Abstract
A procedure is presented for the purification of Factor IX from human plasma. The final product is homogeneous as judged by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Furthermore, it is completely free of other coagulation component activities. Factor IX is converted to its enzymatically active form by the addition of small quantities of Factor IXa in the presence of calcium ions. This activated species is added to purified antithrombin-heparin cofactor and the interaction is studied in the presence and absence of heparin. Antithrombin-heparin cofactor is found to be a progressive, time-dependent inhibitor of Factor IXa and neutralizes approximately 57% of this enzyme's proteolytic activity within 30 min. The addition of heparin dramatically accelerates the rate of this interaction with virtually complete inhibition of Factor IXa occurring within 15 s. Sodium dodecyl sulfate gel electrophoresis of reduced and nonreduced proteins indicates that antithrombin-heparin cofactor functions as a potent inhibitor of Factor IXa by forming an undissociable complex with the enzyme which is stable in the presence of denaturing or reducing agents (or both). This complex represents a 1:1 stoichiometric combination of enzyme and inhibitor. Heparin increases the rate of formation of this complex without affecting its dissociability or stoichiometry.
Highlights
Factor IX is converted to its enzymatically active form by the addition of small quantities of Factor IX, in the presence of calcium ions
Health Heart well as Factor X, we predicted that most, if not all, serine proteases of the coagulation-fibrinolytic mechanism would be neutralized by this inhibitor and that heparin would accelerate each of these interactions [6]
We have previously examined the interactions of human antithrombin-heparin cofactor with human Factor XI. as well as human plasmin and have shown that these serine proteases are inactivated in this, heretofore unsuspected, fashion [6, 7]
Summary
All chemicals employed were reagent grade or better. Human plasma was obtained by plasmaphoresis utilizing (0.44 g of citric acid/l.32 g of t&sodium dextrose/100 ml of H,O) as anticoagulant. Protein initiated within 2 hours of plasma collection. Fibrinogen was obtained from AB Kabi, Stockholm, Sweden. Mine sulfate was provided by Eli Lilly Co. Heparin employed to activate antithrombin was Organon. Heparin utilized to prepare heparin-Sepharose in crude form (Stage 14) from the Wilson Chemical purified as described by Lindahl et al [8]. P-150 were purchased from Bio-Rad. Sepharose 4B and Sephadex G-25 were obtained from Pharmacia Fine Chemicals. Heparin-Sepharose was prepared as previously described [5], except that this matrix was washed extensively with 0.6 M sodium chloride in 0.05 M Tris-HCI
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