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Thromb Haemost 1996; 75(02): 313-317
DOI: 10.1055/s-0038-1650267
Original Article
Schattauer GmbH Stuttgart

Characterization of Recombinant Human Coagulation Factor XFriuli

D J Kim
1   The Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas, USA
,
A Girolami
2   The Institute of Semeiotica Medica, University of Padua Medical School, Padua, Italy
,
H L James
1   The Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas, USA
› Author Affiliations
Further Information

Publication History

Received: 17 August 1995

Accepted after revision27 October 1995

Publication Date:
26 July 2018 (online)

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Summary

Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

 

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