgel D, Gandrille S, Gouault-Heilmann M, Aiach M. First frameshift mutation in the active protein S gene associated with a quantitative hereditary deficiency. Blood Coag Fibrinol 1994; 5: 593-600.
• 12 Brennan SO, Borg JY, George PM. et al. New carbohydrate site in mutant antithrombin (7Ile-Asn) with decreased heparin affinity. FEBS Lett 1990; 237: 118-22.
• 13 Carrell RW, Evans DL, Stein PE. Mobile reactive centre of serpins and the control of thrombosis. Nature 1991; 353: 576-8.
• 14 Caso R, Lane DA, Thompson EA. et al. Antithrombin Vicenza, Ala 384 to Pro (GCA to CCA) mutation transforming the inhibitor into a substrate. Br J Haematol 1991; 77: 87-92.
• 15 Chandra T, Stackhouse R, Kidd VJ, Woo SLC. Isolation and sequence characterization of a cDNA clone of human antithrombin III. Proc Natl Acad Sci USA 1983; 80: 1845-8.
• 16 Chang JY. Binding of heparin to antithrombin III activates selective chemical modification at lysine 236. Lys-107, Lys-125 and Lys-136 are situated within the heparin binding site of antithrombin III. J Biol Chem 1989; 264: 3111-5.
• 17 Chang JY, Tran TH. Antithrombin Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity. J Biol Chem 1986; 261: 1174-6.
• 18 Choay J, Petitou M, Lormeau JC. et al. Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting anti-factor Xa activity. Biochem Biophys Res Commun 1983; 116: 492-9.
• 19 Chowdhury V, Lane DA, Mille B. et al. Homozygous antithrombin deficiency: Report of two new cases (99 Leu to Phe) associated with arterial and venous thrombosis. Thromb Haemost 1994; 72: 198-202.
• 20 Crabtree GR, Plutzky J, Marlar R. et al. The range of genotypes underlying human protein C deficiency. Thromb Haemost 1985; 54: 56 (S 331).
• 21 Dahlbäck B, Stenflo J. A natural anticoagulant pathway: proteins C, S, C4b-binding protein and thrombomodulin. In: Bloom AL, Forbes ChD, Thomas DP, Tuddenham EGD. (eds). Haemostasis and Thrombosis. Edinburgh, London: Churchill Livingstone; 1994: 671-717.
• 22 Diepstraten CM, Ploos van Amstel JK, Reitsma PH, Bertina RM. A CCA/CCG neutral dimorphism in the codon for Pro 626 of the human protein S gene PSα (PROS 1). Nucl Acid Res 1991; 19: 5091.
• 23 Edenbrandt CM, Lundwall A, Wydro R, Stenflo J. Molecular analysis of the gene for vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization. Biochemistry 1990; 29: 7861-8.
• 24 Erdjument H, Lane DA, Ireland H. et al. Antithrombin Milano, single amino acid substitution at the reactive site, Arg 393 to Cys. Thromb Haemost 1988; 60: 471-5.
• 25 Erdjument H, Lane DA, Ireland H. et al. Formation of a covalent disulfide-linked antithrombin complex by an antithrombin variant, antithrombin Northwick Park. J Biol Chem 1987; 262: 13381-4.
• 26 Erdjument H, Lane DA, Panico M. et al. Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow. J Biol Chem 1988; 263: 5589-93.
• 27 Erdjument H, Lane DA, Panico M. et al. Antithrombin Chicago, amino acid substitution of arginine 393 to histidine. Thromb Res 1989; 54: 613-9.
• 28 Fair DS, Marlar RA. Biosynthesis and secretion of factor VII, protein C, protein S, and the protein C inhibitor from a human hepatoma cell line. Blood 1986; 67: 64-70.
• 29 Fair DS, Marlar RA, Levin EG. Human endothelial cells synthesize protein S. Blood 1986; 67: 1168-71.
• 30 Foster DC, Holly RO, Sprecher CA. et al. Endoproteolytic processing of the human protein C precursor by the yeast kex 2 endopeptidase coexpressed in mammalian cells. Biochemistry 1991; 30: 367-72.
