Albertini A, Lenfant CL, Mannuccí PM, Sixma JJ (eds): Biotechnology of Plasma Proteins. Curr Stud Hematol Blood Transf. Basel, Karger, 1991, No 58, pp 94-99
Molecular Basis of Hereditary Protein C and Protein S Deficiency' Pieter H. Reitsma, Hans K. Ploos van Amstel, Bert R. Poort, Pieter A. van der Velden, Rogier M. Eertin University Medical Center Leiden, Hemostasis and Thrombosis Research Unit, Department of Hematology, Leiden, The Netherlands
One of the major inhibitory mechanisms operating in the blood coagulation cascade is the so-called `protein C anticoagulant pathway' [ 1]. This pathway is initiated when the circulating zymogen protein C is converted to activated protein C (APC) by a thrombin-thrombomodulín complex on the surface of endothelial cells. The formed APC, in the presence of its co-factor protein S, Cat ± ions, and phospholipids downregulates the coagulation cascade by proteolytic inactivation of the procoagulant cofactors Va and VIIIa. The physiological importance of the protein C pathway is most dramatically illustrated by the rarely occurring cases of severe protein C deficiency [2]. In these patients, who are either homozygotes or compound heterozygotes for a defect in the protein C gene, very low or undetectable levels of protein C coincide with severe thromboembolic disease as early as 1-6 days after birth. More mildly affected are patients that are heterozygotes for protein C or protein S deficiency [3, 4]. These deficiencies are frequently encountered (: 15-20%) in familial thrombophilia [5]. Thromboembolic symptoms in affected family members typically occur initially between the ages of 15 and 40 years. The diagnosis of protein C and protein S deficiency is performed with immunological and functional assays. The isolation of cDNA and genomic clones for both the protein C and protein S genes now makes it possible to
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
This work was supported by grant No. 28-1728 from the Praeventíefonds and by grant No. 88.002 from the Thrombose Stichting Nederland. We wish to thank D€nise van Ruiten for her help in preparing this manuscript.
Molecular Basis of Hereditary Protein C and Protein S Deficiency
95
further improve the detection of heterozygotes by DNA analysis. In addition, this type of analysis will enable us to get more insight in the molecular basis of the protein C and protein S deficiencies. In this short review we will focus on these developments.
The gene for protein C is localized on chromosome 2 [6]. The gene is approximately 11 kb long and consists of nine exons [7, 8]. The intron/exon organization of the protein C gene is very similar to that of the coagulation factors VII, IX and X, suggesting that they originate from one ancestral gene. Genetic defects have been described for the protein C gene in 4 patients. The first report by Romeo et al. [9] describes the genetic defect in a Dutch and Spanish family with type I (i.e. low antigen and activity levels) protein C deficiency. In the Dutch family a 306Arg — Stop substitution was found in the catalytic domain of the protein C molecule. The Spanish family was characterized by a 402-Trp — Cys substitution, also in the catalytic domain. Given the type I protein C deficiency, these two mutatuns apparently give rise to an unstable gene product. A second report by Matsuda et al. [ 10] describes the genetic findings in a compound heterozygote (protein C Tochigi) with a combined type I and type II (i.e. a variant protein with decreased activity) protein C deficiency. Analysis of the DNA indicated that the complete deletion of one protein C gene was responsible for the type I deficiency, whereas in the remaining gene a 169Arg —> Trp substitution had occurred. Residue 169Arg is located at the thrombin activation site of protein C and it is plausible that the substitution has led to a molecule that can not be activated, thereby giving rise to a type II deficiency. Grundy et al. [ 11 ] recently reported the same 169Arg — Trp substitution (protein C London 1) in a British subject with protein C deficiency and thrombosis. The geographical separation between protein C Tochigi and Protein C London 1 makes it unlikely that the two mutations are identical-by-descent. More likely is that the CpG dinucleotide involved in the substitution is a `hotspot' for mutation as has been reported for other hemostatic disorders [ 12]. In their analysis of protein C London 1, Grundy et al. [11] were the first to report the use of the polymerise chain reaction [ 13] followed by direct sequencing in the analysis of genetic defects in the protein C gene. This method works much faster than the conventional cloning procedure employed by Romeo et al. [9] and Matsuda et al. [ 10] and allows a large
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
Protein C
Reitsma et al.
