Prothrombotic factors and the risk of ac (1)
Thrombosis Research 124 (2009) 397–402
Contents lists available at ScienceDirect
Thrombosis Research
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Prothrombotic factors and the risk of acute onset non-cardioembolic stroke in young
Asian Indians
Arijit Biswas a, Ravi Ranjan a, Arvind Meena a, Suhail Akhter a, Vinita Sharma a, Birendra Kumar Yadav a,
Madhuri Behari b, Renu Saxena a,⁎
a
b
Department of Hematology
Department of Neurology
a r t i c l e
i n f o
Article history:
Received 29 September 2008
Received in revised form 25 February 2009
Accepted 25 February 2009
Available online 27 June 2009
Keywords:
Factor V Leiden mutation
Factor V HR2 Haplotype
Coagulation Inhibitors
Factor VIII levels
Acute phase
Asian-Indians
a b s t r a c t
Introduction: Several prothrombotic factors – both hereditary and acquired – are known to cause stroke.
Commonly investigated causes are activated protein C resistance, factor V Leiden mutation, factor VIII levels,
prothrombin 20210 G-to-A mutation, coagulation inhibitors such as proteins C and S, and antiphospholipid
antibodies such as β2-glycoprotein.
Objective: The literature on the prevalence of hematological defects pertaining to these variables in the Asian
Indian stroke population is limited to a few isolated reports. In the current study we investigate the abovementioned variables in 120 stroke patients (non-cardioembolic acute-onset stroke) and compare their status
with the hematological profile of an equal number of healthy age- and sex-matched controls.
Material and Methods: Plasma and blood leukocytes were collected from all patients and controls for
performing hematological assays and molecular tests respectively. The mutations were detected using
standard polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) procedures.
Statistical analysis was done using SPSS version 12.0.
Results: Factor V Leiden (prevalence 8.3% in patients) and activated protein C resistance (prevalence 19.6% in
patients) both showed a high degree of association (P b 0.01) with the disease condition. However, contrary to
common expectations, factor V Leiden was observed much less frequently in patients showing activated
protein C resistance (10 out of 23; 43.4%) than is commonly observed in the Caucasian population (almost 90%).
Post-acute-phase factor VIII levels were also found to be significantly associated with stroke: 125.6 + 21.1%
number of profitable positions (NPP) for controls and 136.2 + 28.8% NPP for patients (P = 0.001).
Conclusion: factor V mutations, such as factor V Leiden, may be important risk factors for stroke in an Asian
Indian population. Activated protein C resistance has a stronger association with stroke than factor V Leiden
and may be caused by other factors such as elevated factor VIII levels in the Asian Indian population apart from
factor V Leiden itself.
© 2009 Elsevier Ltd. All rights reserved.
Introduction
Prothrombotic risk factors often work together to give rise to
thrombosis, which in turn may lead to stroke. These risk factors may
be inherited, like the factor V Leiden mutation, or acquired – e.g.
resistance to activated protein C (APCr) or antiphospholipid antibodies – in origin. The detection of these defects could lead to relevant
modifications in clinical treatment and patient management. APCr is
one of the defects of the hemostatic system which has been commonly
⁎ Corresponding author. Department of Hematology, I.R.C.H. Building (1st floor), All
India Institute of Medical Sciences, Ansari Nagar, New Delhi – 110 029, India. Tel.: +91
011 26593642; fax: +91 011 26588663.
E-mail address: [email protected] (R. Saxena).
0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2009.02.015
observed in stroke conditions. A meta-analysis has shown that the
prevalence of APCr can vary from 0 to 38% in stroke conditions [1]. A
majority of APCr-positive Caucasian patients (more than 95%) carry
the factor V Leiden mutation. This mutation is an Arg506Gln change,
which decreases the susceptibility of factor Va to inactivation by
activated protein C [2,3] and impairs the ability of factor V to stimulate
the inactivation of factor VIIIa mediated by activated protein-C [4,5].
However, these studies are primarily limited to the Caucasian
population. APCr can also be seen in the absence of factor V Leiden
[6]. It is now a well-documented fact that factor V Leiden prevalence
rates vary from population to population.
The other factor V mutations which are commonly screened, some
of which have also been known to contribute towards APCr, are factor
V Hong Kong/Cambridge and factor V HR2 haplotype. Factor V Hong
Kong/Cambridge are two mutations occurring at the Arg306 activated
protein C cleavage site of factor V. The first mutation is a G-to-C
398
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
Table 1
Primer and polymerase chain reaction (PCR) details for prothrombotic mutations.
Polymorphism/mutation
Primer sequence (5′→3′)
Tm
(°C)
Restriciton enzyme
PCR product size
before digest
PCR product size after digest
Factor V Leiden or 1691 G→A or
506 Arg→Gly (= rs6025)
F: CATGAGAGAACATCGCCTCTG
58
MnlI
147 bp
Normal: 85, 37, 25 bp
703 bp
Heterozygous: 122, 85, 37,25 bp
Mutant: 122, 25 bp
Normal (R1R1): 703 bp
252 bp
Heterozygous (R1R2): 703, 492, 211 bp
Mutant (R2R2): 492, 211 bp
For Cambridge: Normal: 173, 53 bp
345
Heterozygous: 226, 173, 53 bp
Mutant: 226 bp
For Hong Kong: Normal: 252 bp
Heterozygous: 252,198, 54 bp
Mutant: 198, 54 bp
Normal GG: 345 bp
R: GACCTAACATGTTCTAGCCAGAAG
Factor V HR2 haplotype
or 4070 A→G
F: CAAGTCCTTCCCCACAGATATA
58
RsaI
R: AGATCTGCAAAGAGGGGCAT
Factor V Hong Kong/Cambridge or
306 Arg→Gly/ 306 Arg→Thr
F:TCCCACCTCTTCATGTGCCGCCTCTG
56
R:CCAAACTAAAATGTTCAAAAATTGCCTGGGCATTA
Prothrombin mutation
or 20210 GNA
F: TCTAGAAACAGTTGCCTGGC
For Cambridge: MvaI
For Hong Kong: HpaII
58
HindIII
R: ATAGCACTGGGAGCATTGAAGC
Heterozygous GA: 345, 322 and 23 bp
Mutant AA: 322 and 23 bp
F, forward primer; R, reverse primer; bp, base pair.
transversion at nucleotide position 1091 resulting in an Arg306Thr
replacement; this mutation is known as the factor V Cambridge
mutation [7]. The second mutation (called the factor V Hong Kong
mutation) is caused by an A-to-G transition at nucleotide position
1090 resulting in an Arg306Gly replacement [8]. The third genetic
variant is the factor V HR2 haplotype, also referred to as A4070G
mutation, and is another genetic cause for mild APCr [9]. The A4070G
mutation is present in exon 13 of human clotting factor V gene
(A4070G; His1299Arg; mutant genotype named R2R2) [10–12]. It is
found to be in tight association with more than 12 polymorphisms of
the factor V gene, the combination collectively known as the HR2
haplotype. Factor V mutation studies on the Asian Indian population
are slightly under-represented, especially in stroke conditions. Most of
them originate from US or European studies where the investigator
has dealt with small groups of Asian Indians residing in the places
where the studies were conducted. It is therefore likely that frequent
mixing with the local population might have given a diluted result in
these studies and may not have been truly representative of the
genetic character of the Asian Indian population.
