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A novel amino acid substitution in the reactive site of a congenital variant antithrombin. Antithrombin pescara, ARG393 to pro, caused by a CGT to CCT mutation

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Antithrombin is a plasma protein inhibitor that can be grouped within a serine proteinase inhibitor superfamily. Antithrombin Pescara is a functional variant of antithrombin found in a family with a high incidence of thrombosis. Preliminary
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  THE OURNAL F BIOLOGICAL CHEMISTRY 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 264, No. 17, ssue of June 15, p 10200-10204,1969 Printed n U S A A Novel Amino Acid Substitution in the Reactive ite of a Congenital Variant Antithrombin ANTITHROMBIN PESCARA, ARG393 TO PRO, CAUSED BY A CGT TO CCT MUTATION* (Received for publication, November 23, 1988 David A. Lane, Hediye Erdjument, Elizabeth Thompson, Maria Panico, Vincenzo Di Marzo, Howard R. Morris, Giuseppe Leone, Valerio De Stefano, and Swee Lay Thein From the Department o Haematology, Chming ross and Westminster Medical School, Hammersmith, Great Britain, the Department o Biochemistry, Imperial College, South Kensington, London, Great Britain, the Zstituto di Semewtica Medica, Uniuersita Cattolica, Rome. Ztalv. and the Nuffield Deuartment o Clinical Medicine, ohn Radcliffe Hospital, Oxford, Great Britain Antithrombin is a plasma protein inhibitor that can be grouped within a serine proteinase inhibitor super- family. Antithrombin Pescara is a functional variant of antithrombin found in a family with a high incidence of thrombosis. Preliminary functional analysis has suggested that the abnormality esides in the reactive site rather than in the heparin binding omain of the molecule. Accordingly, we have isolated the variant from plasma using heparin-Sepharose chromatogra- phy, followed by chromatography upon thrombin- Sepharose to remove the normal antithrombin that is present (the propositus is heterozygous for the var- iant). The variant protein was reduced, S-carboxy- methylated, nd ragmented with CNBr. A pool (“CNBr pool 4” containing the reactive ite region was isolated by reverse-phase high performance iquid chromatography and sequentially treated with trypsin and VS protease. Fast atom bombardment-mass spec- trometric analysis of this subdigest identified a novel peptide of mass 1708 our steps of Edman degradation together with further analysis by fast atom bombard- ment-mass spectroscopy identified the NH2-terminal sequence of this peptide as Ala-Ala-Ala-Ser. The mass of the novel peptide and its changing mass in response to Edman degradation are only compatible with its identity as AlaS82-Arg39g, ith the reactive site Arg39a replaced by Pro. sing specific oligonucleotide hybrid- ization, we demonstrated that the molecular defect of antithrombin Pescara is caused by a CGT to CCT mu- tation in codon 393 These findings may be oFbroad interest, as other members of the serine protease nhib- itor superfamily contain arginine t their reactive sites and may be expected to undergo a similar mutation. The proteinase inhibitor antithrombin plays a central role in the regulation of proteinase activity generated when the blood coagulation system is activated (1, 2). Inherited func- tional deficiency of this inhibitor is mostly associated with a high incidence of familial thrombosis 3). Patients with in- herited deficiency can be classified according to whether there * This work was supported by a British Heart Foundation grant (to D. A. L. and H. R. M.), a Medical Research Council grant (to H. R. M.), and a Wellcome Trust grant (to S. L. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely o indicate this fact. . Wellcome Senior Research Fellow in Clinical Science. is a reduced level of protein (type I deficiency) or whether there is a normal level of functionally abnormal variant protein (type I1 deficiency) 4). Antithrombin has two distinct functional domains, a heparin binding domain located near to the NH2 terminus 5-7) and a reactive site domain near to the COOH terminus (8). Structural and functional character- izations of inherited variants have permitted their further subclassification. There are two main sub groups of this classification, one with inherited abnormalities affecting the heparin binding domain and the other with abnormalities of reactive site domain (9, 10). Antithrombin Pescara is a variant antithrombin