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Met 358 to Arg Mutation of Alpha 1-Antitrypsin Associated with Protein C Deficiency in a Patient with Mild Bleeding Tendency

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Met 358 to Arg Mutation of Alpha 1-Antitrypsin Associated with Protein C Deficiency in a Patient with Mild Bleeding Tendency Met 358 to Arg mutation of alpha 1-antitrypsin associated with protein C deficiency in a patient with mild bleeding tendency. D Vidaud, … , J N Fiessinger, M Aiach J Clin Invest. 1992;89(5):1537-1543. https://doi.org/10.1172/JCI115746. Research Article The molecular defect responsible for a dramatic prolongation of all standard clotting tests discovered in a 15-yr-old boy has been identified. Initial investigations revealed the presence of an activated Factor X (Factor Xa) and thrombin inhibitor which copurified with alpha 1-antitrypsin (alpha 1-AT), thereby suggesting the occurrence of an alpha 1-AT variant similar to alpha 1-AT Pittsburgh. This was confirmed by dot-blot analysis and direct sequencing after amplification by the polymerase chain reaction. A G to T transition at nucleotide 10038 results in the substitution of Met to an Arg, converting alpha 1-AT into an Arg-Ser protease inhibitor (serpin) that inhibited thrombin and Factor Xa more effectively than antithrombin III. Surprisingly, there was no bleeding history in the proband. The common mutation Z, which may explain a reduced expression of the allele bearing the Arg 358 Met mutation, was not observed in the propositus' DNA. To exclude the presence of another mutation, the coding regions and intron/exon junctions were sequenced. No other mutation was found. Recently, the patient experienced his first hemorrhagic episode at the age of 17. The level of the abnormal inhibitor had increased twofold 2 mo before. The large decrease in protein C concentration may account for the mild bleeding tendency in this case, despite the presence of the alpha 1-AT Pittsburgh mutation. An abnormal protein C […] Find the latest version: https://jci.me/115746/pdf Met 358 to Arg Mutation of Alpha1-Antitrypsin Associated with Protein C Deficiency in a Patient with Mild Bleeding Tendency D. Vidaud, J. Emmerich, M. Alhenc-Gelas, J. Yvart,* J. N. Fiessinger, and M. Aiach Laboratoire d'Hemostase and Centre Claude Bernard de Recherche sur les Maladies Vasculaires, H6pital Broussais, Paris; and *Laboratoire d'Hematologie, H6pital St. Joseph, Paris, France Abstract tor (1). This mutant, designated a,-AT Pittsburgh, or recombi- nant 358 was shown to inhibit other The molecular Arg a,-AT, clotting pro- defect responsible for a dramatic prolongation teases (2-4), thus displaying strong functional analogy with the of all standard clotting tests discovered in a 15-yr-old boy has physiologic clotting inhibitor antithrombin III (AT III), whose been identified. Initial investigations revealed the presence of reactive site consists of an Arg-Ser bond. This mutation pro- an activated X and Factor (Factor Xa) thrombin inhibitor which vided an explanation for the patient's recurrent and ultimately copurified with alpha1-antitrypsin (a,-AT), thereby suggesting fatal hemorrhagic diathesis (1, 5), and confirmed the critical the occurrence of an a,-AT variant similar to a,-AT Pittsburgh. role played by a single amino acid at the reactive site of serine- was This confirmed by dot-blot analysis and direct sequencing protease inhibitors (serpins). Serpins are a superfamily of pro- after amplification by the polymerase chain reaction. A G to T teins that have evolved from a primitive ancestral inhibitor and transition at nucleotide 10038 results in the substitution of Met to which a,-AT and AT III both belong (6). to an Arg, converting a,-AT into an Arg-Ser protease inhibitor Recently, was described as a physiological inhibitor more a,-AT (serpin) that inhibited thrombin and Factor Xa effectively of activated protein C (7), a natural anticoagulant playing a than antithrombin III. Surprisingly, there was no bleeding his- major role in the regulation of blood coagulation (for review in tory the proband. The common mutation Z, which may ex- see 8). Moreover, the Met 358 to Arg substitution was shown to plain a reduced expression of the allele bearing the Arg 358 multiply by 4,400 the rate of activated protein C inhibition (9). Met mutation, was not observed in the propositus' DNA. To A protease responsible for the intracellular processing of pre- exclude the presence of another mutation, the coding regions cursor polypeptides is also inhibited by the mutated a,-AT and intron/exon junctions were sequenced. No other mutation (10). Its coexpression with complement C3 and albumin in was found. Recently, the patient experienced his first hemor- transfected cells led to the inhibition of propeptide cleavage rhagic episode at the age of 17. The level of the abnormal inhibi- ( 11). Such an inhibition confirmed the mechanism proposed to tor had increased twofold 2 mo before. The large decrease in explain the presence of proalbumin in the Pittsburgh patient's protein C concentration may account for the mild bleeding ten- plasma (12). dency in this case, despite the presence of the a,-AT Pittsburgh We suspected the presence of a similar abnormality in a mutation. An