96
number of defects to be analysed in the near future. Recently, our laboratory has also developed the protocols for the selective amplification of each of the protein C exons and is currently analyzing point mutations in the protein C gene. To date, preliminary evidence has been collected on 24 mutations including splice site mutations, nonsense mutations, missense mutations and a frameshift mutation [Reitsma et al. unpubl.].
The primary amino acid sequence of human protein S has been deduced from the nucleotide sequence of cloned cDNAs [ 14-16]. This sequence shows that protein S, like protein C, is a mosaic protein. Mature protein S is composed of a Gla region, a small disulfide loop that is sensitive to thrombin leavage, four EGF-like domains and a 392 residues long carboxy-terminal domain that shows weak but significant homology with the sex-hormone-binding globulin [ 17]. A detailed analysis of the human genome has shown the presence of two protein S genes, PSa and PSß, both of which are located near the centromere of chromosome 3 [ 18, 19]. To date, the cloning of only a part of the two genes has been reported [20, 21]. The partial nucleotide sequence analysis showed that the PSβ gene is highly homologous to the PSa gene but contains several mutations (stop codons and a deletion) that prohibit proper translation. Moreover, no transcription product of the PSβ gene could be identified [20]. Together these data indicate that the PSa gene is responsible for protein S synthesis whereas the PSß gene is a genuine pseudogene. Presently, the available data on a molecular analysis of the protein S genes is limited to a Southern blot analysis in a panel of 25 probands with type I deficiency [22, 23]. This survey revealed genetic changes in two families. In one thrombophilic family a major deletion of the middle part of the active PSa gene was observed [22]. This gene defect cosegregated with a reduced level of total protein S and should be considered the cause of the disease. In a second family the PSß pseudogene showed an altered Mspl restriction pattern in the proband [23]. Also this gene alteration cosegregated in the family with a reduced protein S antigen level. However, since the altered restriction site is located in the pseudogene rather than the active gene, it cannot be the cause of the protein S deficiency. In a screening of 50 normal individuals the alteration has not been observed and therefore it probably represents at most a very rare polymorphism.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
Protein S
Molecular Basis of Hereditary Protein C and Protein S Deficiency
97
The mutation in the PSß gene provided an opportunity to estimate the genetic distance between the two protein S genes by combining the data on plasma protein S levels and the altered restriction pattern. Since these two always cosegregated in 23 meioses the PSa and PSß gene should be located within a distance of 4 cM [23]. Recently, an interesting immunological variant protein S molecule (PS Heerlen) has been reported [24]. DNA analysis showed that the protein polymorphism is due to a T -+ C transition in the codon for 460Ser of the PSa gene. Consequently, 460Ser is substituted by Pro. The substitution has occurred in the consensus sequence for the N-linked glycosylation of 458Asn and most likely the PS Heerlen molecule is not glycosylated at this site. In this repect the PS Heerlen variant may be identical with a recently reported variant protein S molecule that was shown to have a reduced carbohydrate content [25]. The frequency of the PS Heerlen variant in the normal population and in patients with unexplained thrombosis is the same (x 0.5%). The molecule also has a normal APC cofactor activity and it is therefore unlikely that it plays a role in the etiology of thrombophilia.
Conclusion It has taken only a few years to go from the first case reports on protein C and protein S deficiency to the underlying molecular defects in the genes. With the advent of the PCR technique it is now expected that numerous mutations will be identified in the next few years. This will teach us how heterogeneous the gene defects in these deficiencies are.