• 31 Foster DC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985; 82: 4673-7.
• 32 Gandrille S, Aiach M, Lane DA. et al. Important role of Arg 129 in heparin binding site of antithrombin III: identification of novel mutation Arg 129 to GIn. J Biol Chem 1990; 265: 18997-19001.
• 33 Gandrille S, Goossens M, Aiach M. Scanning method to establish the molecular basis of protein C deficiencies. Hum Mutat 1994; 4: 20-30.
• 34 Gandrille S, Vidaud M, Aiach M. et al. Two novel mutations responsible for hereditary type I protein C deficiency: characterization by denaturing gradient gel electrophoresis. Hum Mutat 1992; 1: 491-500.
• 35 Gershagen S, Fernlund P, Lundwall A. A cDNA coding for human sex hormone binding globulin. Homology to vitamin K-dependent protein S. FEBS Lett 1987; 220: 129-35.
• 36 Gladson CL, Scharrer I, Hach V. et al. The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients with venous thrombosis. Thromb Haemost 1988; 59: 18-22.
• 37 Gomez E, Ledford MR, Pegelow CH. et al. Homozygous protein S deficiency due to a one base pair deletion that leads to a stop codon in exon III of the protein S gene. Thromb Haemost 1994; 71: 723-6.
• 38 Greengard JS, Fisher CL, Villoutreix B, Griffin JH. Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: patterns revealed by three-dimensional molecular modelling of mutations of the protease domain. Proteins 1994; 18: 367-80.
• 39 Heeb MJ, Griffin JH. The biochemistry of protein S. In: Bertina RM. (ed). Protein C and related proteins. Edinburgh, London, Melbourne, New York: Churchill Livingstone; 1988: 55-70.
• 40 Hernandez A, Uhrberg M, Enczmann J, Witt I, Reitsma PH, Wernet P. Rapid identification of gene defects in protein C deficiency by temperature gradient gel electrophoresis. Blood Coagul Fibrinolysis 1995; 6: 23-30.
• 41 Hogg PJ, Öhlin A-K, Stenflo J. Identification of structural domains in protein C involved in its interaction with thrombinthrombomodulin in the surface of endothelial cells. J Biol Chem 1992; 267: 703-6.
• 42 Hoskins J, Norman DK, Beckmann RJ, Long GL. Cloning and characterization of human liver cDNA encoding a protein S precursor. Proc Natl Acad Sci USA 1987; 84: 349-53.
• 43 Ireland H, Lane DA, Thompson E. et al. Antithrombin Frankfurt I: arginine to cysteine substitution at the reactive site and formation of a variant antithrombin-albumin covalent complex. Thromb Haemost 1991; 65: 913.
• 44 Ireland H, Lane DA, Thompson FA. et al. Antithrombin Glasgow II: alanine to threonine mutation in the serpin P 12 position, resulting in a substrate reaction with thrombin. Br J Haematol 1991; 79: 70-4.
• 45 Jorgensen AM, Borders CL, Fish WW. Arginine residues are critical for the heparincofactor activity of antithrombin III. Biochem J 1985; 231: 59-65.
• 46 Jorgensen MJ, Cantor AB, Furie BC. et al. Recognition site directing vitamin K dependent γ-carboxylation resides on the propeptide of factor IX. Cell 1987; 48: 185-91.
• 47 Koeleman BPC, Reitsma PH, Allaart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C deficient families. Blood 1994; 84: 1031-5.
• 48 Koide T, Odani S, Takahashi K. et al. Antithrombin III Toyama: replacement of Arginine 47 by Cysteine in hereditary abnormal antithrombin III that lacks heparin-binding ability. Proc Natl Acad Sci USA 1984; 81: 289-93.
• 49 Lane DA, Erdjument H, Flynn A. et al. Antithrombin Sheffield: amino acid substitution at the reactive site (Arg 393 to His) causing thrombosis. Br J Haematol 1989; 71: 91-6.
• 50 Lane AD, Erdjument H, Thompson E. et al. A novel amino acid substitution in the reactive site of a congenital variant antithrombin. Antithrombin Pescara, Arg 393 to Pro, caused by CGT to CCT mutation. J Biol Chem 1989; 264: 10200-4.
