Dysprothrombinaemia is a rare, congenital cause of bleeding. Fewer than 25 families who express a functional prothrombin (factor II) defect have been reported. The original patient with prothrombin Denver had a severe haemophilia-like bleeding disorder treated with weekly prophylactic factor replacement. Analysis of factor II activity and antigen in the patient showed a factor II activity of 5 units/dl and factor II antigen of 21 units/dl. Genomic DNA from the patient, mother and brother was obtained from peripheral blood white cells. Oligonucleotides were constructed, and prothrombin exons were amplified via polymerase chain reaction (PCR). The entire sequence of the thrombin portion of the molecule (exons VIII-XIV) and that of exons I-II and IV-VII was determined. This moderately severe dysprothrombinaemia was found to be associated with compound heterozygosity for two different Glu-->Lys point mutations, at amino acid positions 300 and 309. Assays of plasma from the prothrombin Denver proband suggested that the functional defect was in the activation of zymogen to enzyme.

SummaryHemophilia B is a sex-linked recessive bleeding disorder characterized by the presence of either a decreased amount of normal factor IX (FIX) or the presence of a dysfunctional FIX. We have identified a unique mutation in a family with mild hemophilia B. DNA analysis of family members revealed a single base transition in the 8th exon of the FIX gene predicting an amino acid change of Asn 346→Asp in the catalytic domain. The FIX variant, named FIX Denver, was purified from proband plasma. Kinetic studies of factor X (FX) interactions with normal FIXa or FIXa Denver and phospholipid (PL) showed little difference in kcat, but a significant difference when factor VIIIa (FVIIIa) was included in the reaction. Using kinetic assays to infer the Kd of FIXa for FVIIIa, normal FIXa had a Kd of 0.095 nM while that of FIXa Denver was 9.85 nM. The major defect caused by this point mutation is a marked decrease in the affinity of FIXa Denver for factor VIIIa.