1 Esmon CT; Protein C: Biochemistry, physiology and clinical implications. Blood 1983;62:1155-1158. 2 Marlar RA, Montgomery RR, Broekmans AW: Diagnosis and treatment of homozygous protein C deficiency. J Pediatr 1989;114:5287-5304. 3 Grime JH, Evatt B, Zimmermann TS, Kleiss AJ, Wideman C: Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981;68:1370-1373. 4 Broekmans AW, Veltkamp JJ, Bertina RI: Congenital protein C deficiency and venous thrombo-embolism. A study of three Dutch families. N Engl J Med 1983;309:340-344. 5 Bertinm RI: Prevalance of hereditary thrombophilia and the identification of genetic risk factors. Fibrínolysis 1988;2(suppl 2):7-13.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
References
98
6 Patracchini P, Mello V, Pallazzi P, Calzolarí E, Bernardi F: Sublocalization of the human protein C gene on chromosone 2813—q14. Hum Genet 1989;81:191-492. 7 Foster DC, Yoshitake S, Davie EW: The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985;82:4673-4677. 8 Plutzky J, Hoskins JA, Long GL, Crabtree GR: Evolution and organization of the human protein C gene. Proc Natl Acad Sc USA 1986;83:546-550. 9 Romeo G, Hassan HJ, Staempfli S, Roncuzzi L, Cianetti L, Leonardi A, Vincente V, Mannucci PM, Bertína R, Peschle C, Cortese R: Hereditary thrombophília: Identification of nonsense and missense mutations in the protein C gene. Proc Nati Acad Sc USA 1987;84:2829-2832. 10 Matsuda M, Sugo T, Sakota Y, Murayama H, Mimuro J, Tenebe S, Yoshitake S: A thrombotic state due to abnormal protein C. N Engl J Med 1988;316:1265-1268. 11 Grundy C, Chitolic A, Talbot S, Bevan N, Kakkar V, Cooper DI: Protein C London 1: Recurrent mutation at Arg 169 (CGG —' TGG) in the protein C gene causing thrombosis. Nucl Acids Res 1989;17:10513. 12 Youssoufian H, Kazazían HH, Phillips DG, Aronis S, Tsiftis G, Brown VA, Antonarakis SE: Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. Nature 1986;324:380-382. 13 Kogan SC, Doherty M, Gitschier J: An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 1987;317:985990. 14 Lundwall A, Dackowski W, Cohen E, Shaffer M, Mahr A, Dahlbäck B, Stenflo J, Wydro R: Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation. Proc Nati Acad Sci USA 1986;83:6716-6720. 15 Hoskins JA, Norman DK, Beckmann Ri, Long GL: Cloning and characterization of human liver cDNA encoding a protein S precursor. Proc Nati Acad Sci USA 1987;84:349-353. 16 Ploos van Amstel HK, Van der Zanden AL, Reitsma PH, Bertína RΜ: Human protein S cDNA encodes Phe-16 and Tyr 222 in consensus sequences for the post-translational processing. FEES Lett 1987;222:186-190. 17 Gershagen S, Femlund P, Lundwall A: A cDNA coding for human sex hormone binding globulin. Homology to vitamin K-dependent protein S. FERS Lett 1987;220:129-135. 18 Ploos van Amstel HK, Van der Zanden AL, Bakker E, Reitsma PH, Bertína RM: Two genes homologous with human protein S cDNA are located on chromosome 3. Thromb Haemost 1987;58:982-987. 19 Watkins PC, Eddy R, Fukushima Y, Byers MG, Cohen EH, Dackowski WR, Wydro RΜ, Shows TB: The gene for protein S maps near the centromere of human chromosome 3. Blood 1988;71:238-241. 20 Ploos van Amstel HK, Reitsma PH, Bertina RΜ: The human protein S locus: Identification of the PSa gene as a site of liver protein S messenger RNA synthesis. Biochem Biophys Res Commun 1988;157:1033-1038. 21 Ploos van Amstel HK, Reitsma PH, Bertina RM: Identification of the protein Sβ gene as a pseudogene (abstract). Thromb Haemostas 1989;873. 22 Ploos van Amstel HK, Huisman MV, Reitsma PH, Ten Cate JW, Bertin RM: Partial protein S gene deletion in a family with hereditary thrombophilia. Blood 1989;73:479483. 23 Ploos van Amstel HK, Reitsma PH, Ηamulyák Κ, DeDie-Smulders CEl, Mannucci PM, Bertína RM: A mutation in the protein S pseudogene is linked to protein S deficiency in a thrombophiiic family. Thromb Haemost 1989;62:897-901.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
Reitsma et al.
Molecular Basis of Hereditary Protein C and Protein S Deficiency
99
Pieter H. Reitsma, MD, University Medical Center Leiden, Hemostasis and Thrombosis Research Unit, Department of Hematology, Building 1:C2-R, PO Box 9600, NL-2300 RC Leiden (The Netherlands)
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
24 Bertina RI, Ploos van Amstel HK, Van Wijngaarden A, Coenen J, Deutz-Terlouw PP, Reitsma PH, Van der Linden IK: The Heerlen polymorphism of protein S, an immunologic polymorphism due to dimorphism of residue 460. (abstract). Thromb Haemost 1989;62:975. 25 Schwartz HP, Heeb MJ, Lottenberg R, Roberts H, Grimn JH: Familial protein S deficiency with a variant protein S molecule in plasma and platelets. Blood 1989;74:213221.
