The N-terminal EGF Module of Coagulation factor IX. Studies of Calcium Binding and Module Interactions
Abstract: Coagulation factor IX (FIX) is a vitamin K-dependent serine protease zymogen that circulates in plasma. Defects in FIX cause the bleeding disorder hemophilia B. FIX contains a Gla module, two Epidermal Growth Factor (EGF) -like modules and a serine protease region. In this thesis, studies have been performed on the N-terminal EGF module, which binds one Ca2+. — The present results show that binding of Ca2+ to EGF1 of FIX was 10-fold stronger when the Gla module was added to EGF1 (KD = 160 µM), indicating that binding of Ca2+ is important for stabilizing the structure of FIX. The mutations Val46Glu and Gln50Glu caused only a marginal increase in the affinity of Ca2+. — The mab AW was carefully characterized, and its non-Ca2+-dependent epitope was found to be in the C-terminal part of EGF1 of FIX. This mab has a 10-fold higher affinity for FIXa than for FIX, a difference that is maintained when residue 55 is mutated, indicating the existence of intramolecular communication between the serine protease region and EGF1. — The mab AW did not affect the amidolytic activity of FIXa, and it was used to study activation of FIX. The antibody had very little effect on activation induced by TF/FVIIa, whereas it almost completely blocked activation caused by FXIa, indicating that the C-terminal part of EGF1 (where the epitope recognized by the mab is located) can interact with FXIa, but not with TF/FVIIa. — The mab AW caused a marginal reduction in the kcat,app for activation of FX, both in the presence and absence of FVIIIa. This information was used in the production of a computer model of the FIXa-FVIIIa complex, in which it can be seen that EGF1 of FIXa does not interact directly with FVIIIa. The model also shows that the salt bridge proposed to link Glu78 and Arg94 is not important for maintenance of the structure of FIX, which is supported by normal behavior of FIX Arg94Asp in clotting assays. — Two patients with different mutations in EGF1 of FIX (Pro55Ser and Pro55Leu) that gave rise to mild hemophilia were studied. These patients exhibited 10–12% of FIX coagulant activity and 50% of FIX antigen levels compared to normal subjects. Binding of Ca2+ to the isolated mutated EGF modules was investigated, and only minor changes were found in the affinity for this ion. When FIX Pro55Leu was activated to FIXa, it was degraded soon after activation, with cleavage at Arg318 and a concomitant loss of amidolytic activity. These findings indicate the existence of intramolecular communication between EGF1 and the serine protease region. In contrast, wild-type FIX and the Pro55Ser variant showed no such degradation. In FX activation reactions, there was a small decrease in kcat,app for FIXa Pro55Ser, and this reduction was in the same range regardless of whether FVIIIa was included in the assay. These results support the above-mentioned computer model in which EGF1 of FIXa does not interact directly with FVIIIa in the activation of FX.
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