Characterization of plasminogen variants in healthy subjects and plasminogen mutants in patients with inherited plasminogen deficiency by isoelectric focusing gel electrophoresis

Katrin Tefs; Maria Georgieva; Stefan Seregard; Campbell R. Tait; Lori Luchtman-Jones; Maike Ziegler; Boris Hügle; Volker Schuster

doi:10.1160/TH04-01-0041

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 92(02): 352-357
DOI: 10.1160/TH04-01-0041

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Characterization of plasminogen variants in healthy subjects and plasminogen mutants in patients with inherited plasminogen deficiency by isoelectric focusing gel electrophoresis

Authors

Katrin Tefs

¹Children´s Hospital, University of Leipzig, Germany
Maria Georgieva

¹Children´s Hospital, University of Leipzig, Germany
Stefan Seregard

²Ophthalmic Pathology and Oncology Service, St Erik’s Eye Hospital and Karolinska Institute, Stockholm, Sweden
Campbell R. Tait

³Department of Haematology, Glasgow Royal Infirmary, UK
Lori Luchtman-Jones

⁴St. Louis Children’s Hospital, Washington University School of Medicine, St. Louis, Missouri, USA
Maike Ziegler

¹Children´s Hospital, University of Leipzig, Germany
Boris Hügle

¹Children´s Hospital, University of Leipzig, Germany
Volker Schuster

¹Children´s Hospital, University of Leipzig, Germany

Abstract

Summary

Plasmin(ogen) plays an important role in fibrinolysis and wound healing. Severe hypoplasminogenemia has recently been linked to ligneous conjunctivitis. Plasminogen (plg) is known as a polymorphic protein and most of these variants have been identified using isoelectric focusing (IEF) gel electrophoresis. Here, we studied common plg variants from healthy subjects and plg mutants from three patients with hypoplasminogenemia and three subjects with dysplasminogenemia by molecular genetic analysis and IEF. Analysis of 24 healthy subjects showed that subjects with the most common IEF plg phenotype A (n = 12) were homozygous for aspartate at position 453 (453D), while both subjects with IEF plg phenotype B were homozygous for asparagine at this position (453N). Subjects with IEF plg phenotype AB (n = 10) were compound-heterozygous for 453D/453N. Three patients with severe hypoplasminogenemia and different plg gene mutations exhibited characteristic “abnormal” IEF band patterns when compared with IEF plg phenotypes A and B. In all heterozygous family members the observed IEF plg phenotype was derived from the wild type plg molecule only, probably due to low concentration of the mutant plg molecule in plasma. In contrast, in three unrelated subjects with heterozygous dysplasminogenemia an equal “mixture” of wild type and mutant plg was found by IEF analysis. In conclusion, plg phenotyping by IEF in combination with molecular analysis of the plg gene seems to be a useful method for characterization of plg variants and mutants.

Keywords

Isoelectric focusing (IEF) gel electrophoresis - plasminogen (plg) - hypoplasminogenemia - dysplasminogenemia - ligneous conjunctivitis