Molecular Basis of Hereditary Protein C and Protein S Deficiency' Pieter H. Reitsma, Hans K. Ploos van Amstel, Bert R. Poort, Pieter A. van der Velden, Rogier M. Eertin University Medical Center Leiden, Hemostasis and Thrombosis Research Unit, Department of Hematology, Leiden, The Netherlands
One of the major inhibitory mechanisms operating in the blood coagulation cascade is the so-called `protein C anticoagulant pathway' [ 1]. This pathway is initiated when the circulating zymogen protein C is converted to activated protein C (APC) by a thrombin-thrombomodulín complex on the surface of endothelial cells. The formed APC, in the presence of its co-factor protein S, Cat ± ions, and phospholipids downregulates the coagulation cascade by proteolytic inactivation of the procoagulant cofactors Va and VIIIa. The physiological importance of the protein C pathway is most dramatically illustrated by the rarely occurring cases of severe protein C deficiency [2]. In these patients, who are either homozygotes or compound heterozygotes for a defect in the protein C gene, very low or undetectable levels of protein C coincide with severe thromboembolic disease as early as 1-6 days after birth. More mildly affected are patients that are heterozygotes for protein C or protein S deficiency [3, 4]. These deficiencies are frequently encountered (: 15-20%) in familial thrombophilia [5]. Thromboembolic symptoms in affected family members typically occur initially between the ages of 15 and 40 years. The diagnosis of protein C and protein S deficiency is performed with immunological and functional assays. The isolation of cDNA and genomic clones for both the protein C and protein S genes now makes it possible to
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
This work was supported by grant No. 28-1728 from the Praeventíefonds and by grant No. 88.002 from the Thrombose Stichting Nederland. We wish to thank D€nise van Ruiten for her help in preparing this manuscript.
Molecular Basis of Hereditary Protein C and Protein S Deficiency
95
further improve the detection of heterozygotes by DNA analysis. In addition, this type of analysis will enable us to get more insight in the molecular basis of the protein C and protein S deficiencies. In this short review we will focus on these developments.
The gene for protein C is localized on chromosome 2 [6]. The gene is approximately 11 kb long and consists of nine exons [7, 8]. The intron/exon organization of the protein C gene is very similar to that of the coagulation factors VII, IX and X, suggesting that they originate from one ancestral gene. Genetic defects have been described for the protein C gene in 4 patients. The first report by Romeo et al. [9] describes the genetic defect in a Dutch and Spanish family with type I (i.e. low antigen and activity levels) protein C deficiency. In the Dutch family a 306Arg — Stop substitution was found in the catalytic domain of the protein C molecule. The Spanish family was characterized by a 402-Trp — Cys substitution, also in the catalytic domain. Given the type I protein C deficiency, these two mutatuns apparently give rise to an unstable gene product. A second report by Matsuda et al. [ 10] describes the genetic findings in a compound heterozygote (protein C Tochigi) with a combined type I and type II (i.e. a variant protein with decreased activity) protein C deficiency. Analysis of the DNA indicated that the complete deletion of one protein C gene was responsible for the type I deficiency, whereas in the remaining gene a 169Arg —> Trp substitution had occurred. Residue 169Arg is located at the thrombin activation site of protein C and it is plausible that the substitution has led to a molecule that can not be activated, thereby giving rise to a type II deficiency. Grundy et al. [ 11 ] recently reported the same 169Arg — Trp substitution (protein C London 1) in a British subject with protein C deficiency and thrombosis. The geographical separation between protein C Tochigi and Protein C London 1 makes it unlikely that the two mutations are identical-by-descent. More likely is that the CpG dinucleotide involved in the substitution is a `hotspot' for mutation as has been reported for other hemostatic disorders [ 12]. In their analysis of protein C London 1, Grundy et al. [11] were the first to report the use of the polymerise chain reaction [ 13] followed by direct sequencing in the analysis of genetic defects in the protein C gene. This method works much faster than the conventional cloning procedure employed by Romeo et al. [9] and Matsuda et al. [ 10] and allows a large
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
Protein C
Reitsma et al.
96
number of defects to be analysed in the near future. Recently, our laboratory has also developed the protocols for the selective amplification of each of the protein C exons and is currently analyzing point mutations in the protein C gene. To date, preliminary evidence has been collected on 24 mutations including splice site mutations, nonsense mutations, missense mutations and a frameshift mutation [Reitsma et al. unpubl.].
