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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 176-183
Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit
Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa
In Vivo
S.W. Pipe,
A.N. Eickhorst,
S.H. McKinley,
E.L. Saenko, and
R.J. Kaufman
From the Departments of Pediatrics and Biological Chemistry, Howard
Hughes Medical Institute, University of Michigan Medical Center, Ann
Arbor, MI; and the Holland Red Cross Laboratory, Rockville, MD.
Approximately 5% of hemophilia A patients have normal amounts of a
dysfunctional factor VIII (FVIII) protein and are termed cross-reacting
material (CRM)-positive. FVIII is a heterodimer (domain structure
A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit
procoagulant activity. Thrombin-activated FVIII is a heterotrimer with
the A2 subunit (amino acid residues 373 to 740) in a weak ionic
interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2
subunit correlates with inactivation of FVIII. Recently, a phenotype of
CRM-positive hemophilia A patients has been characterized whose plasma
displays a discrepancy between their FVIII activities, where the
one-stage clotting assay displays greater activity than the two-stage
clotting assay. One example is a missense mutation where
ARG531 has been substituted by HIS531. An FVIII
cDNA construct was prepared containing the
ARG531HIS mutation and the protein was
expressed in COS-1 monkey cells by transient DNA transfection.
Metabolic labeling with [35S]-methionine demonstrated
that ARG531HIS was synthesized at an equal rate
compared with FVIII wild-type (WT) but had slightly reduced antigen in
the conditioned medium, suggesting a modest secretion defect. A time
course of structural cleavage of ARG531HIS
demonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes,
ARG531HIS had discrepant activity as measured
by a one-stage activated partial thromboplastin time (aPTT) clotting
assay (36% ± 9.6% of FVIII WT) and a variation of the two-stage
assay using a chromogenic substrate (COAMATIC; 19% ± 6.9% of FVIII
WT). Partially purified FVIII WT and ARG531HIS
proteins were subjected to functional activation by incubation with
thrombin. ARG531HIS demonstrated significantly
reduced peak activity and was completely inactivated after 30 seconds,
whereas FVIII WT retained activity until 2.5 minutes after activation.
Because the ARG531HIS missense mutation
predicts a charge change to the A2 subunit, we hypothesized that the
ARG531HIS A2 subunit could be subject to more
rapid dissociation from the heterotrimer. The rate of A2 dissociation,
using an optical biosensor, was determined to be fourfold faster for
ARG531HIS compared with FVIII WT. Because the
two-stage assay involves a preincubation phase before assay
measurement, an increased rate of A2 dissociation would result in an
increased rate of inactivation and reduced specific activity.
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