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The factor V B-domain provides two functions to facilitate thrombin
cleavage and release of the light chain
KA Marquette, DD Pittman and RJ Kaufman
Genetics Institute, Cambridge, MA, USA.
Blood coagulation factors V and VIII are homologous proteins that have the
domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, the
B-domains of both molecules are released. Previous studies on factor VIII
showed that the B-domain was not required for thrombin cleavage or
activity. In contrast, deletion of the factor V B-domain (residues 709 to
1545) yielded a molecule with sevenfold reduced procoagulant activity that
was not cleaved by thrombin. However, this factor V B- domain deletion
molecule was activated by factor Xa, although the fold- activation was 85%
that of wild-type factor V. Thrombin cleavage of factor V occurs initially
after residue 709 and subsequently after residues 1018 and 1545. The
requirement for thrombin cleavage within the B-domain at residue 1018 was
evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant,
the rate of thrombin activation and appearance of maximal cofactor activity
was delayed and was consistent with delayed cleavage of the light chain at
residue 1545. In contrast, the rate of factor Xa activation in the R1018I
mutant was not altered. This finding suggests that thrombin cleavage at
1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis
was used to study the requirement for sequences within the factor V B-
domain for thrombin cleavage at residue 1545. Whereas the factor V deletion
molecule removing residues 709 to 1545 was not cleaved by thrombin, a
smaller B-domain deletion molecule (residues 709 to 1476) containing an
acidic amino acid-rich region (residues 1490 to 1520) was effectively
cleaved by thrombin. These results show that residues 1476 to 1545, which
contain an acidic amino acid-rich region, were required for thrombin
cleavage of the light chain. Introduction of an acidic amino acid-rich
region from factor VIII (residues 337 to 372) into the factor V 709 to 1545
deletion also restored thrombin cleavage of the light chain. In contrast,
similar replacement with the acidic region from the factor VIII light chain
(residues 1649 to 1689) was significantly less effective in promoting
thrombin cleavage of the light chain. This finding suggests that the
different acidic regions in factors V and VIII are not functionally
equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400
WORDS)
Volume 86,
Issue 8,
pp. 3026-3034,
10/15/1995
Copyright © 1995 by The American Society of Hematology
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