The primary amino acid sequence of human protein S has been deduced from the nucleotide sequence of cloned cDNAs [ 14-16]. This sequence shows that protein S, like protein C, is a mosaic protein. Mature protein S is composed of a Gla region, a small disulfide loop that is sensitive to thrombin leavage, four EGF-like domains and a 392 residues long carboxy-terminal domain that shows weak but significant homology with the sex-hormone-binding globulin [ 17]. A detailed analysis of the human genome has shown the presence of two protein S genes, PSa and PSß, both of which are located near the centromere of chromosome 3 [ 18, 19]. To date, the cloning of only a part of the two genes has been reported [20, 21]. The partial nucleotide sequence analysis showed that the PSβ gene is highly homologous to the PSa gene but contains several mutations (stop codons and a deletion) that prohibit proper translation. Moreover, no transcription product of the PSβ gene could be identified [20]. Together these data indicate that the PSa gene is responsible for protein S synthesis whereas the PSß gene is a genuine pseudogene. Presently, the available data on a molecular analysis of the protein S genes is limited to a Southern blot analysis in a panel of 25 probands with type I deficiency [22, 23]. This survey revealed genetic changes in two families. In one thrombophilic family a major deletion of the middle part of the active PSa gene was observed [22]. This gene defect cosegregated with a reduced level of total protein S and should be considered the cause of the disease. In a second family the PSß pseudogene showed an altered Mspl restriction pattern in the proband [23]. Also this gene alteration cosegregated in the family with a reduced protein S antigen level. However, since the altered restriction site is located in the pseudogene rather than the active gene, it cannot be the cause of the protein S deficiency. In a screening of 50 normal individuals the alteration has not been observed and therefore it probably represents at most a very rare polymorphism.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
Protein S
Molecular Basis of Hereditary Protein C and Protein S Deficiency
97
The mutation in the PSß gene provided an opportunity to estimate the genetic distance between the two protein S genes by combining the data on plasma protein S levels and the altered restriction pattern. Since these two always cosegregated in 23 meioses the PSa and PSß gene should be located within a distance of 4 cM [23]. Recently, an interesting immunological variant protein S molecule (PS Heerlen) has been reported [24]. DNA analysis showed that the protein polymorphism is due to a T -+ C transition in the codon for 460Ser of the PSa gene. Consequently, 460Ser is substituted by Pro. The substitution has occurred in the consensus sequence for the N-linked glycosylation of 458Asn and most likely the PS Heerlen molecule is not glycosylated at this site. In this repect the PS Heerlen variant may be identical with a recently reported variant protein S molecule that was shown to have a reduced carbohydrate content [25]. The frequency of the PS Heerlen variant in the normal population and in patients with unexplained thrombosis is the same (x 0.5%). The molecule also has a normal APC cofactor activity and it is therefore unlikely that it plays a role in the etiology of thrombophilia.
Conclusion It has taken only a few years to go from the first case reports on protein C and protein S deficiency to the underlying molecular defects in the genes. With the advent of the PCR technique it is now expected that numerous mutations will be identified in the next few years. This will teach us how heterogeneous the gene defects in these deficiencies are.