that has been the subject of an initial report 11). The investigation of the family with the variant has recently been extended. Fam- ily members have a predisposition to thrombosis. Out of 34 family members from 2 generations, 13 have had deep venous thrombosis, carotid artery occlusion, or pulmonary embolism. Of these 13 amily members, only the propositus and 2 ousins are still alive for investigation. The variant protein has been detected in 9 out of 32 family members available for investi- gation. Five of these 9 are in a 3rd generation, are under 25 years, and are as yet free from thrombosis. From the initial report 11) it appeared that antithrombin Pescara had normal ability to bind heparin-Sepharose and reduced ability to inactivate the coagulation proteinases thrombin and factor Xa, determined in assays performed in the presence of heparin (heparin cofactor assays). This sug- gested an abnormality in the reactive site region, rather than in the heparin binding domain. Furthermore, the variant was compared to other variants y crossed immunoelectrofocusing and appeared to have an abnormal pattern found in anti- thrombin variants with defective inactivation of serine pro- teinases 12). In this report, we demonstrate that antithrom- bin Pescara has a single amino acid substitution at the reac- tive site, to Pro. We demonstrate by oligonucleotide hybridization that this amino acid substitution results from a point mutation, CGT to CCT, in the codon for Arp. MATERIALS AND METHODS Protein Analysis-Antithrombin was isolated from plasma of nor- mal subjects and the propositus (with antithrombin Pescara) 11) by stepwise elution from heparin-Sepharose followed by anion exchange chromatography (fast protein liquid chromatography, Phamacia LKB Biotechnology Inc.) (13, 14). Antithrombin obtained in this way from the propositus was further purified to remove normal antithrombin (this patient is heterozygous) by thrombin-Sepharose chromatogra- phy 15). This was performed as follows. CNBr-activated Sepharose (Pharmacia) was swollen and washed in 1 mMHCl according to the manufacturer’s instructions. To 7 ml of swollen gel (equivalent to -2 10200  Antithrombin Pescara t Pro 10201 g of dried gel) was added 20 mg of thrombin (kindly supplied by Dr. J.-M. Freyssinet, Centre de Transfusion Sanguine de Strasbourg, France), dissolved in 20 ml of NaHC03, pH 8.3, containing 0.5 M NaC1. The suspension was gently stirred for 2 h at room temperature and then washed extensively with 0.1 M Tris-HC1 buffer, pH 8.3 containing 0.5 M NaCl. Antithrombin was applied very slowly to the column, which had been previously equilibrated with 0.1 M NHdHC03, pH 8.3, containing 0.15 M NaCI. Preliminary experiments with a small aliquot of the thrombin-Sepharose established that at least 5 mg of normal antithrombin would bind if all of the matrix was to be used. Approximately 5 mg of antithrombin from the propositus was therefore applied to -6.5 ml of the column, and the protein that was eluted (variant component) was recovered for further study. The reaction of thrombin and antithrombin was studied at 25 C (in the absence of heparin) under pseudo-first order reaction condi- tions. Thrombin (20 nM) was incubated with increasing concentra- tions (50-350 nM) of normal antithrombin and variant antithrombin (eluted from the thrombin-Sepharose column) in 0.15 M NaCI, 0.05 M Tris-HC1, pH 7.4, containing polyethylene glycol 6000. At ifferent times, the incubation mixtures were subsampled into cuvettes con- taining chromogenic substrate (S2238, KabiVitrum), and residual thrombin was determined. Relative velocities of substrate hydrolysis, VIVO where V is the velocity at a given time and Vo the initial velocity, were plotted (semilog) against time (for example, see Ref. 13). S-Carboxymethylation of normal and variant antithrombins was carried out as described elsewhere (14). Following fragmentation with CNBr, the major pool CNBr pool 4 containing the regions of the reactive site was recovered by reverse-phase high performance liquid chromatography (15). Sequential trypsin and V8 protease digestions of normal and variant CNBr pool 4 were performed in 50 mMNH4HC03, pH 8.4, using trypsin (Sigma) at an enzymembstrate ratio of 1:50 (w/w) and Staphylococcus aureus V8 protease (ICN) at an enzyme:substrate ratio of 1:20 (w/w). Sequential Edman degradation was carried out on the enzyme subdigests using, at each stage, phenylisothiocyanate (Rathburn Chemicals) at 45 C for 1 h, followed by