The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

Patrick H.N Celie,Gunny Van Stempvoort,Caroline Fribourg,Leon J Schurgers,Peter J Lenting,Koen Mertens

doi:10.1074/jbc.m108446200

Patrick H.N Celie, Gunny Van Stempvoort + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m108446200

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Abstract

The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu(84)-Thr(87) and the factor IX-specific loop Asn(89)-Lys(91) were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn(89)-Lys(91) resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu(84)-Thr(87) between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa.

Highlights

Factor IX (FIX)1 is a vitamin K-dependent serine protease precursor that participates in the process of blood coagulation
The salt bridge is crucial for interaction of factor IXa (FIXa) with its cofactor, factor VIIIa (FVIIIa) [15], whereas the hydrophobic contact contributes to FIXa enzymatic activity [16]
We focused on other FIXa-specific elements that are exposed at the interface between both epidermal growth factor (EGF)-like domains, including the sequence Leu84-Thr87 that connects both domains and the loop Asn89-Lys91 at the N-terminal part of the second EGF-like domain

Summary

Introduction

Factor IX (FIX)1 is a vitamin K-dependent serine protease precursor that participates in the process of blood coagulation. Activated FIX chimeras were characterized with particular reference to enzymatic activity and response to FVIIIa. Our results demonstrate that in particular the junction Leu84-Thr87 between the two EGF-like modules and to some extent the loop Asn89-Lys91 contribute to respond to the cofactor FVIIIa. For chimeras comprising the complete spacer sequence of FX or FVII, impaired sensitivity to intact FVIIIa was accompanied by a reduced stimulation by the isolated FVIII A2 domain.

Results

Conclusion