More recently, factor VIII levels in prothrombotic conditions have
been under investigation. Factor VIII has unique characteristics as a
risk factor. It is well established as an acute-phase reactant [13], and
therefore one could argue that high levels of factor VIII following a
thrombotic event may be more a result of the acute phase rather than
the cause of the event. However, there have been reports which do
establish it to be predictive for thrombosis and not just the result of an
acute-phase event [14]. The dichotomy therefore still remains. While
most of these studies have looked at venous thrombosis [14,15], stroke
(which can have both arterial and venous origins) has been less
thoroughly investigated in this regard, especially when it comes to
investigations conducted after the acute phase. Abnormalities in
natural anticoagulants such as protein C and protein S, and elevated
Fig. 1. A histogram showing the distribution of Protein C and S in Patients and Controls.
399
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
Table 2
Comparative protein C and protein S levels in patients and controls.
Patients
(n = 120)
Controls
(n = 120)
P value*
Protein C
Median levels in % (range)
Number of subjects with deficiency
(as per the kit definition i.e. b 65%)
78 (53–95)
8
78 (65.5–95)
0
0.360
0.003
Protein S
Median levels in % (range)
Number of subjects with deficiency
(as per the kit definition i.e. b 50%)
67 (43–89)
10
67.5 (51–89)
0
0.578
b 0.001
* P values were determined using the Mann–Whitney test for comparison of median
levels of proteins C and S, and Chi-square for number of subjects in patient and control
groups showing protein C and S deficiency.
levels of antiphospholipid antibodies, have also been shown to be
associated with ischemic stroke.
In the present study we look at the impact of the mutants of factor V
(factor V Leiden, factor V Hong Kong/Cambridge and HR2 haplotype)
and prothrombin gene (factor II G20210A mutation) on the Asian Indian
stroke population. Our prevalence study has been done against the
background of other hematological assays such as the levels of natural
anticoagulants (proteins C and S), factor VIII, and β2-glycoprotein IgG
levels in order to obtain a more comprehensive picture. This kind of
study is limited in the Asian Indian population, which has a very diverse
genetic background which is different from that of Caucasian and other
Asian populations (i.e. oriental populations), and should give a fair idea
to clinicians and future investigators as to the hematological status of
Asian Indian stroke patients.
reported for at least a single follow-up were chosen as the subjects for
the study. Initially 231 patients matching the inclusion and exclusion
criteria reported for the study. However, only 120 patients returned
for a follow-up, and these were the subjects of study. The remaining
111 patients were either lost to follow-up or refused consent during
the study period. For controls, 120 age- and sex-matched healthy
subjects were collected from hospital staff members or attendants of
patients not related to them.
Patients
Inclusion Criteria
All patients below 40 years of age presenting with a diagnosis of
acute-onset stroke (cerebrovascular event) and showing evidence of
ischemic infarct were included in the study. The diagnosis of stroke
was confirmed by a combination of objective imaging methods such as
computed tomography/magnetic resonance imaging (CT/MRI) brain/
Doppler studies. Patients with stroke of non-cardioembolic origin
were included in the study. Patients who presented within 4 weeks of
onset of illness were included in the study. Only patients of northern
Indian origin (Jammu and Kashmir, Uttar Pradesh, Haryana, Himachal
Pradesh, Bihar, West Bengal) were included in the study.
Exclusion Criteria
Patients with cardioembolic strokes or having past history of
cardiovascular disease were excluded from the study. Patients on oral
anticoagulants during the first sample collection were excluded from
the study. Patients suffering from diabetes mellitus, hyperlipoproteinemias, cancer, sickle-cell anemia, and liver disease were excluded
from the study.
Material and Methods
Controls
Place of study
Patient collection was done from the Outpatient Departments and
Wards of the Departments of Neurosciences and Hematology at the All
India Institute of Medical Sciences. Control sample collection was
done from Hospital staffers and their relatives or from unrelated
attendants of the patients.
Ethical Clearance
The local ethics committee of All India Institute of Medical Sciences
approved this study, and written informed consent for the study was
obtained.
Sample size
Patients matching the requisite inclusion and exclusion criteria
were asked to report for a 3–6-month follow-up. Only patients who
Inclusion Criteria
Apparently healthy men and women were selected as controls. All
controls were of northern Indian origin.
Exclusion Criteria
Controls taking any form of medication or having had surgery or
suffered trauma in the past 30 days were excluded from the study.
Controls with a history of bleeding, thrombotic, or cardiac disorders
were also excluded from the study, as were pregnant women.
Method
Fasting blood samples were collected in siliconized glass containers containing 1 part sodium citrate solution (0.11 mol/l) with 9 parts
venous blood; care was taken to avoid foam formation during
collection. Platelet-poor plasma was prepared from citrated blood by
Table 3
Genotype and resistance to activated protein C (APCr) details of patients and controls.
Total number
Factor V Leiden
Factor V Leiden homozygous mutant form
Factor V Leiden heterozygous form
Factor V HR2 haplotype
Factor V HR2 haplotype mutant form (R2R2)
Factor V HR2 haplotype heterozygous form (R1R2)
Factor V Hong Kong/Cambridge
APCr
Controls (%)
Patients (%)
Chi-square, P value*
120
1 (0.8)
0 (0)
1 (0.8)
16 (13.3)
0 (0)
16 (13.3)
0 (0)
1 (0.83)
120
10 (8.3)
4 (3.3)
6 (8.3)
18 (17.5)
2 (1.2)
16 (13.3)
0 (0)
23 (20)
–
7.72, 0.005
0.121
0.059
0.034, 0.853
0.248
1.00
–
22.47, b 0.001
OR (range)
10.8 (1.3–229.5)
1.14 (0.61–2.01)
28.21 (3.74–212.7)
*P value b0.05 was considered significant. Pearson's corrected P value has been used for P-value calculations except where numbers b 5 were encountered, in which case Fisher exact
test was used. All P-values are two-tailed.
400
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
Table 4
Resistance to activated protein C (APCr) results for patients in the presence and absence
of factor V Leiden and HR2 haplotype.