1 Esmon CT; Protein C: Biochemistry, physiology and clinical implications. Blood 1983;62:1155-1158. 2 Marlar RA, Montgomery RR, Broekmans AW: Diagnosis and treatment of homozygous protein C deficiency. J Pediatr 1989;114:5287-5304. 3 Grime JH, Evatt B, Zimmermann TS, Kleiss AJ, Wideman C: Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981;68:1370-1373. 4 Broekmans AW, Veltkamp JJ, Bertina RI: Congenital protein C deficiency and venous thrombo-embolism. A study of three Dutch families. N Engl J Med 1983;309:340-344. 5 Bertinm RI: Prevalance of hereditary thrombophilia and the identification of genetic risk factors. Fibrínolysis 1988;2(suppl 2):7-13.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
References
98
6 Patracchini P, Mello V, Pallazzi P, Calzolarí E, Bernardi F: Sublocalization of the human protein C gene on chromosone 2813—q14. Hum Genet 1989;81:191-492. 7 Foster DC, Yoshitake S, Davie EW: The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985;82:4673-4677. 8 Plutzky J, Hoskins JA, Long GL, Crabtree GR: Evolution and organization of the human protein C gene. Proc Natl Acad Sc USA 1986;83:546-550. 9 Romeo G, Hassan HJ, Staempfli S, Roncuzzi L, Cianetti L, Leonardi A, Vincente V, Mannucci PM, Bertína R, Peschle C, Cortese R: Hereditary thrombophília: Identification of nonsense and missense mutations in the protein C gene. Proc Nati Acad Sc USA 1987;84:2829-2832. 10 Matsuda M, Sugo T, Sakota Y, Murayama H, Mimuro J, Tenebe S, Yoshitake S: A thrombotic state due to abnormal protein C. N Engl J Med 1988;316:1265-1268. 11 Grundy C, Chitolic A, Talbot S, Bevan N, Kakkar V, Cooper DI: Protein C London 1: Recurrent mutation at Arg 169 (CGG —' TGG) in the protein C gene causing thrombosis. Nucl Acids Res 1989;17:10513. 12 Youssoufian H, Kazazían HH, Phillips DG, Aronis S, Tsiftis G, Brown VA, Antonarakis SE: Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. Nature 1986;324:380-382. 13 Kogan SC, Doherty M, Gitschier J: An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 1987;317:985990. 14 Lundwall A, Dackowski W, Cohen E, Shaffer M, Mahr A, Dahlbäck B, Stenflo J, Wydro R: Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation. Proc Nati Acad Sci USA 1986;83:6716-6720. 15 Hoskins JA, Norman DK, Beckmann Ri, Long GL: Cloning and characterization of human liver cDNA encoding a protein S precursor. Proc Nati Acad Sci USA 1987;84:349-353. 16 Ploos van Amstel HK, Van der Zanden AL, Reitsma PH, Bertína RΜ: Human protein S cDNA encodes Phe-16 and Tyr 222 in consensus sequences for the post-translational processing. FEES Lett 1987;222:186-190. 17 Gershagen S, Femlund P, Lundwall A: A cDNA coding for human sex hormone binding globulin. Homology to vitamin K-dependent protein S. FERS Lett 1987;220:129-135. 18 Ploos van Amstel HK, Van der Zanden AL, Bakker E, Reitsma PH, Bertína RM: Two genes homologous with human protein S cDNA are located on chromosome 3. Thromb Haemost 1987;58:982-987. 19 Watkins PC, Eddy R, Fukushima Y, Byers MG, Cohen EH, Dackowski WR, Wydro RΜ, Shows TB: The gene for protein S maps near the centromere of human chromosome 3. Blood 1988;71:238-241. 20 Ploos van Amstel HK, Reitsma PH, Bertina RΜ: The human protein S locus: Identification of the PSa gene as a site of liver protein S messenger RNA synthesis. Biochem Biophys Res Commun 1988;157:1033-1038. 21 Ploos van Amstel HK, Reitsma PH, Bertina RM: Identification of the protein Sβ gene as a pseudogene (abstract). Thromb Haemostas 1989;873. 22 Ploos van Amstel HK, Huisman MV, Reitsma PH, Ten Cate JW, Bertin RM: Partial protein S gene deletion in a family with hereditary thrombophilia. Blood 1989;73:479483. 23 Ploos van Amstel HK, Reitsma PH, Ηamulyák Κ, DeDie-Smulders CEl, Mannucci PM, Bertína RM: A mutation in the protein S pseudogene is linked to protein S deficiency in a thrombophiiic family. Thromb Haemost 1989;62:897-901.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
Reitsma et al.
Molecular Basis of Hereditary Protein C and Protein S Deficiency
99
Pieter H. Reitsma, MD, University Medical Center Leiden, Hemostasis and Thrombosis Research Unit, Department of Hematology, Building 1:C2-R, PO Box 9600, NL-2300 RC Leiden (The Netherlands)
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 4:40:14 PM
24 Bertina RI, Ploos van Amstel HK, Van Wijngaarden A, Coenen J, Deutz-Terlouw PP, Reitsma PH, Van der Linden IK: The Heerlen polymorphism of protein S, an immunologic polymorphism due to dimorphism of residue 460. (abstract). Thromb Haemost 1989;62:975. 25 Schwartz HP, Heeb MJ, Lottenberg R, Roberts H, Grimn JH: Familial protein S deficiency with a variant protein S molecule in plasma and platelets. Blood 1989;74:213221.
