Evidence for a prevalent dimorphism in the activation peptide of human coagulation factor IX.

Abstract

We have independently isolated and characterized cDNA and genomic clones for the human coagulation factor IX. Sequence analysis in both cases indicates that threonine is encoded by the triplet ACT as the third residue of the activation peptide. This is in agreement with some earlier reports but in disagreement with others that show the alanine triplet GCT at this position. The discrepancy can thus be accounted for by natural variation of a single nucleotide in the normal population. Amino acid sequence analyses of activated factor IX from plasma samples of four individuals yielded two cases of alanine and two cases of threonine at the third position of the activation peptide. In factor IX from pooled plasma and in factor IX from a heterozygous individual, however, both alanine and threonine were found. Taken together, the findings show that a prevalent nondeleterious dimorphism exists in the activation peptide of human coagulation factor IX.Factor IX is the plasma protein that is missing or defective in individuals afflicted with the X-chromosome-linked bleeding disorder hemophilia B. Its role in the blood coagulation cascade is to activate factor X through interactions with calcium, membrane phospholipids, and factor VIII. Factor IX circulates as an inactive zymogen until proteolytic release of its "activation peptide" allows it to assume the conformation of an active serine protease.At the molecular level, it is known that hemophilia B may result from a variety of genetic changes (1, 2). Partial and/or complete deletions of the factor IX gene have been shown to be responsible for the disease in some cases (3,4). Several hemophilia B variants have also been described that show normal levels of the factor IX protein by immunological methods but have reduced or negligible activity in clotting assays. These variants have been designated CRM+ (crossreacting material positive). One of the CRM+ variants, factor IX Chapel Hill, results from an amino acid substitution at one of the proteolytic activation sites, blocking cleavage and subsequent activation (5). A change affecting the other cleavage site is likely to be involved in the variant factor IXDeventer (6).The molecular defect in another CRM+ variant, factor IXAlabama (7), is presently under study in our laboratories. The dimorphism described in this report, however, appears to be the result of a nondeleterious mutation which has been fixed in the normal population.As early as 1978, a partial amino acid sequence was reported for the amino-terminal region of the activation peptide of human factor IX (8). This analysis, apparently done on material from pooled plasma, showed an aminoterminal sequence Ala-Glu-Thr-Val-Phe-for the activation peptide, in agreement with a previously determined sequence for the corresponding region from bovine factor IX (9). No mention was made of alanine at the third position. Several years later, however, the same laboratory did report a cDNA sequence for human factor IX which indicated the presence of an alanine codon at the th...

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

“…The interaction of this factor IX with phospholipids was normal, but the activation of factor X either in the presence or absence of factor VIII was found to occur at a reduced rate, relative to the normal protein (Briet et al, 1982;Jones et al, 1985). The level of (3-hydroxyaspartate was found to be normal in this variant (McGraw et al, 1985). Thus, the clotting activities of the patient factor IX and the mutant factor IX 220 generated in this work are essentially similar, although we have not compared their properties in factor X activation.…”

Creation of a mouse expressing defective human factor IX

“…9 The polyclonal antibodies used above recognize both the alanine and threonine forms. However, the monoclonal antibody A-1 recognizes the threonine isoform preferentially.…”

“…To make the mutant mouse more useful we have expressed the human factor IX R333Q gene as the alanine form of the Ala148Thr dimorphism. 9 Because the A-1 antibody binds well to factor IX with threonine at residue 148 but weakly to the alanine isoform, the effectiveness of gene therapy can be evaluated by expressing the threonine isoform and detecting its presence with the A-1 antibody. We anticipate that this mouse model will be an important tool for gene therapy and for studying the function of mutant factor IX in vivo.…”

Hemophilia B (Christmas disease) variants and carrier detection analyzed by DNA probes.

“…In kindred A, a 1.8-kb fragment was identified in place ofthe 1 (27). The change in Taq I fragment size in each case without loss of other constant fragments and the absence of changes with Xmn I (Fig.…”