SummaryInherited antithrombin deficiency is associated with an increased risk of thrombosis, primarily venous rather than arterial. Most affected individuals have inherited only a single copy of an abnormal antithrombin (AT) gene. Homozygously affected individuals, although rare, have a severe thrombotic history of early onset and often affecting the arteries. We report two new cases of type II HBS (heparin binding site) deficiency in which the propositi are homozygous for the previously reported mutation 99 Leu to Phe, and who have a severe thrombotic history. These cases are considered alongside existing homozygote and compound heterozygote cases.

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We report the characterization of three variant antithrombins with reduced heparin binding as the primary abnormality. Two of these variants, antithrombin Southport (Leu 99 to Val, 2759 C to G) and antithrombin Vienna (Gln 118 to Pro, 5349 A to C) were novel, whereas the third, Pro 41 to Leu, has been previously described as antithrombin Basel. All three variants exhibited reduced binding for heparin on crossed immunoelectrophoresis and in a quantitative monoclonal antibody-based assay. The mutations were characterized by direct sequence analysis of enzymatically amplified genomic DNA and all affected individuals were heterozygous for the mutations. These three mutations do not occur at the sites of the basic amino acids directly involved in heparin binding nor do they result in a change in charge of the affected residue. It seems probable that they reduce heparin affinity either by perturbing the initial contact site involved in the heparin-binding domain (Arg 47, Arg 129 and possibly Arg 24), or by preventing the subsequent heparin-induced conformational change.

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.

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