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Molecular Basis of Fibrinogen Naples Associated with Defective Thrombin Binding and Thrombophilia

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Molecular Basis of Fibrinogen Naples Associated with Defective Thrombin Binding and Thrombophilia Molecular basis of fibrinogen Naples associated with defective thrombin binding and thrombophilia. Homozygous substitution of B beta 68 Ala----Thr. J Koopman, … , J Grimbergen, P M Mannucci J Clin Invest. 1992;90(1):238-244. https://doi.org/10.1172/JCI115841. Research Article In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G----A) in the B beta chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the B beta chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human alpha-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound less than 10% of active site inhibited alpha-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound approximately 50% of alpha-thrombin. These results suggest that the mutation of B beta Ala 68----Thr affects the binding of alpha-thrombin […] Find the latest version: https://jci.me/115841/pdf Molecular Basis of Fibrinogen Naples Associated with Defective Thrombin Binding and Thrombophilia Homozygous Substitution of BB 68 Ala -- Thr Jaap Koopman,** Frits Haverkate,t Susan T. Lord,1 Jos Grimbergen,* and Pier Mannuccio Mannuccill * University Hospital and *Gaubius Laboratory Instituut voor Verouderings-en Vaatziekte Onderzoek, 2300 AK Leiden, The Netherlands; ODepartment of Pathology, University of North Carolina, Chapel Hill, North Carolina 27599-7525; and 1A. Bianchi Bonomi Hemophiliaand Thrombosis Centre and Institute ofInternal Medicine, University ofMilan, 20122 Milan, Italy Abstract tides A and B (FpA and FpB)' from the amino terminus of the Aa. and B,3 chains, respectively. The interaction of thrombin In an abnormal fibrinogen (fibrinogen Naples) associated with with fibrin(ogen) involves multiple sites on both thrombin and congenital thrombophilia we have identified a single base sub- fibrin(ogen). An important domain of fibrinogen that interacts stitution (G - A) in the B,8 chain gene that results in an amino with the catalytic'site of thrombin is located in the Aa chain acid substitution of alanine by threonine at position 68 in the between amino acids 1 and 23 (2-6). The interaction ofthrom- BB chain of fibrinogen. The propositus and two siblings were bin with fibrin through a site independent of the catalytic site found to be homozygous for the mutation, whereas the parents (anion-binding exosite) (7-12) is located in a cyanogen bro- and another sibling were found to be heterozygous. Individuals mide (CNBr)-derived fragment of the amino-terminal part of homozygous for the defect had a severe history of both arterial the fibrin(ogen) molecule amino-terminal disulfide knot and venous thrombosis; heterozygous individuals had no clini- (NDSK) (13-15). Binding of a-thrombin to fibrin by the an- cal symptoms. The three homozygotes had a prolonged throm- ion-binding exosite results in the removal of thrombin from bin clotting time in plasma, whereas the heterozygotes had a solution (7, 9). It has been proposed that this interaction plays normal thrombin clotting time. Fibrinopeptide A and B (FpA an important role in the regulation of thrombus formation in and FpB) release from purified fibrinogen by human a-throm- vivo by limiting the amount of free active thrombin in the bin was delayed in both the homozygous propositus and a het- circulation (7). Congenitally abnormal fibrinogens are valuable erozygous family member. Release of FpA from the normal and tools for structure-function studies of human fibrinogen. They abnormal amino-terminal disulfide knot (NDSK) corresponded also provide a basis for correlating the molecular defect with to that found with the intact fibrinogens, indicating a decreased the clinical symptoms of affected individuals. Many abnormal interaction of thrombin with the NDSK part of fibrinogen Na- fibrinogens have been described (16), of which a large number ples. Binding studies showed that fibrin from homozygous ab- show defective release of fibrinopeptides due to a mutation at normal fibrinogen bound < 10% of active site inhibited a- or near the thrombin cleaved bond (17-20). The structural thrombin as compared with normal fibrin, while fibrin formed defect of an abnormal fibrinogen (fibrinogen New York I) with from heterozygous abnormal fibrinogen bound - 50% of a- impaired thrombin binding to fibrin (21) has been determined. thrombin. These results suggest that the mutation of BB Ala This fibrinogen lacks amino, acids 9-72 in the Bfl-chain (22). 