Nonreducing SDS-PAGE analysis of the stable factor Xa and thrombin complexes with plasma or mutant antithrombin.

(A) Human plasma or mutant antithrombin (2 μM) was incubated with an equimolar concentration of factor Xa for 30 minutes in the absence of heparin or for 5 minutes in the presence of pentasaccharide or approximately 70-saccharide high-affinity heparin (4 μM) in 25 μL reactions in TBS buffer at room temperature. Five microliters 5× nonreducing sample buffer was added to each reaction, and the samples were boiled for 5 minutes. Twenty-five microliters of each reaction was loaded on 10% gel. The stable proteinase-antithrombin complexes migrated as the highest molecular weight bands (HAT-FXa), followed by degraded complexes. Lane 1, factor Xa (FXa in α and β forms). Lane 2, human plasma antithrombin (HAT). Lanes 3 to 5, plasma antithrombin incubated with factor Xa in the absence of heparin, in the presence of pentasaccharide, or in the presence of approximately 70-saccharide heparin, respectively. Lane 6, mutant antithrombin. Lanes 7 to 9, mutant antithrombin incubated with factor Xa in the absence of heparin, in the presence of pentasaccharide, or in the presence of approximately 70-saccharide heparin, respectively. MW, molecular weight marker. (B) Identical to panel A, except that thrombin (IIa) was used to initiate the reactions.