A novel amino acid substitution in the reactive site of a congenital variant antithrombin

Lane, David A.; Erdjument, H; Thompson, Elizabeth A.; Panico, Maria; Marzo, Vincenzo Di; Morris, HR; Leone, Giuseppe; Stefano, V. De; Thein, Swee Lay

doi:10.1016/s0021-9258(18)81785-9

Cited by 33 publications

(2 citation statements)

References 38 publications

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“…As with most other serpins, a principal determinant of antithrombin specificity appears to be the P1 residue localized within the substrate recognition sequence of the reactive center loop (). The importance of the P1 residue, arginine 393, in antithrombin is indicated from the observation that natural mutations of this residue appear to abolish antithrombin's inhibitory activity toward its target trypsin-like proteinases ( − ). In addition to determining specificity, the P1 residue has been proposed to play a critical role in heparin activation of antithrombin.…”

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confidence: 99%

The Antithrombin P1 Residue Is Important for Target Proteinase Specificity but Not for Heparin Activation of the Serpin. Characterization of P1 Antithrombin Variants with Altered Proteinase Specificity but Normal Heparin Activation

et al. 2001

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Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.

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confidence: 99%

The Antithrombin P1 Residue Is Important for Target Proteinase Specificity but Not for Heparin Activation of the Serpin. Characterization of P1 Antithrombin Variants with Altered Proteinase Specificity but Normal Heparin Activation

et al. 2001

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“…Several genetic variations (Table 16.2) have been identifi ed in genes coding for components of the coagulation system that infl uence the quantity or function of these proteins and have been shown to be signifi cant risk factors for thrombosis [198,200]. These include variations in genes coding for factor V [201], prothrombin [202][203][204][205], antithrombin [206][207][208][209][210], protein C [211][212][213][214][215], protein S [216][217][218][219][220][221], methylentetrahydrofolate reductase [222], endothelial nitric oxide synthase [223][224][225], α-fi brinogen [226][227][228][229], and factor XIII [228,[230][231][232][233][234][235]. However, no researchers have reported the relative risks of these various genetic polymorphisms in the development of thrombosis in children in PICUs.…”

Section: Genetic Polymorphisms In Thrombosismentioning

confidence: 99%

Genetic Polymorphisms in Critical Care and Illness

Dahmer

Quasney

2008

Science and Practice of Pediatric Critical Care Medicine

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pair substitution. In addition, polymorphisms within genes may also be caused by insertions or deletions of fragments of DNA or to the presence of a variable number of tandem repeats (VNTR) of short, repetitive DNA sequences.Polymorphic sites can exist in coding and noncoding regions of the gene. They can have no effect, or they can infl uence the activity and/or level of the resulting protein, thereby affecting cell function. When present in the coding sequences of the gene, these variations can result in an alteration in the amino acid sequence of the protein that can affect the structure and function of the protein. When the polymorphic site exists in a noncoding region of the gene, it can affect the regulation of gene transcription, resulting in altered levels of protein product in the cell. Genotyping of Polymorphic SitesAlthough biochemical analyses of proteins have indicated that protein products are polymorphic, the fi rst demonstration of the extent of polymorphism in the human genome was demonstrated using restriction enzymes that recognize and cut DNA at specifi c nucleotide sequences. Analyses of the DNA fragments generated by the action of a specifi c restriction enzyme on human DNA demonstrated that the size of the cleavage products differed among individuals. These restriction fragment length polymorphisms (RFLPs) are generally caused by an SNP within restriction enzyme recognition sites. After the realization that many SNPs were present in the human genome, a number of other methods were used to identify SNPs within genes [1,2].Once a polymorphic site within a gene is identifi ed, there are a number of methods that can be used to determine the genotype of individuals at that polymorphic site. As individuals have two copies of each gene, at any given polymorphic site an individual can be homozygous for one or the other polymorphism found at that site; or the individual may be heterozygous. Almost all genotyping techniques require amplifi cation of the fragment of DNA containing the site of interest by the polymerase chain reaction (PCR) technique. This technique allows for the amplifi cation of a specifi c region of the genome (in this case a region containing the polymorphic site) using small fragments of DNA that fl ank the polymorphic site as primers for the PCR. For insertions or deletions and most VNTRs, the genotype can be determined by examining the size of the PCR products by gel electrophoresis.

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Molecular genetics of human antithrombin deficiency

Perry

Carrell

1996

Hum. Mutat.

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A novel amino acid substitution in the reactive site of a congenital variant antithrombin

Cited by 33 publications

References 38 publications

The Antithrombin P1 Residue Is Important for Target Proteinase Specificity but Not for Heparin Activation of the Serpin. Characterization of P1 Antithrombin Variants with Altered Proteinase Specificity but Normal Heparin Activation

The Antithrombin P1 Residue Is Important for Target Proteinase Specificity but Not for Heparin Activation of the Serpin. Characterization of P1 Antithrombin Variants with Altered Proteinase Specificity but Normal Heparin Activation

Genetic Polymorphisms in Critical Care and Illness

Molecular genetics of human antithrombin deficiency

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