• 51 Lane DA, Ireland H, Olds RJ. et al. Antithrombin III: A database of mutations. Thromb Haemost 1991; 66: 657-61.
• 52 Lane DA, Olds RJ, Boisclair M. et al. Antithrombin III mutation database: First update. Thromb Haemost 1993; 70: 361-9.
• 53 Lane DA, Olds RJ, Thein SL. Antithrombin and its deficiency states. Blood Coag Fibrinol 1992; 3: 315-41.
• 54 Liu CS, Chang JY. The heparin binding site of human antithrombin III. Selective chemical modification at Lys 114, Lys 125 and Lys 287 impairs its heparin cofactor activity. J Biol Chem 1987; 262: 17356-61.
• 55 Lundwall A, Dackowski W, Cohen E. et al. Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation. Proc Natl Acad Sci USA 1986; 83: 6717-20.
• 56 Mahasandana C, Suvatte V, Marlar RA. et al. Neonatal purpura fulminans associated with homozygous protein S deficiency. Lancet 1990; 335: 61-2.
• 57 Marlar RA. Plasma single chain protein C is functionally similar to the two chain form of plasma protein C. Thromb Haemost 1985; 54: 1275.
• 58 Marlar RA, Neumann A. Neonatal purpura fulminans due to homozygous protein C or protein S deficiency. Sem Thromb Haemost 1990; 16: 229-309.
• 59 Melissari E, Monte G, Lindo VS. et al. Congenital thrombophilia among patients with venous thromboembolism. Blood Coag Fibrinol 1993; 3: 749-58.
• 60 Miletich JP, Leykam JF, Broze jr GJ. Detection of single chain protein C in human plasma. Blood 1983; (Suppl. 01) 62: 306a.
• 61 Miletich J, Sherman L, Broze jr G. Absence of thrombosis in subjects with heterozygous protein C deficiency. N Engl J Med 1987; 317: 991-6.
• 62 Mustafa S, Pabinger I, Heistinger M, Mannhalter Ch. New mutations in the protein S gene. Ann Hematol 1994; 68 (Suppl. 02) A 58 Abstr 64.
• 63 Ogura M, Tanabe N, Nishioka J. et al. Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01). Blood 1987; 70: 301-6.
• 64 Okajima K, Abe H, Maeda S. et al. Antithrombin III Nagasaki (Ser 116-Pro): a heterozygous variant with defective heparin binding associated with thrombosis. Blood 1993; 81: 1300-5.
• 65 Olds RJ, Lane DA, Boisclair M. et al. Antithrombin Budapest 3: an antithrombin variant with reduced heparin affinity resulting from the substitution L99F. Fed Eur Biochem Soc Lett. 1992; 300: 241-6.
• 66 Olds RJ, Lane DA, Chowdhury V. et al. Complete nucleotide sequence of the antithrombin gene. Evidence for homologous recombination causing thrombophilia. Biochemistry 1993; 32: 4216-24.
• 67 Olds RJ, Lane DA, Chowdhury V. et al. (ATT) trinucleotide repeats in the antithrombin gene and their use in determining the origin of repeated mutations. Hum Mutat 1994; 4: 31-41.
• 68 Petersen TE, Dudek-Wojciechowska G, Sottrup-Jensen L, Magnusson S. Primary structure of antithrombin III (heparin cofactor). Partial homology between α1-anti-trypsin and antitrombin III. In: Collen D, Wiman B, Verstraete M. (eds). The physiological inhibitors of blood coagulation and fibrinolysis. Amsterdam: Elsevier Science; 1979: 43-54.
• 69 Peterson CR, Noyes CM, Pecon JM. et al. Identification of a lysyl residue in antithrombin which is essential for heparin binding. J Biol Chem 1987; 262: 8061-5.
• 70 Ploos van Amstel HK, Reitsma PH, Hamulyak K. et al. A mutation in the protein S pseudogene is linked to protein S deficiency in a thrombophilic family. Thromb Haemost 1989; 62: 897-901.