S. No. Genotype
1
2
3
4
5
6
7
8
9
10
Patients carrying only factor V Leiden
homozygous mutant form
Patients carrying only factor V HR2 haplotype
mutant form (R2R2)
Patients carrying only factor V Leiden
heterozygous form
Patients carrying only factor V HR2 haplotype
heterozygous form (R1R2)
Patients carrying both factor V Leiden homozygous
mutant and HR2 haplotype homozygous mutant
forms(R2R2)
Patients carrying both factor V Leiden heterozygous
and HR2 haplotype homozygous mutant forms (R2R2)
Patients carrying both factor V Leiden homozygous
mutant and HR2 haplotype heterozygous form (R1R2)
Patients carrying the factor V Leiden heterozygous
form and HR2 haplotype heterozygous form (R1R2)
Patients carrying neither factor V Leiden nor HR2
haplotype mutant or heterozygous forms
Total number of patients
APCr
Kong/Cambridge, prothrombin 20210 G→A and HR2 haplotype were
detected using a PCR/RFLP procedure using the primer and restriction
enzymes listed in the Table 1.
Non-APCr Total
4
0
4
2
0
2
3
0
3
10
3
13
0
0
0
0
0
0
0
0
0
3
0
3
1
94
95
23
97
120
Statistical Analysis
Descriptive and frequency statistical analyses were obtained, and
comparisons were performed by use of the SPSS statistical package,
version 12.0. A difference of P b 0.05 was considered statistically
significant. A non-parametric Mann–Whitney test was used for
comparison of variables which were skewed. Variables which were
normally distributed were compared using the Student t-test.
Result
centrifugation at 1500 g for 10 min. The supernatant plasma was
frozen rapidly in a well closed plastic container, at – 70 °C. Genomic
DNA was extracted from peripheral-blood leukocytes. Thrombinactivatable fibrinolysis inhibitor (TAFI) antigen assay was performed
using the TAFI antigen assay kit (STAGO diagnostica). Total homocysteine concentration was evaluated using enzyme immunoassay
(EIA) (Axis shield kit). Normal levels for homocysteine were 5–
16 μmol/L (as per kit instructions). Fibrinogen levels were measured
using the Clauss method (Kit from STAGO Diagnostica). Normal levels
for fibrinogen were 200–450 mg/dl (as per kit instructions). Protein C
and S antigen levels were measured using enzyme-linked immunosorbent assay (ELISA; DADE Behring kit). Activated protein C
resistance assays were performed using kits from STAGO diagnostica.
Normal levels for proteins C and S were 65–140% and 70–130%
respectively (as per kit instructions). Factor V Leiden, factor V Hong
Coagulation Inhibitors
Since the distribution of proteins C and S in the entire study
population was negatively skewed (Fig. 1), the median levels in patients
and controls were compared using the Mann–Whitney test. No
significant difference was observed in the median levels of proteins C
and S (Table 2) between the patient and control populations. No
difference in median levels was seen between arterial (origin) and
venous (origin) strokes (P = 0.3556 and 0.665 for proteins C and S
respectively) or between recurrent and non-recurrent strokes
(P = 0.770 and 0.502 for proteins C and S respectively). No difference
in median levels of proteins C and S was observed between males and
females (P = 0.389 and 0.647 respectively). When the cut-offs (protein
C b 65% and protein S b 50%) defined by the kit were used as reference,
eight (6.6%) and ten (8.3%) patients were seen to be carrying proteins C
and S deficiency respectively (Table 2). Four of these patients carried
combined protein C and S deficiency; however, this may be explained by
the fact that all these four were on anticoagulants when their sample
was taken (since both protein C and protein S were evaluated after the
acute phase of stroke, i.e., when the second sample was taken).
Therefore effectively only four (3.3%) and six patients (3.3% and 5%
respectively) carried protein C and protein S deficiency for non-acquired
reasons. None of the controls were carrying either protein C or protein S
deficiency.
Fig. 2. Illustration depicting the observed relationship between Activated Protein C resistance and Factor V mutations in Patients. The shaded region represents Activated Protein C
resistance. The number of subjects in individual groups can be calculated by counting the respective bars.
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
401
Activated Protein C resistance
APCr was seen in 23 out of 120 of patients (19.16%) (Table 3). Of
these, 43.4% (10 out of 23) were seen to carry factor V Leiden mutation
(Table 4; see also Fig. 2 for a better understanding of the relationship
between APCr and factor V mutations in this study). Eighteen out of 23
(78.2%) were seen to carry factor V HR2 haplotype. Three patients
showed both factor V Leiden and factor V HR2 haplotype. All patients
positive for factor V Leiden were APCr-positive. Three patients positive
for HR2 haplotype (all heterozygous) did not show APCr. In the
patient's positive for APCr and factor V HR2 (n = 12), but not carrying
factor V Leiden, elevated factor VIII, β2-glycoprotein IgG and
homocysteine levels were seen in three, three and three patients
respectively. Only one sample showing APCr was not positive for
either factor V Leiden or factor V HR2 haplotype. Sixteen control
samples (13.3%) showed factor V HR2 haplotype (although only in
heterozygous form) APCr, but only one control sample was found to be
carrying factor V Leiden (in heterozygous state).
Factor V and other prothrombotic mutations
Factor V Leiden mutation was seen in 8.3% (10 out of 120) of patients
and 0.83% (1 out of 120) of controls. Factor V Leiden mutation was seen
to be strongly associated with the disease phenotype (P = 0.005).
Carriers of the factor V Leiden mutation were ~10 times more likely to
develop stroke (Table 3). Factor V HR2 haplotype on the other hand was
not independently associated with stroke (P = 0.371) (Table 3). Factor V
HR2 haplotype was seen in 17.5% (21 out of 120) of patients and 13.3%
(16 out of 120) of controls. None of the patients or controls carried the
factor V Hong Kong or Cambridge mutations. None of the patient or
controls carried the prothrombin 20210 G→A mutation either.
Factor VIII levels
The distribution of factor VIII levels in the control population was
seen to be normal (Kolmogorov–Smirnov test, P = 0.283). Post-acutephase mean factor VIII levels (125.6 + 21.1% NPP for controls and
136.2 + 28.8% NPP for patients) were significantly raised in patients
compared with controls (P = 0.001; Fig. 3). When the numbers of
patients and controls – eight (6.6%) and two (1.6%) respectively – with
high factor VIII levels (i.e. 177% or N95th percentile of the studied
population) in our study were compared, the difference was only
Fig. 3. Comparison of Factor VIII levels in Patients and Controls.
Fig. 4. Rise and Odds Ratio in the 4h Quartile of Factor VIII levels. Q refers to Quartile.
modestly significant (Fisher exact test, P = 0.041). There isn't much of
a change in risk in the first three quartiles of factor VIII levels, nor is
there a definitive increasing or decreasing trend; however, there is an
awkward jump in odds ratio (OR) to 4.05 as we enter the fourth and
last quartile of the factor VIII level (Fig. 4).