cleavage with trifluoroacetic acid (Rathburn Chemicals) for 10 min at 45 C. The exact molecular mass (M H)+ of remaining shortened peptides (after n cycles) was then determined by high field FAB-MS.' This was performed using a VGZAB HF instrument equipped with an Scan FAB gun operated at a 20-hA beam current at keV (16). Oligonucleotide Analysis-According to the genetic code for amino acids and he cDNA sequence of antithrombin (17-19), the only possible single base substitution in codon 393 that could produce an Arg to Pro substitution is CGT to CCT. A 22-base-long oligonucleo- tide (AT-Pescara) homologous to thepredicted ariant gene sequence in the region of codon 393 (Fig. 3) was synthesized on an Applied Biosystems synthesizer. Other oligonucleotides corresponding to the normal antithrombin gene (AT-Normal) and to the mutant equences present in antithrombins Northwick Park (AT-NWP) and Glasgow (AT-Glasgow) were synthesized as described (20). The sequences of these oligonucleotides were designed to obtain the maximum desta- bilizing effect between the single base mismatched hybrids. Previous applications have shown that the mismatched nucleotides should be as central as possible and that G-T mismatches should be avoided 21). The oligonucleotide probes, AT-NWP and AT-Glasgow, were included in this study as further controls for the specificity of the oligonucleotide AT-Pescara. DNA was xtracted from peripheral blood cell buffy coats according to standard rocedures. Peripheral blood was only available from the propositus and not from the remainder of the family with antithrom- bin Pescara. Ten micrograms of genomic DNA from the propositus, individuals I1,12, and I12 from family A with antithrombin Northwick Park, D. W with antithrombin Glasgow (see Ref. 20 for a summary of the family trees), and a normal individual, were digested with PstI and the ragments were separated by electrophoresis in a 0.7% agarose gel. Hybridization experiments were performed using 32P-labeled oli- gonucleotides as described in detail elsewhere (20). The gel was hybridized at 50 C overnight to each of the labeled oligonucleotides consecutively. After washing twice for 15 min each in 6 SSC 1 SSC is 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0) at room temperature and then wice for 20 min in 3 M tetramethylammonium chloride, 50 mMTris at pH 8.0, 2 mMEDTA, 0.1% sodium dodecyl The abbreviations used are: FAB-MS, fast atom bombardment- mass spectroscopy; kb, kilobase pairs. sulfate at 60-63 C, the gels were autoradiographed between two intensifying screens at -70 'C. RESULTS Antithrombin from the propositus, which was solated from plasma by heparin-Sepharose and thrombin-Sepharose chro- matography, was reacted with thrombin (in the absence of heparin) under pseudo-first order conditions. It had minimal ability to inactivate thrombin under circumstances in which normal antithrombin had a second order rate constant of inhibition of approximately 1.0 ' min (result not shown). This result was consistent with the view that anti- thrombin Pescara s a variant with a reactive site abnormality 11). V8 protease subdigestion of the tryptic digest of peptides contained in CNBr pool 4 of normal antithrombin produced peptide mixtures that were analyzed by FAB-MS. Results were entirely consistent with those previously reported in detail (15). Trypsin digestion of CNBr pool 4 from normal antithrombin produces more than 20 peptides that can be assigned to its primary structure. Subdigestion with V8 pro- tease cleaves these peptides after Glu residues. Of particular interest in the present context are he two tryptic peptides of mass 2290 and 700 that correspond to the normal sequence Ala371-Ar$93 and Se~~'~-Arg399, espectively. Following addi- tion of V8 protease, the peptide of mass 2290 is reduced to a mass 1086, corresponding to the sequence Ala382-Arg393 15). It has been shown previously that amino acid substitutions at the reactive site (Arg o Cys and to His, in antithrom- bins Northwick Park and Glasgow, respectively) can prevent trypsin cleavage of the peptide Ala371-Arg3w t the 393-394 bond 15). ubsequent addition of V8 protease can result in novel peptides corresponding to Ala3s2-Ar$w containing the amino acid substitutions. These novel peptides can be iden- tified on the basis of their unique masses (M H + 772 and 1748, detectable by FAB-MS analysis. A novel peptide of unique mass was indeed found when the V8 protease subdigest of CNBr pool 4 of antithrombin Pescara