Hemophilia Bm is characterized by a strikingly prolonged plasma ox brain prothrombin time. In an attempt to find an explanation for this phenomenon we have analyzed various aspects of the Bm variants factor IX Deventer, factor IX Milano, factor IX Novara, and factor IX Bergamo. Proteolytic cleavage by factor XIa was normal in two Bm variants, but absent at the Arg180-Val bond in the other two. In the latter variants Arg180 was replaced by either Trp or Gln, whereas Val181→ Phe and Pro368 → Thr replacements have occurred in the variants that were normally cleaved by factor XIa. In all four variants the Bm effect could be neutralized with a single monoclonal antibody against factor IX. Also, after treatment with factor XIa, none of the Bm variants reacted with antithrombin III (in contrast to normal factor IXa). Purified factor IX Deventer (one of the variants with a replacement of Arg180), either with or without pretreatment with factor XIa, was found to be a more effective competitive inhibitor of the factor VIIa-tissue factor-induced factor X activation than similarly treated normal factor IX. In addition, this inhibitory effect was much more pronounced when bovine tissue factor was used instead of human tissue factor. We propose that the normal activation of factor IX not only produces a conformational change around the active site serine that allows efficient substrate binding and catalysis, but that the same conformational change is instrumental in effectively dissociating factor IXa from the activating factor VIIa-tissue factor complex. Amino acid replacements that disrupt this conformational transition directly (e.g. Pro368 → Thr near the catalytic center) or indirectly (mutations at the Arg180-Val activation site) therefore lead to a combination of 1) the loss of coagulant activity and 2) an inhibitory effect in the ox brain prothrombin time assay.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jul 5 1990|
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