Antiphospholipid antibodies
A significantly higher number of patients – nine of 120 patients
(7.5%) – were seen to have raised levels of IgG (i.e. N15 U/ml, kit
instructions, which incidentally was also the 90th percentile of the
studied population) compared with controls (one in 120, 0.83%;
P = 0.009). Median IgG levels in patients were also significantly raised
compared with those in controls (median levels: patients 8.2 U/ml,
range 2.2–21.2; controls 7 U/ml, range: 2–18; P b 0.001).
Discussion and Conclusions
Our study showed only 3.3 and 5% (after accounting for the
number of patients on anticoagulants when the second sample was
taken) of the patient population to be carrying type-I protein C and S
deficiency respectively. This, coupled with the fact that antigenic
levels of proteins C and S did not vary significantly in our stroke
population (from the healthy control population; P N 0.05), led us to
conclude that inherited type-I protein C and S deficiencies per se may
not be independent risk factors for stroke in our study population.
APCr, on the other hand, showed a strong association with stroke in
our population (P b 0.001). However, unlike the Caucasian population,
in which almost 90% of patients positive for APCr carry the factor V
Leiden mutation, only 43.4% of the patients in our study population
who were positive for APCr carried the factor V Leiden mutation
(Table 4, Fig. 2). Rather the factor V HR2 haplotype was seen at higher
frequency (78.2%) in patients showing APCr. Earlier, our group had
reported somewhat similar results in patients with deep vein
thrombosis [16]. However, there were three patients as well as all
controls (except one) who carried factor V HR2 haplotype (and not
factor V Leiden mutation) but did not show APCr, indicating that the
mere presence of factor V HR2 haplotype alone may not cause APCr. It
would require the simultaneous presence of other defects in the
background. Earlier reports have shown raised homocysteine and
factor VIII levels as well as β2-glycoprotein to contribute to activated
protein C resistance [17,18]. Also the fact that factor V HR2 haplotype is
402
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
not independently associated with stroke in our study is supported by
previous similar reports from the Caucasian population [19]. Therefore
one of the important finding of our study was that APCr (with or
without factor V Leiden mutation) might be an important risk factor
for stroke in our population.
Post-acute-phase mean factor VIII levels were significantly raised
in patients compared with controls (P = 0.001) in our study. This was
also true for the number of patients and controls – 12 (10%) and 4
(3.3%) respectively – with high factor VIII (N177%), although the P
value was only modestly significant (P = 0.041). Borderline P values
do indicate that a bigger population size might reflect a different
result. One recent study conducted on pediatric stroke showed no
difference in levels of factor VIII post acute phase [20]. The ARIC Study
had earlier shown that, per SD increase in factor VIII and in von
Willebrand factor (vWF), the risk for stroke increased 1.34-fold (95%
CI 1.2–1.5) and 1.36-fold (95% CI 1.2–1.5), respectively [21]. In our
study there isn't much of a change in risk in the first three quartiles of
factor VIII levels, nor is there a definitive increasing or decreasing
trend; however, there is an awkward jump in OR to 4.05 as we enter
the fourth and last quartile of the factor VIII level (Fig. 4.). This trend
may be explained by the fact that there is a great deal of heterogeneity
in factor VIII levels in the general population, and a significant
difference in patients and controls is observed only in the topmost
quartile of the factor VIII level. The presence of high factor VIII levels
post acute phase is also an interesting finding, as this has previously
been reported in deep vein thrombosis patients also [22]. Earlier
studies have shown that high factor VIII levels may prevent cleavage of
factor V by protein C, thereby creating conditions of activated protein
C resistance [23] or may even lead to exaggerated levels of thrombin.
Therefore, from the preliminary results, we conclude that factor VIII
would be a worthwhile variable to investigate in a typically Asian
Indian prothrombotic condition.
Limitations
Despite some interesting results, our study does have serious
limitations. One of the major limitations of this entire exercise is that,
in spite of selecting patients of north Indian origin, we might not have
selected a genetically homogenous population. India is a huge country,
and even within the same geographical location the marriage patterns
of different ethnic groups is a complicated affair which gives rise to
incredible genetic diversity. Future investigators may look to identify
population subgroups (using genetic, phenotypic, behavorial or
lifestyle markers) for such studies. Also a larger population size may
be more informative as to the exact role of some of the mutations
studied. Apart from providing more informative genetic content, a
larger population size would also be helpful in statistically analyzing
clinical subgroups (with respect to symptoms or the presence or
absence of other covariates such as smoking, hypertension etc) which
we felt could not be adequately adjusted for while performing the
statistical analyses owing to the smaller sample size of the study. Such
adjustments and more might give better information on the gene–
environment interaction, and may also help in establishing important
clinical correlations. All in all, future studies might consider a large
cross-sectional multicentric study that is more detailed in the clinical
aspects.
Conflict of interest statement
No conflict of interest declared.
References
[1] Bushnell CD, Goldstein LB. Diagnostic testing for coagulopathies in patients with
ischemic stroke. Stroke 2000;31:3067–78.
[2] Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular
defect in factor VR506Q. J Biol Chem 1995;270:4053–7.
[3] Nicolaes GA, Tans G, Thomassen MC, Hemker HC, Pabinger I, Varadi K, Schwarz HP,
et al. Peptide bond cleavages and loss of functional activity during inactivation of
factor Va and factorVaR506Q by activated protein C. J Biol Chem 1995;270:21158–66.
[4] Varadi K, Rosing J, Tans G, Pabinger I, Keil B, Schwarz HP. Factor V enhances the
cofactor function of protein S in the APC-mediated inactivation of factor VIII:
influence of the factor VR506Q mutation. Thromb Haemost 1996;76:208–14.
[5] Thorelli E, Kaufman RJ, Dahlback B. Cleavage of Factor V at Arg 506 by activated
protein C and the expression of anticoagulant activity of factor V. Blood 1999;93:
2552–8.
[6] Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in
patients homozygous for factor V Leiden (activated protein C resistance). Blood
1995;85:1504–8.
[7] Williamson D, Brown K, Luddington R, Baglin C. Factor V Cambridge. A new
mutation (Arg306 to Thr) associated with resistance to activated protein C. Blood
1998;91:1140.
[8] Chan WP, Lee CK, Kwong YL, Lam CK, Liang R. A novel mutation of Arg306 of factor
V gene in Hong Kong Chinese. Blood 1998;91:1135.
[9] Bernardi F, Faioni EM, Castoldi E, Lunghi B, Castaman G, Sacchi E, Mannucci PM, et
al. A factor V genetic component differing from factor V R506Q contributes to the
activated protein C resistance phenotype. Blood 1997;90: 1552–7.
[10] Lunghi B, Iacoviello L, Gemmati D, Dilasio MG, Castoldi E, Pinotti M, Castaman G, et
al. Detection of new polymorphic markers in the factor V gene: association with
factor V levels in plasma. Thromb Haemost 1996;75:45–8.
[11] Pepe G, Rickards O, Vanegas OC, Brunelli T, Gori AM, Giusti B, Attanasio M, et al.
