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Blood, 15 September 2001, Vol. 98, No. 6, pp. 1988-1990
CORRESPONDENCE
To the editor:
Phenotype and genotype expression in pseudohomozygous
R2 factor V
Factor Va is the important cofactor for prothrombinase. Factor
Va is inactivated by activated protein C (APC) following 3 cleavages of
the heavy chain at Arg306, Arg506, and Arg679.1 Cleavage of normal factor Va by APC at these sites results in the
production of Mr 45 000, Mr 30 000,
Mr 22 000 and 20 000 fragments.1 In
plasma, following the addition of APC and a synthetic membrane surface
(phosphatidylcholine phosphatidylserine [PCPS]), appearance of the
Mr 30 000 fragment demonstrates cleavage of normal factor V at Arg306 and Arg506.2 The Mr 30 000
fragment can be detected in plasma by using an anti-human factor V
monoclonal antibody that recognizes an epitope located between residues
307 and 506 of the molecule.2,3 The R2 haplotype in factor V is characterized by an A4070G substitution
(His1299Arg) in the factor V molecule and is associated with mild APC
resistance,4 but the underlying molecular mechanism remains unclear. We have recently described a thrombotic family with 4 symptomatic members.5 One of them (I3) was doubly
heterozygous for the factor V HR2 haplotype and for the factor V
Tyr1702Cys mutation, causing CRM-factor V deficiency. Since the factor
V allele predicting the Tyr1702Cys substitution is not expressed at the
protein level,5 the plasma of this patient contains only
R2 factor V, in accordance with her reduced factor V levels (FV:Ag
43%; FV:C 36%; normal range 70%-130%) and mild APC resistance (nAPC-sr 0.72, normal values > 0.84). We have concluded that while the His1299Arg substitution in factor V induces APC resistance, the presence of the Tyr1702Cys mutation was responsible for absence of
expression of the corresponding allele (previously reported to result
in CRM-factor V deficiency5). This particular
condition (pseudohomozygosity for the factor V HR2 haplotype) offers
the opportunity to study the APC-mediated inactivation of R2 factor Va
in plasma, which is otherwise possible only in the rare (~0.4% of
the general population) homozygous individuals. Molecular investigations were undertaken in this thrombotic patient
(I3, see Castoldi et al5). The patient was found to be
heterozygous, at both the DNA and messenger RNA (mRNA) levels, for the
His1299Arg and Asp2194Gly substitutions, characterizing the HR2
haplotype4 as well as for the Tyr1702Cys mutation in factor V. No mutation, in addition to the polymorphic changes previously described4,5 in the factor V gene, were found in this patient at the DNA and/or RNA levels and in particular in exon
7 (Arg306 cleavage site), exon 10 (Arg506 cleavage site), and exon 18 (interaction with APC). Factor Va cleavage in the patient's plasma was
investigated by analyzing the presence or absence of the Mr
30 000 fragment following incubation of the plasma with APC and PCPS
as described.2 Slow cleavage of factor Va at Arg506/Arg306
could be detected in the plasma of the propositus as compared with the
rapid cleavage of factor Va in normal plasma and generation of the
Mr 30 000 fragment (Figure
1A, lanes 2-5). Substantial amounts of
uncleaved heavy chain remained in the patient's plasma following
extended incubation with APC and PCPS, and low amounts of the
Mr 30 000 fragment were generated (Figure 1B, lanes 2-5).
These data demonstrate an APC resistance in this individual. Since the
allele carrying the Tyr1702Cys substitution is not
expressed,5 this individual can be defined as a
pseudohomozygous R2 genotype. Thus, because of the absence of normal
factor V in the patient's plasma, the factor V molecules resistant to
APC inactivation are characterized by the mutations included in the HR2
haplotype.7 These findings suggest the possibility that 1 or more of the amino acid substitutions predicted by the HR2 haplotype
might impair factor Va cleavage at Arg506/Arg306, possibly by
preventing optimal interaction with APC. Alternatively, impaired factor
Va inactivation may result from suboptimal interaction with
phospholipid membranes. Overall, our data confirm previous findings
that the Tyr1702Cys substitution causes CRM-factor V
deficiency and strongly suggest that the amino acid substitutions in R2
factor V confer APC resistance by impairing APC-factor Va interaction,
cleavage at Arg506 or Arg306, and subsequent inactivation of factor Va.
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| Figure 1.
Identification of the factor V molecule present in
plasma.