was examined by A) 698 1086 1104 1708 698 FIG 1. FAB mapping analysis of antithrombin Pescara. Normal antithrombin and antithrombin Pescara were reduced, S- carboxymethylated, and treated with CNBr. The pools (CNBr pool 4) containing the sequence Gly339-Met423 ere then recovered by reverse-phase high performance liquid chromatography. These nor mal and variant pools were sequentially treated with trypsin and V8 protease, and the peptides generated were studied by FAB mapping. Only the regions of the spectra where differences were observed between the two digests, normal antithrombin (A) and antithrombin Pescara B) are shown. That is, the regions corresponding to the quasimolecular ions at m z 700, 1086, and 1708.  10202 Antithrombin Pescara, Ar 93 to Pro \ V M+H)+ 2912 V8 Protease 371 Ala-phe-Leu-Glu ............................................. Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-lle-Ala-Gly- Pro -Ser-Leu-Asn-Pro-Asn-Arg 74 382 399 (M+H)+ 479 (M+H)+ 1708 1,2,3,4 tep Edman degradation Ala, Ala, Ala, Ser -Ser-Leu-Asn-Pro-Asn-Arg 386 399 (M+H)+ 1408 FIG. 2. Strategy employed for the characterization of the amino acid abnormality of antithrombin Pescara. A tryptic digest of CNBr pool 4 obtained from antithrombin Pescara was subdigested with V8 protease. The (presumed) tryptic eptide of sequence Ala37 -Ar299, resent in the CNBr pool because trypsin does not cleave the Pro393-Ser394 ond, is cleaved at the Gl~p-Ala~~~ ond to produce the novel peptide Ala382-Ar299 containing Pro393. This peptide has a unique mass of 1708 which is readily detectable by FAB-MS (see Fig. 1). Four steps of Edman degradation performed on the ubdigest resulted in progressive reduction in the mass of this peptide (1637, 1566, 1495, and 1408), corresponding to loss of the NHn-terminal amino acids Ala, Ala, Ala, and Ser. The mass of the peptide produced by V8 protease digestion (1708), its NHz-terminal sequence, and its changing mass with Edman degradation are all only compatible with its sequence Ala382-Arp, with Argg3 substituted by Pro (as shown). 389 90 91 92 A B Normal al Ile la ly rg er eu Asn Pro 5 - GTG ATT GCT GGC CGT TCG CTA AAC CCC -3 3 - CAC TAA CGA CCG GCA AGC GAT TTG GGG-5 Antithrombin escara 5 - GTG ATT GCT GGCCCT TCG CTA AAC CCC -3 3 - CAC TAA CGA CCG GGA AGC GAT TTG GGG -5 kb Antithrombin Northwick Park 5 - GTG ATT GCT GC GT CG TA AC CC -3 CY 5 3 - CAC AA CGA CCG CA AGC GAT TTG GGG -5 2 5 1 2 3 4 N P kb .l .. 23 94 6.5 4.3 2.3 2.0 kb 425 Antithrombin Glasgow 5 - GTG ATT GCT GC AT CG TA AC CC -3 His 3 - CAC AA GA CG LA AGC GAT TTG GGG -5 Oligonucleotide Probes AT-Noma1 5 -GTTTAGCWACGGCCAGCAATC-3 AT-Pescara 5 -GATTGCTGGCCsTTCGCTAAAC-3 AT-NWP 5 -GTTTAGCGAACAGCCAGCAATC-3 AT-Glasgow 5 -GATTGCTGGCCATTCGCTAAAC-3 FIG. 3. Sequences of the oligonucleotide probes used in the analysis of antithrombin Pescara. The oligonucleotide probes were synthesized according to the sense or antisense strands of the normal and variant genes in the region of codon 393. The choice of sense or antisense strand was made in order that there would be a maximum destabilizing effect between the single base mismatched hybrids, G-T mismatches being avoided. FAB-MS. Fig. shows the regional FAB-MS of V8 protease subdigests of normal antithrombin and of antithrombin Pes- cara. The normal antithrombin FAB-MS clearly shows mass signals from the peptides of 1086 and 700, while that of antithrombin Pescara is characterized by the virtual absence of these mass signals and the presence of a novel mass signal corresponding to a peptide of mass 1708. These results were only compatible with a substitution of Pro at Arg?93 see Fig. 2). To further substantiate the assignment of this amino acid substitution, additional analysis of the V8 protease subdigest of antithrombin Pescara was undertaken. Sequential Edman degradation in combination with FAB-MS was carried out. Amino acids Ala, Ala, Ala, Ser were sequentially lost from the AT Normal AT Pescara D 1234NP 234NP kb , 23 9.4 6.5 4.3 b 42.5 kb 2 5 2.3 2.0 AT Northwick Park AT Glasgow FIG. 4. Analysis of the antithrombin variants using syn thetic oligonucleotide probes. Hybridization was carried out with the normal (AT-Normal, A). AT-Pescara B), AT-NWP (C), and AT- Glasgow 0) ligonucleotide probes. Lunes 1 2, and refer to indi- viduals I1,12, and I12 of family A with antithrombin Northwick Park; lune 4 refers to D. W. of family B with antithrombin Glasgow (see Ref. 20 for summary of family trees); N, normal individual; and P the