Prevalence of factor V Leiden mutation in non-European populations. Thromb
Haemost 1997;77:329–31.
[12] Cushman M. Inherited risk factors for venous thrombosis. Hematol Am Soc
Hematol Educ Program 2005:452–7.
[13] Noe DA, Murphy PA, Bell WR, Siegel JN. Acute-phase behavior of factor VIII
procoagulant and other acute-phase reactants in rabbits. Am J Physiol 1989;257:
R49–56.
[14] Tsai AW, Cushman M, Rosamond WD, Heckbert SR, Tracy RP, Aleksic N, Folsom AR.
Coagulation factors, inflammation markers, and venous thromboembolism: the
longitudinal investigation of thromboembolism etiology (LITE). Am J Med
2002;113:636–42.
[15] Christiansen SC, Cannegieter SC, Koster T, Vandenbroucke JP, Rosendaal FR.
Thrombophilia, clinical factors, and recurrent venous thrombotic events. JAMA
May 18 2005;293:2352–61.
[16] Biswas A, Bajaj J, Ranjan R, Meena A, Akhter MS, Yadav BK, Sharma V, et al. Factor V
Leiden: Is it the chief contributor to activated protein C resistance in Asian-Indian
patients with deep vein thrombosis? Clin Chim Acta 2008;392:21–4.
[17] Undas A, Williams EB, Butenas S, Orfeo T, Mann KG. Homocysteine inhibits
inactivation of factor Va by activated protein C. J Biol Chem 2001;276:4389–97.
[18] Graf LL, Welsh CH, Qamar Z, Marlar RA. Activated protein C resistance assay detects
thrombotic risk factors other than factor V Leidenq. Am J Clin Pathol
2003;119:52–60.
[19] Lecumberri R, Ceberio I, Montes R, López ML, Alberca I, Rocha E, et al. qEvaluation
of the factor V HR2 haplotype as a risk factor for ischemic cerebrovascular diseaseq.
Haematologica 2003;88:236–7.
[20] Duran R, Biner B, Demir M, Celtik C, Karasalihoğlu S. Factor V Leiden mutation and
other thrombophilia markers in childhood ischemic stroke. Clin Appl Thromb
Hemost 2005;11:83–8.
[21] Folsom AR, Rosamond WD, Shahar E, Cooper LS, Aleksic N, Nieto FJ, Rasmussen ML,
et al. Prospective study of markers of hemostatic function with risk of ischemic
stroke. The Atherosclerosis Risk in Communities (ARIC) Study Investigators.
Circulation 1999;100:736–42.
[22] O'Donnell J, Mumford AD, Manning RA, Laffan M. Elevation of FVIII: C in venous
thromboembolism is persistent and independent of the acute phase response.
Thromb Haemost 2000;83: 10–3.
[23] Henkens CM, Bom VJ, van der Meer J. Lowered APC-sensitivity ratio related to
increased factor VIII-clotting activity. Thromb Haemost 1995;74:1198–9.
Contents lists available at ScienceDirect
Thrombosis Research
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Prothrombotic factors and the risk of acute onset non-cardioembolic stroke in young
Asian Indians
Arijit Biswas a, Ravi Ranjan a, Arvind Meena a, Suhail Akhter a, Vinita Sharma a, Birendra Kumar Yadav a,
Madhuri Behari b, Renu Saxena a,⁎
a
b
Department of Hematology
Department of Neurology
a r t i c l e
i n f o
Article history:
Received 29 September 2008
Received in revised form 25 February 2009
Accepted 25 February 2009
Available online 27 June 2009
Keywords:
Factor V Leiden mutation
Factor V HR2 Haplotype
Coagulation Inhibitors
Factor VIII levels
Acute phase
Asian-Indians
a b s t r a c t
Introduction: Several prothrombotic factors – both hereditary and acquired – are known to cause stroke.
Commonly investigated causes are activated protein C resistance, factor V Leiden mutation, factor VIII levels,
prothrombin 20210 G-to-A mutation, coagulation inhibitors such as proteins C and S, and antiphospholipid
antibodies such as β2-glycoprotein.
Objective: The literature on the prevalence of hematological defects pertaining to these variables in the Asian
Indian stroke population is limited to a few isolated reports. In the current study we investigate the abovementioned variables in 120 stroke patients (non-cardioembolic acute-onset stroke) and compare their status
with the hematological profile of an equal number of healthy age- and sex-matched controls.
Material and Methods: Plasma and blood leukocytes were collected from all patients and controls for
performing hematological assays and molecular tests respectively. The mutations were detected using
standard polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) procedures.
Statistical analysis was done using SPSS version 12.0.
Results: Factor V Leiden (prevalence 8.3% in patients) and activated protein C resistance (prevalence 19.6% in
patients) both showed a high degree of association (P b 0.01) with the disease condition. However, contrary to
common expectations, factor V Leiden was observed much less frequently in patients showing activated
protein C resistance (10 out of 23; 43.4%) than is commonly observed in the Caucasian population (almost 90%).
Post-acute-phase factor VIII levels were also found to be significantly associated with stroke: 125.6 + 21.1%
number of profitable positions (NPP) for controls and 136.2 + 28.8% NPP for patients (P = 0.001).
Conclusion: factor V mutations, such as factor V Leiden, may be important risk factors for stroke in an Asian
Indian population. Activated protein C resistance has a stronger association with stroke than factor V Leiden
and may be caused by other factors such as elevated factor VIII levels in the Asian Indian population apart from
factor V Leiden itself.
© 2009 Elsevier Ltd. All rights reserved.
Introduction
Prothrombotic risk factors often work together to give rise to
thrombosis, which in turn may lead to stroke. These risk factors may
be inherited, like the factor V Leiden mutation, or acquired – e.g.
resistance to activated protein C (APCr) or antiphospholipid antibodies – in origin. The detection of these defects could lead to relevant
modifications in clinical treatment and patient management. APCr is
one of the defects of the hemostatic system which has been commonly
⁎ Corresponding author. Department of Hematology, I.R.C.H. Building (1st floor), All
India Institute of Medical Sciences, Ansari Nagar, New Delhi – 110 029, India. Tel.: +91
011 26593642; fax: +91 011 26588663.
E-mail address: [email protected] (R. Saxena).
0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2009.02.015
observed in stroke conditions. A meta-analysis has shown that the
prevalence of APCr can vary from 0 to 38% in stroke conditions [1]. A
majority of APCr-positive Caucasian patients (more than 95%) carry
the factor V Leiden mutation. This mutation is an Arg506Gln change,
which decreases the susceptibility of factor Va to inactivation by
activated protein C [2,3] and impairs the ability of factor V to stimulate
the inactivation of factor VIIIa mediated by activated protein-C [4,5].
However, these studies are primarily limited to the Caucasian
population. APCr can also be seen in the absence of factor V Leiden
[6]. It is now a well-documented fact that factor V Leiden prevalence
rates vary from population to population.
