Process for producing activated thrombin-activatable fibrinolysis inhibitor

1. A process for purifying TAFIa from a serum sample comprising the sequential steps of a) adding a reversible inhibitor of TAFIa to the serum sample to form a first mixture; b) adding a coagulation initiator to the first mixture to convert TAFI to TAFIa and form a second mixture; c) applying the second mixture to a heparin-sepharose column to bind TAFI and TAFIa to the column; d) washing the heparin-sepharose column to remove proteins not bound to the heparin-sepharose column; e) eluting the bound TAFI and bound TAFIa from the heparin-sepharose column to form a third mixture; f) applying the third mixture to a TAFIa isolation column to bind TAFIa to the isolation column; g) washing the TAFIa isolation column to remove proteins not bound to the TAFIa isolation column; and h) eluting the bound TAFIa from the TAFIa isolation column.

2. The method of claim 1 wherein the reversible inhibitor is ε-aminocaproic acid, 2-mercaptomethyl-3-guanidinoethyl-propanoic acid, potato tuber carboxypeptidase inhibitor, leech carboxypeptidase inhibitor, or tomato carboxypeptidase inhibitor.

3. The method of claim 1 wherein the TAFIa isolation column is a potato carboxypeptidase inhibitor affinity column or a Con-A column.

4. The method of claim 1 wherein the coagulation initiator is selected from the group consisting of calcium chloride, thrombomodulin, or thrombin.

5. The method of claim 2 wherein the reversible inhibitor is ε-aminocaproic acid.

6. A method of measuring the activity of TAFIa in the presence of a reversible inhibitor comprising the sequential steps of: a) contacting a peptide, comprising a C-terminal carboxylate moiety masked by an amino acid, with TAFIa, in the presence of a reversible inhibitor of TAFIa, to form a first mixture; b) unmasking the C-terminal carboxylate moiety by hydrolyzing the amino acid with TAFIa; c) adding a labeled antibody or other labeled protein, which recognizes the unmasked C-terminal carboxylate moiety on the peptide, to the first mixture, to form a second mixture; and d) measuring the extent of hydrolysis by determining the amount of labeled antibody or other labeled protein bound to the unmasked C-terminal carboxylate moiety on the peptide of the second mixture.

7. The method of claim 6 wherein the reversible inhibitor is selected from the group consisting of ε-aminocaproic acid, 2-mercaptomethyl-3-guanidinoethyl-propanoic acid, potato tuber carboxypeptidase inhibitor, leech carboxypeptidase inhibitor, or tomato carboxypeptidase inhibitor.

8. The method of claim 6 wherein the peptide includes an N-terminal biotin.

9. The method of claim 7 wherein said reversible inhibitor is ε-aminocaproic acid.