Full Text

References

References
1 Robbins KC, Summaria L. Plasminogen and plasmin. Methods Enzymol 1976; 45: 257-273.
2 Schuster V, Seregard S. Ligneous conjunctivitis. Surv Ophthalmol 2003; 48: 369-388.
3 Robbins KC. Dysplasminogenemias. Prog Cardiovasc Dis 1992; 34: 295-308.
4 Skoda U, Klein A, Lubcke I. et al. Application of plasminogen polymorphism to forensic hemogenetics. Electrophoresis 1988; 09: 422-426.
5 Aoki N, Moroi M, Sakata Y. et al. Abnormal plasminogen. A hereditary molecular abnormality found in a patient with recurrent thrombosis. J Clin Invest 1978; 61: 1186-1195.
6 Shigekiyo T, Kanazuka M, Aihara K. et al. No increased risk of thrombosis in heterozygous congenital dysplasminogenemia. Int J Hematol 2000; 72: 247-252.
7 Tait RC, Walker ID, Conkie JA. et al. Isolated familial plasminogen deficiency may not be a risk factor for thrombosis. Thromb Haemost 1996; 76: 1004-1008.
8 Okamoto A, Sakata T, Mannami T. et al. Population-based distribution of plasminogen activity and estimated prevalence and relevance to thrombotic diseases of plasminogen deficiency in Japanese: the Suita Study. J Thromb Haemost 2003; 01: 2397-2403.
9 Hobart MJ. Genetic polymorphism of human plasminogen. Ann Hum Genet 1979; 42: 419-423.
10 Raum D, Marcus D, Alper CA. Genetic polymorphism of human plasminogen. Am J Hum Genet 1980; 32: 681-689.
11 Skoda U, Bertrams J, Dykers D. et al. Proposal for the nomenclature of human plasminogen (PLG) polymorphism. Vox Sang 1986; 51: 244-248.
12 Pytlik C, Rand S, Brinkmann B. Immunochemical demonstration of plasminogen phenotypes using electroblotting. Int J Legal Med 1990; 104: 21-23.
13 Skoda U, Goldmann SF, Handler C. et al. Plasminogen hemizygosity. Detection of a silent allele in 7 members of a family by determination of plasminogen phenotypes, anti-genic levels, and functional activity. Vox Sang 1988; 54: 210-214.
14 Kikuchi S, Yamanouchi Y, Li L. et al. Plasminogen with type-I mutation is polymorphic in the Japanese population. Hum Genet 1992; 90: 7-11.
15 Li L, Kikuchi S, Arinami T. et al. Plasminogen with type-I mutation in the Chinese Han population. Clin Genet 1994; 45: 285-287.
16 Schuster V, Seidenspinner S, Zeitler P. et al. Compound-heterozygous mutations in the plasminogen gene predispose to the development of ligneous conjunctivitis. Blood 1999; 93: 3457-3466.
17 Schuster V, Zeitler P, Seregard S. et al. Homozygous and compound-heterozygous type I plasminogen deficiency is a common cause of ligneous conjunctivitis. Thromb Haemost 2001; 85: 1004-1010.
18 Tefs K, Tait RC, Walker ID. et al. AK19E missense mutation in the plasminogen gene is a common cause of familial hypoplasminogenaemia. Blood Coagul Fibrinolysis 2003; 14: 411-416.
19 Petersen TE, Martzen MR, Ichinose A. et al. Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system. J Biol Chem 1990; 265: 6104-6111.
20 Geserick G, Patzelt D, Schröder H. Zum Polymorphismus des menschlichen Plasminogens. Z Klin Med 1988; 43: 587-589.
21 Tait RC, Walker ID, Conkie JA. et al. Plasminogen levels in healthy volunteers - influence of age, sex, smoking and oral contraceptives. Thromb Haemost 1992; 68: 506-510.
22 Summaria L, Arzadon L, Bernabe P. et al. Studies on the isolation of the multiple molecular forms of human plasminogen and plasmin by isoelectric focusing methods. J Biol Chem 1972; 247: 4691-4702.
23 Summaria L, Arzadon L, Bernabe P. et al. Characterization of the NH 2-terminal glutamic acid and NH 2-terminal lysine forms of human plasminogen isolated by affinity chromatography and isoelectric focusing methods. J Biol Chem 1973; 248: 2984-2991.
24 Hayes ML, Castellino JF. Carbohydrate of the human plasminogen variants. I. Carbohydrate composition, glycopeptide isolation, and characterization. J Biol Chem 1979; 254: 8768-8771.
25 Hayes ML, Castellino JF. Carbohydrate of the human plasminogen variants. II. Structure of the asparagine-linked oligosaccharide unit. J Biol Chem 1979; 254: 8772-8776.
26 Molgaard L, Ponting CP, Christensen U. Glycosylation at Asn-289 facilitates the ligand-induced conformational changes of human Gluplasminogen. FEBS Lett 1997; 405: 363-368.
27 Scherz R, Rohner R, Pflugshaupt R. et al. Genetic polymorphism of plasminogen in the Swiss population. In: Advances in forensic haemogenetics. Brinkmann Henningse. eds: Springer, Berlin; 1986: 279-281.
28 Dykes DD, Polesky HF. Incidence of the PLG*Q0 Allele in Human Populations. In: Advances in forensic haemogenetics. Mayr: Springer, Berlin; 1988: 261-264.
29 Schuster V, Mingers AM, Seidenspinner S. et al. Homozygous mutations in the plasminogen gene of two unrelated girls with ligneous conjunctivitis. Blood 1997; 90: 958-966.
30 Azuma H, Mima N, Shirakawa M. et al. Molecular pathogenesis of type I congenital plasminogen deficiency: expression of recombinant human mutant plasminogens in mammalian cells. Blood 1997; 89: 183-190.
31 Ichinose A, Espling ES, Takamatsu J. et al. Two types of abnormal genes for plasminogen in families with a predisposition for thrombosis. Proc Natl Acad Sci USA 1991; 88: 115-119.
32 Tsutsumi S, Saito T, Sakata T. et al. Genetic diagnosis of dysplasminogenemia: detection of an Ala601-Thr mutation in 118 out of 125 families and identification of a new Asp676-Asn mutation. Thromb Haemost 1996; 76: 135-138.
33 Heidemann DG, Williams GA, Hartzer M. et al. Treatment of ligneous conjunctivitis with topical plasmin and topical plasminogen. Cornea 2003; 22: 760-762.