The other factor V mutations which are commonly screened, some
of which have also been known to contribute towards APCr, are factor
V Hong Kong/Cambridge and factor V HR2 haplotype. Factor V Hong
Kong/Cambridge are two mutations occurring at the Arg306 activated
protein C cleavage site of factor V. The first mutation is a G-to-C
398
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
Table 1
Primer and polymerase chain reaction (PCR) details for prothrombotic mutations.
Polymorphism/mutation
Primer sequence (5′→3′)
Tm
(°C)
Restriciton enzyme
PCR product size
before digest
PCR product size after digest
Factor V Leiden or 1691 G→A or
506 Arg→Gly (= rs6025)
F: CATGAGAGAACATCGCCTCTG
58
MnlI
147 bp
Normal: 85, 37, 25 bp
703 bp
Heterozygous: 122, 85, 37,25 bp
Mutant: 122, 25 bp
Normal (R1R1): 703 bp
252 bp
Heterozygous (R1R2): 703, 492, 211 bp
Mutant (R2R2): 492, 211 bp
For Cambridge: Normal: 173, 53 bp
345
Heterozygous: 226, 173, 53 bp
Mutant: 226 bp
For Hong Kong: Normal: 252 bp
Heterozygous: 252,198, 54 bp
Mutant: 198, 54 bp
Normal GG: 345 bp
R: GACCTAACATGTTCTAGCCAGAAG
Factor V HR2 haplotype
or 4070 A→G
F: CAAGTCCTTCCCCACAGATATA
58
RsaI
R: AGATCTGCAAAGAGGGGCAT
Factor V Hong Kong/Cambridge or
306 Arg→Gly/ 306 Arg→Thr
F:TCCCACCTCTTCATGTGCCGCCTCTG
56
R:CCAAACTAAAATGTTCAAAAATTGCCTGGGCATTA
Prothrombin mutation
or 20210 GNA
F: TCTAGAAACAGTTGCCTGGC
For Cambridge: MvaI
For Hong Kong: HpaII
58
HindIII
R: ATAGCACTGGGAGCATTGAAGC
Heterozygous GA: 345, 322 and 23 bp
Mutant AA: 322 and 23 bp
F, forward primer; R, reverse primer; bp, base pair.
transversion at nucleotide position 1091 resulting in an Arg306Thr
replacement; this mutation is known as the factor V Cambridge
mutation [7]. The second mutation (called the factor V Hong Kong
mutation) is caused by an A-to-G transition at nucleotide position
1090 resulting in an Arg306Gly replacement [8]. The third genetic
variant is the factor V HR2 haplotype, also referred to as A4070G
mutation, and is another genetic cause for mild APCr [9]. The A4070G
mutation is present in exon 13 of human clotting factor V gene
(A4070G; His1299Arg; mutant genotype named R2R2) [10–12]. It is
found to be in tight association with more than 12 polymorphisms of
the factor V gene, the combination collectively known as the HR2
haplotype. Factor V mutation studies on the Asian Indian population
are slightly under-represented, especially in stroke conditions. Most of
them originate from US or European studies where the investigator
has dealt with small groups of Asian Indians residing in the places
where the studies were conducted. It is therefore likely that frequent
mixing with the local population might have given a diluted result in
these studies and may not have been truly representative of the
genetic character of the Asian Indian population.
More recently, factor VIII levels in prothrombotic conditions have
been under investigation. Factor VIII has unique characteristics as a
risk factor. It is well established as an acute-phase reactant [13], and
therefore one could argue that high levels of factor VIII following a
thrombotic event may be more a result of the acute phase rather than
the cause of the event. However, there have been reports which do
establish it to be predictive for thrombosis and not just the result of an
acute-phase event [14]. The dichotomy therefore still remains. While
most of these studies have looked at venous thrombosis [14,15], stroke
(which can have both arterial and venous origins) has been less
thoroughly investigated in this regard, especially when it comes to
investigations conducted after the acute phase. Abnormalities in
natural anticoagulants such as protein C and protein S, and elevated
Fig. 1. A histogram showing the distribution of Protein C and S in Patients and Controls.
399
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
Table 2
Comparative protein C and protein S levels in patients and controls.
Patients
(n = 120)
Controls
(n = 120)
P value*
Protein C
Median levels in % (range)
Number of subjects with deficiency
(as per the kit definition i.e. b 65%)
78 (53–95)
8
78 (65.5–95)
0
0.360
0.003
Protein S
Median levels in % (range)
Number of subjects with deficiency
(as per the kit definition i.e. b 50%)
67 (43–89)
10
67.5 (51–89)
0
0.578
b 0.001
* P values were determined using the Mann–Whitney test for comparison of median
levels of proteins C and S, and Chi-square for number of subjects in patient and control
groups showing protein C and S deficiency.
levels of antiphospholipid antibodies, have also been shown to be
associated with ischemic stroke.
In the present study we look at the impact of the mutants of factor V
(factor V Leiden, factor V Hong Kong/Cambridge and HR2 haplotype)
and prothrombin gene (factor II G20210A mutation) on the Asian Indian
stroke population. Our prevalence study has been done against the
background of other hematological assays such as the levels of natural
anticoagulants (proteins C and S), factor VIII, and β2-glycoprotein IgG
levels in order to obtain a more comprehensive picture. This kind of
study is limited in the Asian Indian population, which has a very diverse
genetic background which is different from that of Caucasian and other
Asian populations (i.e. oriental populations), and should give a fair idea
to clinicians and future investigators as to the hematological status of
Asian Indian stroke patients.
reported for at least a single follow-up were chosen as the subjects for
the study. Initially 231 patients matching the inclusion and exclusion
criteria reported for the study. However, only 120 patients returned
for a follow-up, and these were the subjects of study. The remaining
111 patients were either lost to follow-up or refused consent during
the study period. For controls, 120 age- and sex-matched healthy
subjects were collected from hospital staff members or attendants of
patients not related to them.
Patients
Inclusion Criteria
All patients below 40 years of age presenting with a diagnosis of
acute-onset stroke (cerebrovascular event) and showing evidence of
ischemic infarct were included in the study. The diagnosis of stroke
was confirmed by a combination of objective imaging methods such as
computed tomography/magnetic resonance imaging (CT/MRI) brain/
Doppler studies. Patients with stroke of non-cardioembolic origin
were included in the study. Patients who presented within 4 weeks of
onset of illness were included in the study. Only patients of northern
Indian origin (Jammu and Kashmir, Uttar Pradesh, Haryana, Himachal
Pradesh, Bihar, West Bengal) were included in the study.
Exclusion Criteria
Patients with cardioembolic strokes or having past history of
cardiovascular disease were excluded from the study. Patients on oral
anticoagulants during the first sample collection were excluded from
the study. Patients suffering from diabetes mellitus, hyperlipoproteinemias, cancer, sickle-cell anemia, and liver disease were excluded
from the study.