propositus with antithrombin Pescara. The positions of the Hind111 markers correspond to 23-, 9.4-, 6.5-, 4.3-, 2.3-, and 2.0-kb sizes. NH2 terminus of this peptide, as determined by its changing mass 1637,1566,1495, and 1408) with each step of degrada- tion see Fig. 2 . This procedure unequivocally placed the NH2 terminus of the peptide of mass 1708 at Ala382 nd conclusively demonstrates the substitution of Pro393, o other substitution  Antithrombin Pescara, Ar. 93 to Pro 10203 or combination of likely substitutions could produce these changing mass numbers. Having identified the amino acid sequence abnormality of antithrombin Pescara, we sought its genetic srcin using the technique of specific oligonucleotide hybridization. Previous studies have shown that the antithrombin gene has 6 introns and 7 exons and that PstI igestion yields a minimum of three 10.5, 2.5, and 1.8 kb) antithrombin-specific genomic frag- ments 23-25). The reactive site region is contained on the 7th exon. A PstI site polymorphism divides the main 10.5-kb fragment into 5.5 and 5.0 kb. The sequence detected by the oligonucleotides specific for the region around codon 393 is present in the invariant 2.5-kb fragment. Hybridization with the AT-Normal probe showed that the invariant 2.5-kb fragment was present n all the genomic DNAs Figs. 3 and 4A . Rehybridization of the gel with AT- Pescara showed a 2.5-kb fragment present only in the ropos- itus but not n the DNA of the other individuals Figs. 3 and 4B). The results indicate that the propositus is heterozygous for antithrombin Pescara and that the olecular defect is due to a single base substitution, CGT to CCT, at codon 393 of the antithrombin gene. Rehybridization of the gel with AT- NWP Figs. 3 and 4C and AT-Glasgow Figs. 3 and 40), respectively, produced the expected results, that individuals 1 and 3 are heterozygous for antithrombin Northwick Park and that individual 4 is heterozygous for antithrombin Glasgow 15, 20). DISCUSSION The demonstration here of an amino acid substitution at the reactive site, Arp to Pro, n antithrombin Pescara provides an explanation for the inability of this antithrombin to react with proteinases, particularly thrombin; the Pro393- Ser394 ond is not an appropriate substrate for the active site of the enzyme 22). The substitution therefore also accounts for the reduced heparin cofactor activities of plasma from affected members of the family 11). This is because heparin cofactor assays depend upon the ability of antithrombin to interact with both heparin and the proteinase. While the former interaction is presumed normal, the latter is clearly not. Detailed kinetic studies of the interactions of the purified variant inhibitor and thrombin, n the absence or presence of heparin, could not be performed in the present study because of the limited quantities of variant that could be isolated. However, from a consideration of the results of heparin co- factor activities of patient plasma 11) and of the preliminary kinetic experiments noted under “Results,” it might be ex- pected that the variant will have minimal, if any, ability to inactivate thrombin whether or not heparin s present. Antithrombin is the major proteinase inhibitor of thrombin in blood and is known to be important in the regulation of thrombin activity 1-3). Reduction in functional activity of antithrombin is generally associated with thrombotic disor- ders 3). The identification of an Arg to Pro substitution in the reactive site of antithrombin Pescara, resulting in a func- tionally inactive inhibitor, therefore also provides an expla- nation for the high incidence of thrombosis in this family. Other variants with different amino acid substitutions at the same site demonstrate a imilar inability to inhibit thrombin. Inheritance of these other variants, antithrombins orthwick Park and Milano Arg393 o Cys) 15, 26), Glasgow, Sheffield, and Chicago Arg393 to His) 15, 27, 28), are all associated with thrombotic disorders. Inheritance of a recently charac- terized variant, antithrombin Utah, is also associated with thrombosis 25,29). In this latter case, a type I deficiency was srcinally suspected. However, DNA sequence analysis has identified a substitution, Pro407 o Leu, near to the reactive site 25, 29). While those variants with substitutions at cannot react with thrombin, the substitution at Pro407 may also alter the properties of the inhibitor in a different way 25): it may cause a failure in folding of the molecule, as Pro