ALBERT

Description: This report details the precise mapping of a partially deleted human antithrombin III (AT-III) allele, found in a kindred with an inherited type 1 AT-III deficiency. Using truncated AT-III probes generated by polymerase chain reaction (PCR) amplification from a full-length AT-III cDNA, as well as other genomic probes specific for the 5′ upstream region of the AT-III gene, we were able to characterize a partial deletion on an AT-III allele encompassing exons 1 and 2 of the AT-III gene, and a region 5′ to the coding sequences. The absence of the 5′ upstream region in the affected AT-III allele was confirmed directly by the PCR amplification of a 1.5-kb polymorphic fragment of genomic DNA samples from family members. The precise determination of the 5′ breakpoint of the affected allele was made possible by two different approaches: (1) subcloning plus biotin capture PCR, or (2) inverse PCR. This allowed us to confirm the mapping of the deletion obtained by Southern analysis; to show that the 3′ region of the mutant AT-III allele, including exons 3 to 7, was intact; and to sequence approximately 0.7 kb upstream to the breakpoint in the mutant allele. Furthermore, PCR amplification of the region of the breakpoint provided unique products detectable only in affected members of this kindred. The breakpoint in the partially deleted allele is 480 bp upstream from the 5′ boundary of exon 3. No significant homology was found between the 0.7-kb sequence upstream to the breakpoint of the mutant allele and known human sequences.

Description: Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.

Description: Antithrombin-III-Stockholm is a new structural variant of antithrombin- III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT- III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha- thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.

Description: Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.

Description: Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.

Description: This report details the precise mapping of a partially deleted human antithrombin III (AT-III) allele, found in a kindred with an inherited type 1 AT-III deficiency. Using truncated AT-III probes generated by polymerase chain reaction (PCR) amplification from a full-length AT-III cDNA, as well as other genomic probes specific for the 5′ upstream region of the AT-III gene, we were able to characterize a partial deletion on an AT-III allele encompassing exons 1 and 2 of the AT-III gene, and a region 5′ to the coding sequences. The absence of the 5′ upstream region in the affected AT-III allele was confirmed directly by the PCR amplification of a 1.5-kb polymorphic fragment of genomic DNA samples from family members. The precise determination of the 5′ breakpoint of the affected allele was made possible by two different approaches: (1) subcloning plus biotin capture PCR, or (2) inverse PCR. This allowed us to confirm the mapping of the deletion obtained by Southern analysis; to show that the 3′ region of the mutant AT-III allele, including exons 3 to 7, was intact; and to sequence approximately 0.7 kb upstream to the breakpoint in the mutant allele. Furthermore, PCR amplification of the region of the breakpoint provided unique products detectable only in affected members of this kindred. The breakpoint in the partially deleted allele is 480 bp upstream from the 5′ boundary of exon 3. No significant homology was found between the 0.7-kb sequence upstream to the breakpoint of the mutant allele and known human sequences.

Description: Antithrombin-III-Stockholm is a new structural variant of antithrombin- III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT- III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha- thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.

Description: Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.