Citrated plasma (100 µL) from a pool of normal plasma (A) and from
patient I3 (Castoldi et al5, B) was diluted 10-fold in a
buffer containing 5 mM CaCl2 and treated with phospholipid
vesicles and APC (5 nM) as described.2,3 Immunoreactive
fragments were detected using the monoclonal antibody
 HFVaHC17 under nonreducing
conditions.2 (A) Normal pool plasma; (B) plasma from
patient I3 (Catoldi et al5). Lane 1, plasma immediately
following clot formation and the addition of APC (5 nM, ~20 sec);
lanes 3-5 plasma at 5, 10, 20, and 30 min following the addition of
APC. Roman numerals on the right indicate the following: (i) the
heavy chain of factor Va following clot formation; (ii) the
Mr 75 000 fragment deriving from cleavage of the heavy
chain of factor Va at Arg506 (amino acid residues 1-506); (iii) the
Mr 60 000 fragment that derives from factor Va following
cleavage at Arg306 (amino acid residues 307-709); (iv) the
Mr 30 000 fragment derived from normal factor Va following
cleavage at Arg506 and Arg306 (amino acid residues 307-506). The
position of the immunoglobulin G (IgG) molecules (nonreduced) is
also shown.
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Recently, Hoekema et al6 have reported that the rate
constants of APC-catalyzed inactivation of normal factor Va and factor Va from individuals with the R2 haplotype were similar. Our present data using a different methodological approach from the one used by
Hoekema et al6 clearly demonstrate that in a
pseudohomozygous individual the polymorphisms included in the HR2
haplotype confer APC resistance by delaying cleavages at both Arg506
and Arg306. Contribution by other factors present in patient's plasma
is unlikely in our experimental system. At present we cannot generalize
our findings to all patients with the HR2 phenotype since we have studied by this method only patients related to this family (Castoldi et al5 and present data). It is noteworthy that the
patient studied herein is also a carrier of the Asp2194Gly
substitution. In fact all the subjects of this family5
carrying the R2 allele (His1299Arg mutation) are also carriers of the
Asp2194Gly substitution in factor V, and the latter mutation is tightly
associated with the R2 phenotype.7 Since the His1299Arg
substitution is located in the B region of the cofactor, most likely
the APC resistance of factor Va observed in this individual is due to
the Asp2194Gly mutation, which is located on the light chain
of the active cofactor. Interestingly, Kim et al using alanine-scanning
mutagenesis demonstrated that several amino acid residues within the C2
domain of factor V are crucial for the interaction of the cofactor with
phosphatidylserine. Among those, the Asp2194Ala factor V
mutant had impaired binding to phosphatidylserine.8 Thus,
it is possible that factor Va interacts less efficiently with the
membrane surface, due to the Asp2194Gly substitution encoded by the HR2
haplotype resulting in delayed cleavage at Arg306 and/or Arg506 of the
heavy chain of the cofactor and APC resistance.
Michael Kalafatis, Paolo Simioni, and Francesco Bernardi
Correspondence: Michael Kalafatis, Department of Chemistry,
Cleveland State University, Cleveland, OH 44115
References
1.
Kalafatis M, Rand MD, Mann KG.
The mechanism of inactivation of human factor V and human factor Va by activated protein C.
J Biol Chem.
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2.
Kalafatis M, Haley PE, Lu D, Bertina RM, Long GL, Mann KG.
Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay.
Blood.
1996;87:4695-4707[Abstract/Free Full Text].
3.
Kalafatis M, Bernardi F, Simioni P, Lunghi B, Girolami A, Mann KG.
Phenotype and genotype expression in pseudohomozygous factor VLEIDEN: the need for phenotype analysis.
Arterioscler Thromb Vasc Biol.
1999;19:336-342[Abstract/Free Full Text].
4.
Bernardi F, Faioni EM, Castoldi E, et al.
A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype.
Blood.
1997;90:1552-1557[Abstract/Free Full Text].
5.
Castoldi E, Simioni P, Kalafatis M, et al.
Combinations of four mutations (FVR506Q, FV H1299R, FVY1702C, PT20210G/A) affecting the prothrombinase complex in a thrombophilic family.
Blood.
2000;96:1443-1448[Abstract/Free Full Text].
6.
Hoekema L, Castoldi E, Tans G, et al.
Functional properties of factor V and factor Va encoded by the R2-gene.
Thromb Haemost.
2001;85:75-81[Medline]
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7.
Castoldi E, Rosing J, Girelli D, et al.
Mutations in the R2FV gene affect the ratio between the two isoforms in plasma.
Thromb Haemost.
2000;83:362-365[Medline]
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8.
Kim SW, Quinn-Allen MA, Camp JT, et al.
Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis.
Biochemistry.
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