Frank Hallinan

Analysis of antithrombin III microheterogeneity by isoelectric focusing in polyacrylamide gels and immunoblotting

Microheterogeneity of antithrombin III (ATIII) was examined in pooled normal human plasma and serum by IEF/PA and immunoblotting techniques. A pattern consisting of 8 bands varying in isoelectric point from 4.7 to 5.2 was observed in citrated plasma following IEF in a pH range of 4-6. A similar pattern was observed in all 149 healthy adults examined. The reactivity of ATIII with heparin or, thrombin was investigated using this technique. Characteristic shifts of the banding pattern to the anode or, cathode respectively were observed.

Identification and characterization of a new antithrombin III familial variant (AT Dublin) with possible increased frequency in children with cancer

Summary. The antithrombin III (ATIII) isoform pattern of a number of serum and plasma samples was analysed by isoelectric focusing and immuno‐blotting. A novel ATIII isoform pattern which was observed in 4/80 children with acute lymphatic leukaemia (ALL) and in 1/4 children with Ewings sarcoma, has been shown by family studies to be due to a mutant form of ATIII (AT Dublin) in the heterozygous state. The coagulation properties of AT Dublin heterozygotes were normal. In addition the immunological and activity levels of their ATIII were normal. The effects of thrombin and heparin on the mutant ATIII were similar to controls. Neuraminidase treatment reduced the ATIII isoforms to one in controls and two in the mutant. Two‐dimensional gel analysis showed the mutant ATIII to have an identical molecular size distribution to the normal form. This mutant is, thus, most likely due to an amino acid substitution giving a more basic molecule that is clinically silent (at the coagulation level). It may be of interest that the frequency of AT Dublin in the ALL group is significantly higher than in the control group (3/430) studied (P < 0·001).

Identification and characterisation of an antithrombin III mutant (AT Dublin 2) with marginally decreased heparin reactivity

The antithrombin III (ATIII) isoform patterns of plasma and serum samples from cancer patients and controls were analysed by isoelectric focusing and immunoblotting. A novel ATIII banding pattern was identified in two individuals: a patient with breast carcinoma who developed deep venous thrombosis and a blood donor. Family studies in the patient showed the abnormal pattern to be due to a mutant form of ATIII (AT Dublin 2). The coagulation properties of AT Dublin 2 heterozygotes were normal. Immunologic and activity levels of ATIII, measured by standard techniques, were normal. Mutant plasma ATIII showed reduced thrombin reactivity at low concentrations of thrombin and demonstrated decreased reactivity with heparin over a range of heparin concentrations. This was confirmed using a modified ATIII heparin cofactor activity assay with varying heparin concentrations. The abnormal ATIII was also found to elute from heparin agarose at a lower ionic strength than normal ATIII. Two dimensional gel electrophoresis showed the abnormal ATIII to have similar molecular size distribution to normal ATIII. Neuraminidase treatment of normal and mutant plasma reduced the ATIII isoforms to one in both samples. The possible role of AT Dublin 2 in predisposing to hypercoagulation is discussed.