Material and Methods
Controls
Place of study
Patient collection was done from the Outpatient Departments and
Wards of the Departments of Neurosciences and Hematology at the All
India Institute of Medical Sciences. Control sample collection was
done from Hospital staffers and their relatives or from unrelated
attendants of the patients.
Ethical Clearance
The local ethics committee of All India Institute of Medical Sciences
approved this study, and written informed consent for the study was
obtained.
Sample size
Patients matching the requisite inclusion and exclusion criteria
were asked to report for a 3–6-month follow-up. Only patients who
Inclusion Criteria
Apparently healthy men and women were selected as controls. All
controls were of northern Indian origin.
Exclusion Criteria
Controls taking any form of medication or having had surgery or
suffered trauma in the past 30 days were excluded from the study.
Controls with a history of bleeding, thrombotic, or cardiac disorders
were also excluded from the study, as were pregnant women.
Method
Fasting blood samples were collected in siliconized glass containers containing 1 part sodium citrate solution (0.11 mol/l) with 9 parts
venous blood; care was taken to avoid foam formation during
collection. Platelet-poor plasma was prepared from citrated blood by
Table 3
Genotype and resistance to activated protein C (APCr) details of patients and controls.
Total number
Factor V Leiden
Factor V Leiden homozygous mutant form
Factor V Leiden heterozygous form
Factor V HR2 haplotype
Factor V HR2 haplotype mutant form (R2R2)
Factor V HR2 haplotype heterozygous form (R1R2)
Factor V Hong Kong/Cambridge
APCr
Controls (%)
Patients (%)
Chi-square, P value*
120
1 (0.8)
0 (0)
1 (0.8)
16 (13.3)
0 (0)
16 (13.3)
0 (0)
1 (0.83)
120
10 (8.3)
4 (3.3)
6 (8.3)
18 (17.5)
2 (1.2)
16 (13.3)
0 (0)
23 (20)
–
7.72, 0.005
0.121
0.059
0.034, 0.853
0.248
1.00
–
22.47, b 0.001
OR (range)
10.8 (1.3–229.5)
1.14 (0.61–2.01)
28.21 (3.74–212.7)
*P value b0.05 was considered significant. Pearson's corrected P value has been used for P-value calculations except where numbers b 5 were encountered, in which case Fisher exact
test was used. All P-values are two-tailed.
400
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
Table 4
Resistance to activated protein C (APCr) results for patients in the presence and absence
of factor V Leiden and HR2 haplotype.
S. No. Genotype
1
2
3
4
5
6
7
8
9
10
Patients carrying only factor V Leiden
homozygous mutant form
Patients carrying only factor V HR2 haplotype
mutant form (R2R2)
Patients carrying only factor V Leiden
heterozygous form
Patients carrying only factor V HR2 haplotype
heterozygous form (R1R2)
Patients carrying both factor V Leiden homozygous
mutant and HR2 haplotype homozygous mutant
forms(R2R2)
Patients carrying both factor V Leiden heterozygous
and HR2 haplotype homozygous mutant forms (R2R2)
Patients carrying both factor V Leiden homozygous
mutant and HR2 haplotype heterozygous form (R1R2)
Patients carrying the factor V Leiden heterozygous
form and HR2 haplotype heterozygous form (R1R2)
Patients carrying neither factor V Leiden nor HR2
haplotype mutant or heterozygous forms
Total number of patients
APCr
Kong/Cambridge, prothrombin 20210 G→A and HR2 haplotype were
detected using a PCR/RFLP procedure using the primer and restriction
enzymes listed in the Table 1.
Non-APCr Total
4
0
4
2
0
2
3
0
3
10
3
13
0
0
0
0
0
0
0
0
0
3
0
3
1
94
95
23
97
120
Statistical Analysis
Descriptive and frequency statistical analyses were obtained, and
comparisons were performed by use of the SPSS statistical package,
version 12.0. A difference of P b 0.05 was considered statistically
significant. A non-parametric Mann–Whitney test was used for
comparison of variables which were skewed. Variables which were
normally distributed were compared using the Student t-test.
Result
centrifugation at 1500 g for 10 min. The supernatant plasma was
frozen rapidly in a well closed plastic container, at – 70 °C. Genomic
DNA was extracted from peripheral-blood leukocytes. Thrombinactivatable fibrinolysis inhibitor (TAFI) antigen assay was performed
using the TAFI antigen assay kit (STAGO diagnostica). Total homocysteine concentration was evaluated using enzyme immunoassay
(EIA) (Axis shield kit). Normal levels for homocysteine were 5–
16 μmol/L (as per kit instructions). Fibrinogen levels were measured
using the Clauss method (Kit from STAGO Diagnostica). Normal levels
for fibrinogen were 200–450 mg/dl (as per kit instructions). Protein C
and S antigen levels were measured using enzyme-linked immunosorbent assay (ELISA; DADE Behring kit). Activated protein C
resistance assays were performed using kits from STAGO diagnostica.
Normal levels for proteins C and S were 65–140% and 70–130%
respectively (as per kit instructions). Factor V Leiden, factor V Hong
Coagulation Inhibitors
Since the distribution of proteins C and S in the entire study
population was negatively skewed (Fig. 1), the median levels in patients
and controls were compared using the Mann–Whitney test. No
significant difference was observed in the median levels of proteins C
and S (Table 2) between the patient and control populations. No
difference in median levels was seen between arterial (origin) and
venous (origin) strokes (P = 0.3556 and 0.665 for proteins C and S
respectively) or between recurrent and non-recurrent strokes
(P = 0.770 and 0.502 for proteins C and S respectively). No difference
in median levels of proteins C and S was observed between males and
females (P = 0.389 and 0.647 respectively). When the cut-offs (protein
C b 65% and protein S b 50%) defined by the kit were used as reference,
eight (6.6%) and ten (8.3%) patients were seen to be carrying proteins C
and S deficiency respectively (Table 2). Four of these patients carried
combined protein C and S deficiency; however, this may be explained by
the fact that all these four were on anticoagulants when their sample
was taken (since both protein C and protein S were evaluated after the
acute phase of stroke, i.e., when the second sample was taken).
Therefore effectively only four (3.3%) and six patients (3.3% and 5%
respectively) carried protein C and protein S deficiency for non-acquired
reasons. None of the controls were carrying either protein C or protein S
deficiency.
Fig. 2. Illustration depicting the observed relationship between Activated Protein C resistance and Factor V mutations in Patients. The shaded region represents Activated Protein C
resistance. The number of subjects in individual groups can be calculated by counting the respective bars.