68 -- Thr affects the binding of a-thrombin to fibrin, and that The patient, who was shown to be heterozygous, suffered from defective binding results in a decreased release of FpA and FpB thrombotic episodes (23). Fibrinogen Naples (Milano II) (24, in both homozygous and heterozygous abnormal fibrinogens. 25) is another abnormal fibrinogen associated with congenital (J. Clin. Invest. 1992. 90:238-244.) Key words: amino-termi- thrombophilia. Preliminary work on fibrinogen Naples demon- nal disulfide knot * dysfibrinogenemia * fibrinopeptide release. strated a defective interaction between bovine thrombin and polymerase chain reaction * a-thrombin binding fibrin of the propositus (24). In this article we report the struc- tural defect of fibrinogen Naples, inferred from genetic analysis Introduction of patient DNA using the polymerase chain reaction (PCR) (26). The presence of the mutation in the family members, the The fibrinogen molecule is involved in the final phase of blood binding of human a-thrombin to fibrin, and the relationship coagulation and consists of pairs ofAa, Bf, and y chains linked between the defect and clinical symptoms were also deter- by 29 disulphide bonds (1). The conversion of fibrinogen to mined. fibrin is initiated by thrombin catalyzed cleavage of fibrinopep- Methods Patients. The propositus (11.3) developed postoperative deep-vein thrombosis at the age of 33 yr. His sister (11. 1) had a stroke at the age of 25 yr due to thrombotic occlusion of the internal carotid artery and his Address reprint requests to Dr. Haverkate, Gaubius Laboratory, brother (11.2) had a stroke and thrombosis ofthe abdominal aorta at the IVVO-TNO, P. 0. Box 430, 2300 AK Leiden, The Netherlands. age of 21 yr. Another brother (11.4) of the propositus is asymptomatic, Receivedfor publication 19 September 1991 and in revisedform 30 as are his parents (. 1 and 1.2) (who are first cousins). December 1991. J. Clin. Invest. 1. Abbreviations used in this paper: CNBr, cyanogen bromide; DFP, © The American Society for Clinical Investigation, Inc. difluorophosphate; FpA and FpB, fibrinopeptides A and B; NDSK, 0021-9738/92/07/0238/07 $2.00 amino-terminal disulfide knot; PCR, polymerase chain reaction; t-PA, Volume 90, July 1992, 238-244 tissue-type plasminogen activator. 238 Koopman et al. Coagulation studies on plasma. Blood was collected by venipunc- fibrin monomers was obtained by increasing the pH and performed by ture and anticoagulated with 0.1 vol of 0.11 M trisodium citrate. adding 60 ,d of the fibrin monomer solution to 740 ,1 of 0.1 M Tris/ Plasma was prepared by centrifugation at 2,300 g for 30 min at 4°C. HCI, pH 7.5. The increase in absorbance at 350 nm was recorded as a Thrombin clotting times were performed at 37°C with 200 Al ofplasma function of time on a spectrophotometer (SP 1700; Pye Unicam, Cam- and 50 Ml of human a-thrombin (1,000 National Institutes of Health bridge, UK). (NIH) U/mg; Protogen, Lafelfingen, Switzerland), dissolved in 0.15 M Preparation of ["C]difluorophosphate (DFP) a-thrombin. 1 ml of NaCl containing 0.025% (wt/vol) gelatin to a concentration of 25 NIH human a-thrombin, dissolved at 1 mg/ml in 10 mM Tris/HCl, pH 8.3, U/ml. Reptilase (Boehringer Mannheim, Mannheim, FRG) clotting containing 0.75 M NaCl, was incubated with 100 ,d of ['4C]DFP (108 time was performed as described (24). Fibrinogen concentration was mCi/mmol, 1.9 MM/ml; New England Nuclear, s'Hertogenbosch, The determined functionally according to Clauss (27) and immunologically Netherlands) for 90 min at 250C. Subsequently 10 Ml of a 0.1 M DFP according to Mancini et al. (28). solution in dry isopropanol was added, and incubated for 30 min. The Purification offibrinogen and NDSK Fibrinogen was purified from sample was dialyzed against 50 mM Tris/HCl, pH 7.5, 100 mM NaCl plasma as described (29) and dialyzed against PBS for 24 h at 4°C. for 24 h at 40C. Protein concentration was measured spectrophotomet- NDSK was purified after CNBr digestion of purified fibrinogen (30) by rically at 280 nm (A'%"C"' = 18.0 [34]). The amidolytic activity ofinhib- a fast protein liquid chromatography (FPLC) system equipped with a ited thrombin, determined by using the synthetic substrate S-2238 Superose 12 column (Pharmacia, Uppsala, Sweden). The column was (KabiVitrum, Stockholm, Sweden) according to the manufacturer's equilibrated with 10% acetic acid containing 0.1 M NaCl and run at a instructions, was < 0.05% of the original activity. The relative a-, ,B-, flow rate of 1.0 ml/min. The CNBr digest, 6 mg of protein in 0.3 ml, and y-thrombin content was determined by SDS-PAGE (31) followed was injected and 0.5-ml fractions were collected.
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