is conserved at this position in all of members of the serine proteinase inhibitor family 22, 31), and may therefore con- stitute an important structural lement. An incorrectly folded molecule may not be efficiently secreted into the blood, or could be more rapidly catabolized either possibility could explain the observed reduced plasma concentration of this variant. Antithrombin Denver was the first variant reported to have a substitution at the reactive site, Ser394 o Leu 32). Interestingly, thrombin can still react with the variant pro- tein, albeit at a reduced rate. Apparently, the limited inhibi- tory capacity of this variant acting in concert ith the normal antithrombin that is also present in the plasma of this family may almost be sufficient to regulate coagulation proteinase activity, as only the propositus from the family with this variant is known to have had thrombosis. It has been pointed out that while abnormalities affecting the reactive site of antithrombin esult in a similar high incidence of familial thrombosis to that seen in type I defi- ciency, substitutions within the heparin binding domain of antithrombin are not generally associated with this disorder 33-35). Antithrombins Toyama, Tours, and Alger Arg47 o Cys) 36, 37, 45), Base1 Pro41 o Leu) 38), Rouen I Arg47 o His) 39), and Rouen I1 Arg47 o Ser) 40) all have abnormalities that affect heparin binding. It appears that as long as the patients with these abnormalities are heterozygous, they do not have an increased tendency toward thrombosis 35). The molecular basis of antithrombin deficiency has recently been investigated in several studies 20, 23, 30, 37). In a survey of 16 families with type I deficiency using restriction enzymes, only one family was found to have a deletion of the antithrombin gene 30). The antithrombin genes in the other 15 families were grossly normal. The defects could not be identified, but were assumed to be due to point mutations, .e. minor insertions or deletions, or single base substitutions, leading to a reduced level of production of antithrombin. In contrast, type I1 deficiency which is due to a variant protein) can be explained to date) by single base mutations in the coding sequence for antithrombin. Gene cloning and sequenc- ing has directly demonstrated a C o T mutation in antithrom- bin Utah, in codon 407, Tours and Alger, in codon 47 25,29, 37, 45). Using specific oligonucleotide hybridization, anti- thrombins Northwick Park and Glasgow have been shown to be due to C to T and G to mutations, respectively, in codon 393 20). It is likely that other variants with the same amino acid substitutions e.g. Milano, Sheffield, and Chicago) have the same single base substitutions. Similarly, the molecular defect in antithrombin Pescara has been shown here to be due to a G to C mutation in codon 393. It is interesting to note that these mutations of the reactive site codon 393 of antithrombin occur in a CpG dinucleotide. This supports the view that CpG dinucleotides may be a mutation hot spot in he human genome 41-43). Antithrom- bin belongs to a serine proteinase inhibitor uperfamily, other members of which contain arginine at their reactive sites 22, 31). It has recently been shown 44) that one such inhibitor family member, Ci inhibitor which has reactive site Arg444), has mutations similar to those observed for antithrombin. Ci inhibitor is the only known inhibitor of Ci, the first compo- nent of complement. Familial dysfunctional Ci inhibitors have been described, and, recently, two different mutations  10204 Antithrombin Pescara to Pro have been identified in a CpG dinucleotide of the codon for Arg444. he mutations, CGC to C_AC and CGC to TGC, result in amino acid substitutions of Arg to His and to Cys, respec- tively compare these with the mutations of antithrombin Glasgow and Northwick Park, described above). The present results with antithrombin Pescara suggest the possibility of an Arg to Pro substitution in the reactive site of a variant Ci inhibitor, caused by a mutation of the same CpG dinucleotide, CGC to CCC. Similar considerations may apply to the other members of the serine proteinase inhibitor superfamily that contain reactive site arginine residues. Acknowledgments-We thank Professor Sir D. J. Weatherall for support and encouragmsnt, and Dr. S C. Clark for allowing us to see unpublished work on C1 inhibitor. 1 2. 3. 4. 5. 6. 7. a 9. 10. 11 12. 13. 14. 15. 16. 17. 18. 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