Abstract
Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.
Highlights
Human blood coagulation Factor XIa was reduced ing site fohrigh M, kininogen
XIIa as described under “Materialsand Methods.’’Factor XIa reduction and alkylation prior to addition of SDS (6 pg of protein); had an apparentM, of 160,000 (Fig. lA).As seen on SDS gels D,isolated alkylated light chain of Factor XIa (5 pg of protein)
Under reducing conditions inFig. lB, the resulting FactorXIa consisted of heavy chains of M, = 48,000 and light chains of
Summary
All chemicals were the best grade commercially available. Purification of Proteins-Factor XI and high M, kininogen were purified from human plasma using previously published methods' [24]. XI using 8-Factor XIIa. To 7 mlof Factor XI (1.54mg) in 4 mM units by comparison to the clotting activities of serial dilutions of a sodium acetate, 2 mM acetic acid, 0.15 M NaCl, pH 5.3,was added 0.7 standard pool of normal human plasma as described before [8]. A t various log of the concentration of normal plasma, Factor XIa, or its light times, aliquots were removed and assayed for Factor XIa amidolytic chain at the concentrations tested The lines in such plots were activity. Protein concentration of clotting units/ml), 150 p1 of TBS containing 10 mg/ml of BSA, and Factor XIa and its light chain (see below) was calculated from data 24 pl of 0.5 M Tris-C1, pH 7.4,weremixed at 37 "C with 24 pl of obtained from amino acid analysis following 24-h hydrolysis. The Factor IXa procoagulant activity generated upon incubation of 3H-Factor IXwith Factor XIa strongly correlated with the release of trichloroacetic acid-soluble radioactivity
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