The N-terminal Epidermal Growth Factor-like Domain of Coagulation Factor IX: PROBING ITS FUNCTIONS IN THE ACTIVATION OF FACTOR IX AND FACTOR X WITH A MONOCLONAL ANTIBODY

Kristina E.M Persson,Bruno O Villoutreix,Ann-Marie Thämlitz,Karin E Knobe,Johan Stenflo

doi:10.1074/jbc.m205930200

Kristina E.M Persson, Bruno O Villoutreix + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m205930200

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Abstract

The absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor-like (EGF) domains and a serine protease domain. In this study, the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined, and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by activated factor XI, but activation by activated factor FVII-tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent k(cat) for factor X both in the presence and absence of activated factor VIII. Based on these results, we produced a preliminary model of the structure of the activated factor IX-activated factor VIII-AW complex on the surface of phospholipid. The model suggests that in the Xase complex, EGF1 of activated factor IX is not involved in direct binding to activated factor VIII. Studies of the interaction of antibody AW with a mutated FIX molecule (R94D) also suggest that the Glu(78)-Arg(94) salt bridge is not important for maintaining the structure of FIX.

Highlights

The absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B
Characterization of Recombinant and Synthetic FIX Polypeptides—Wild-type FIX and four variants containing the point substitutions D49E or P55S, R94D, and E245K were produced in human kidney 293 cells and used in initial attempts to define the epitope of monoclonal antibody (mAb) AW [21]
We investigated the role of the EGF1 domain of FIX in activation of FIX by FXIa and FVIIa and clarified some of the roles of this domain within the Xase complex

Summary

EXPERIMENTAL PROCEDURES

Proteins—Plasma-derived human FIX, FIXa, and FX were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Clotting Assay—Plasma (100 ␮l) from healthy individuals or from a FIX-deficient patient (FIX Ͻ1%) was incubated for 15 min at room temperature with 2 ␮M mAb AW or its Fab fragment diluted in 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl and 1% (w/v) human serum albumin. Effect of mAb AW on Activation of FX by FIXa—Phospholipid vesicles containing 70% L-␣-phosphatidylcholine (Sigma) and 30% phosphatidyl-L-serine (ICN; Aurora, OH) were prepared as described [28] and added to a final concentration of 50 ␮M in a buffer consisting of 20 mM Hepes, 0.2% (w/v) human serum albumin, 0.15 M NaCl, 10 mM CaCl2, pH 7.4, and incubated for 10 min at 37 °C. Visualization of the molecules during positioning was facilitated by the use of Stereo devices (Crystaleyes)

RESULTS

Without mAb AW

DISCUSSION

Results in the literature have been contradictory concerning