• 71 Ploos van Amstel HK, Reitsma PH, van der Logt PE, Bertina RM. Intron-exon organization of the active human protein S gene PSα and its pseudogene PSβ: duplication and silencing during primate evolution. Biochemistry 1990; 29: 7853-61.
• 72 Ploos van Amstel HK, van der Zanden AL, Bakker E. et al. Two genes homologous with human protein S cDNA are located on chromosome 3. Thromb Haemost 1987; 58: 982-7.
• 73 Plutzky J, Hoskins JA, Long GL, Crabtree GR. Evolution and organization of the human protein C gene. Proc Natl Acad Sci USA 1986; 83: 546-50.
• 74 Reitsma PH, Ploos van Amstel HK, Bertina RM. Three novel mutations in five unrelated subjects with hereditary protein S deficiency type I. J Clin Invest 1994; 93: 486-92.
• 75 Reitsma PH, Poort SR, Bernardi F. et al. Protein C deficiency: A database of mutations. Thromb Haemost 1993; 69: 77-84.
• 76 Reitsma PH, Bernardi F, Doig RG. et al. Protein C deficiency: A database of mutations, 1995 update. Thromb Haemost 1995; 73: 876-89.
• 77 Schmidel DK, Nelson RM, Broxson jr. EH. et al. A 5.3-kb deletion including exon XIII of the protein S a gene occurs in two protein S-deficient families. Blood 1991; 77: 551-9.
• 78 Schmidel DK, Tatro AV, Phelps LG. et al. Organization of the human protein S genes. Biochemistry 1990; 29: 7845-52.
• 79 Soria JM, Brito D, Barcelo J. et al. Severe homozygous protein C deficiency: Identification of a splice site missense mutation (184, Q → H) in exon 7 of the protein C gene. Thromb Haemost 1994; 72: 65-9.
• 80 Spek CA, Poort SR, Bertina RM, Reitsma PH. Determination of the allelic and haplotype frequencies of three polymorphisms in the promoter region of the human protein C gene. Blood Coag Fibrinol 1994; 5: 309-11.
• 81 Stenflo J. The biochemistry of protein C. In: Bertina RM. (ed). Protein C and related proteins. Edinburgh, London, Melbourne, New York: Churchill Livingstone; 1988: 21-54.
• 82 Sun XI, Chang JY. Evidence that arginine-129 and arginine-145 are located within the heparin binding site of human antithrombin III. Biochemistry 1990; 29: 8957-62.
• 83 Thunberg L, Backstrom G, Lindahl U. Further characterization of the antithrombin-binding sequence in heparin. Carbohydr Res 1982; 100: 393-410.
• 84 Vikydal R, Korninger C, Kyrle PA. et al. The prevalence of hereditary antithrombin III deficiency in patients with a history of venous thromboembolism. Thromb Haemost 1985; 54: 744-5.
• 85 Wacey AI, Pemberton S, Cooper DN. et al. A molecular model of the serine protease domain of activated protein C: application to the study of missense mutations causing protein C deficiency. Br J Haematol 1993; 84: 290-300.
• 86 Watkins PC, Eddy R, Fukushima Y. et al. The gene for protein S maps near the centromere of human chromosome 3. Blood 1988; 71: 238-41.
• 87 Witt I, Beck S, Seydewitz HH. et al. A novel homozygous missense mutation (Val 325 → Ala) in the protein C gene causing neonatal purpura fulminans. Blood Coag Fibrinol 1994; 5: 651-3.
• 88 Witt I, Seydewitz HH, Asbeck D. et al. Protein C gene mutations in ten unrelated families with symptomatic protein C deficiency. Ann Hematol 1994; 68 (Suppl. 02) A58 Abstr. 63.
• 89 Witt I, Seydewitz HH. Unveröffentliche Ergebnisse.
• 90 Yamazaki T, Sugiura I, Matsushita T. et al. A phenotypically neutral dimorphism of protein S: The substitution of Lys 155 by Glu in the second EGF domain predicted by an A to G base exchange in the gene. Thromb Res 1993; 70: 395-403.