abnormal protein C pattern was observed in pa- 15-yr-old patient whose routine tests before surgery revealed tient's plasma, suggesting that the circulating deficiency might markedly impaired coagulation, and were able to confirm the be due to a deleterious effect of the abnormal inhibitor on both existence ofa Met 358 to Arg mutation by genomic DNA analy- intracellular processing and catabolism of protein C. (J. Clin. Invest. sis. This patient had, however, never experienced any hemor- 1992.89:1537-1543.) Key words: a1-antitrypsin Pitts- rhagic complication. The association of the mutation - - * a,-AT burgh protein C * bleeding DNA mutation with a profound decrease in plasma protein C concentration suggests that Arg 358 a1-AT interferes with the in vivo metabo- Introduction lism of this anticoagulant regulatory protein. In 1983, a molecular defect responsible for the development of a potent circulating antithrombin activity in a young boy with Methods a severe bleeding disorder was characterized as Met 358 to Arg substitution at the reactive Met-Ser site of alpha1-antitrypsin thereby modifying the target of this protease inhibi- Coagulation factor assays. All plasma clotting assays were performed (a,-AT),' using commercial reagents from Diagnostica Stago (Gennevilliers, France) for Factors XII, XI, IX, VIII, X, and VII, or from bioMerieux Address correspondence and reprint requests to Dr. Martine Aiach, (Lyon, France) for Factors II and V. Laboratoire d'Hemostase, Hopital Broussais, 96, rue Didot, F-75674 Protein C was evaluated by amidolytic assays after Protac activa- Paris Cedex 14, France. tion (Diagnostica Stago), and after thrombin activation of an alumin- Receivedfor publication 20 June 1991 and in revisedform 14 Jan- ium hydroxide eluate according to Bertina (13), using Coatest protein uary 1992. C (Kabi, Mohndal, Sweden). AT III was measured using an amidolytic assay (AT-prest, Diagnostica Stago). Fibrinogen was evaluated in a 1. Abbreviations used in this paper: a,-AT, a1-antitrypsin; a, M, nornal chronometric assay, using Fibrinogen kit from bioMerieux. ASO probe; a, N, wild-type ASO probe; a, P, mutant probe; a, Z, Immunoassays were performed using immune sera from Diagnos- mutant ASO probe; ASO, allele-specific oligonucleotide; AT III, an- tica Stago and rocket immunoelectrophoresis, except for protein C and tithrombin III; Factor Xa, activated Factor X; PCR, polymerase chain Factor VII, which were measured by immunoenzymatic techniques reaction. (Diagnostica Stago). Bleeding time was measured using a simplate device (Organon Tek- J. Clin. Invest. nika, Turnhout, Belgium). © The American Society for Clinical Investigation, Inc. Purification of the variant. Plasma from the patient was first ad- 0021-9738/92/05/1537/07 $2.00 sorbed with bentonite (Sigma Chemical Co., St. Louis, MO), which Volume 89, May 1992, 1537-1543 eliminates numerous proteins, including AT III. The supernatant was Met 358 to Arg a,-Antitrypsin Associated with Protein C Deficiency 1537 then passed through a 50-ml column ofQ Sepharose (Pharmacia, Upp- Results sala, Sweden) at pH 7.4, using a FPLC system (Pharmacia), and eluted by a sodium chloride gradient. The fractions containing both a,-AT Case report. The hemostatic abnormality was discovered dur- and strong thrombin inhibitory activity were applied to the same col- ing a routine preoperative laboratory investigation of a 1 5-yr- umn and eluted using a pH gradient. The two final steps were per- old-boy suffering from kyphoscoliosis. The patient had no per- formed on a Mono column the by chromatography Q (Pharmacia), sonal or family history of spontaneous bleeding. Moreover, a first at pH 7.4 and the second at pH 6. Thrombin and FactorXa inhibition rates. The inhibitory activity of traumatic humeral fracture sustained at the age of 12 was not plasma or purified fractions was evaluated toward purified proteases at associated with any bleeding complication. Apart from ortho- 370C in 0.02 M Tris, pH 7.4, 0.15 M NaCl, PEG 0.5%. Briefly, 0.035 pedic disease, his clinical examination was entirely normal. ml of the sample was added to 0.35 ml of purified human thrombin or Laboratory evaluation included immunoelectrophoresis, Factor Xa (Diagnostica Stago). After a 1-min incubation, the residual complement, tumoral markers, antinuclear factors, immune protease amidolytic activity was determined and compared with a con- complexes, blood and urinary amino acids, and bone marrow trol incubated in the same conditions, but without inhibitor. The chro- aspirate, all ofwhich were normal. No hematuria was detected. mogenic substrates were H-D-phenylalanine-L-pipecolyl-L-arginine-D- Chest x-ray, abdominal echography, bone scintigraphy, and nitroanilide (S 2238; Kabi Vitrum, Stockholm, Sweden) and methane abdominal and thoracic CAT scan were also normal. sulfonyl-D-leucyl-glycyl-arginine-paranitroanilide (CBS 31.39; Diag- The results of the coagulation tests (Table I) suggested the nostica Stago). The first- and second-order rate constants for thrombin presence of a potent coagulation inhibitor, because they and Factor Xa inhibition were determined by incubating less than 10 nM of the protease for 1 min with a large molar excess of the inhibitor.
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