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
401
Activated Protein C resistance
APCr was seen in 23 out of 120 of patients (19.16%) (Table 3). Of
these, 43.4% (10 out of 23) were seen to carry factor V Leiden mutation
(Table 4; see also Fig. 2 for a better understanding of the relationship
between APCr and factor V mutations in this study). Eighteen out of 23
(78.2%) were seen to carry factor V HR2 haplotype. Three patients
showed both factor V Leiden and factor V HR2 haplotype. All patients
positive for factor V Leiden were APCr-positive. Three patients positive
for HR2 haplotype (all heterozygous) did not show APCr. In the
patient's positive for APCr and factor V HR2 (n = 12), but not carrying
factor V Leiden, elevated factor VIII, β2-glycoprotein IgG and
homocysteine levels were seen in three, three and three patients
respectively. Only one sample showing APCr was not positive for
either factor V Leiden or factor V HR2 haplotype. Sixteen control
samples (13.3%) showed factor V HR2 haplotype (although only in
heterozygous form) APCr, but only one control sample was found to be
carrying factor V Leiden (in heterozygous state).
Factor V and other prothrombotic mutations
Factor V Leiden mutation was seen in 8.3% (10 out of 120) of patients
and 0.83% (1 out of 120) of controls. Factor V Leiden mutation was seen
to be strongly associated with the disease phenotype (P = 0.005).
Carriers of the factor V Leiden mutation were ~10 times more likely to
develop stroke (Table 3). Factor V HR2 haplotype on the other hand was
not independently associated with stroke (P = 0.371) (Table 3). Factor V
HR2 haplotype was seen in 17.5% (21 out of 120) of patients and 13.3%
(16 out of 120) of controls. None of the patients or controls carried the
factor V Hong Kong or Cambridge mutations. None of the patient or
controls carried the prothrombin 20210 G→A mutation either.
Factor VIII levels
The distribution of factor VIII levels in the control population was
seen to be normal (Kolmogorov–Smirnov test, P = 0.283). Post-acutephase mean factor VIII levels (125.6 + 21.1% NPP for controls and
136.2 + 28.8% NPP for patients) were significantly raised in patients
compared with controls (P = 0.001; Fig. 3). When the numbers of
patients and controls – eight (6.6%) and two (1.6%) respectively – with
high factor VIII levels (i.e. 177% or N95th percentile of the studied
population) in our study were compared, the difference was only
Fig. 3. Comparison of Factor VIII levels in Patients and Controls.
Fig. 4. Rise and Odds Ratio in the 4h Quartile of Factor VIII levels. Q refers to Quartile.
modestly significant (Fisher exact test, P = 0.041). There isn't much of
a change in risk in the first three quartiles of factor VIII levels, nor is
there a definitive increasing or decreasing trend; however, there is an
awkward jump in odds ratio (OR) to 4.05 as we enter the fourth and
last quartile of the factor VIII level (Fig. 4).
Antiphospholipid antibodies
A significantly higher number of patients – nine of 120 patients
(7.5%) – were seen to have raised levels of IgG (i.e. N15 U/ml, kit
instructions, which incidentally was also the 90th percentile of the
studied population) compared with controls (one in 120, 0.83%;
P = 0.009). Median IgG levels in patients were also significantly raised
compared with those in controls (median levels: patients 8.2 U/ml,
range 2.2–21.2; controls 7 U/ml, range: 2–18; P b 0.001).
Discussion and Conclusions
Our study showed only 3.3 and 5% (after accounting for the
number of patients on anticoagulants when the second sample was
taken) of the patient population to be carrying type-I protein C and S
deficiency respectively. This, coupled with the fact that antigenic
levels of proteins C and S did not vary significantly in our stroke
population (from the healthy control population; P N 0.05), led us to
conclude that inherited type-I protein C and S deficiencies per se may
not be independent risk factors for stroke in our study population.
APCr, on the other hand, showed a strong association with stroke in
our population (P b 0.001). However, unlike the Caucasian population,
in which almost 90% of patients positive for APCr carry the factor V
Leiden mutation, only 43.4% of the patients in our study population
who were positive for APCr carried the factor V Leiden mutation
(Table 4, Fig. 2). Rather the factor V HR2 haplotype was seen at higher
frequency (78.2%) in patients showing APCr. Earlier, our group had
reported somewhat similar results in patients with deep vein
thrombosis [16]. However, there were three patients as well as all
controls (except one) who carried factor V HR2 haplotype (and not
factor V Leiden mutation) but did not show APCr, indicating that the
mere presence of factor V HR2 haplotype alone may not cause APCr. It
would require the simultaneous presence of other defects in the
background. Earlier reports have shown raised homocysteine and
factor VIII levels as well as β2-glycoprotein to contribute to activated
protein C resistance [17,18]. Also the fact that factor V HR2 haplotype is
402
A. Biswas et al. / Thrombosis Research 124 (2009) 397–402
not independently associated with stroke in our study is supported by
previous similar reports from the Caucasian population [19]. Therefore
one of the important finding of our study was that APCr (with or
without factor V Leiden mutation) might be an important risk factor
for stroke in our population.
Post-acute-phase mean factor VIII levels were significantly raised
in patients compared with controls (P = 0.001) in our study. This was
also true for the number of patients and controls – 12 (10%) and 4
(3.3%) respectively – with high factor VIII (N177%), although the P
value was only modestly significant (P = 0.041). Borderline P values
do indicate that a bigger population size might reflect a different
result. One recent study conducted on pediatric stroke showed no
difference in levels of factor VIII post acute phase [20]. The ARIC Study
had earlier shown that, per SD increase in factor VIII and in von
Willebrand factor (vWF), the risk for stroke increased 1.34-fold (95%
CI 1.2–1.5) and 1.36-fold (95% CI 1.2–1.5), respectively [21]. In our
study there isn't much of a change in risk in the first three quartiles of
factor VIII levels, nor is there a definitive increasing or decreasing
trend; however, there is an awkward jump in OR to 4.05 as we enter
the fourth and last quartile of the factor VIII level (Fig. 4.). This trend
may be explained by the fact that there is a great deal of heterogeneity
in factor VIII levels in the general population, and a significant
difference in patients and controls is observed only in the topmost
quartile of the factor VIII level. The presence of high factor VIII levels
post acute phase is also an interesting finding, as this has previously
been reported in deep vein thrombosis patients also [22]. Earlier
studies have shown that high factor VIII levels may prevent cleavage of
factor V by protein C, thereby creating conditions of activated protein
C resistance [23] or may even lead to exaggerated levels of thrombin.
Therefore, from the preliminary results, we conclude that factor VIII
would be a worthwhile variable to investigate in a typically Asian
Indian prothrombotic condition.
Limitations
Despite some interesting results, our study does have serious
limitations. One of the major limitations of this entire exercise is that,
in spite of selecting patients of north Indian origin, we might not have
selected a genetically homogenous population. India is a huge country,
and even within the same geographical location the marriage patterns
of different ethnic groups is a complicated affair which gives rise to
incredible genetic diversity. Future investigators may look to identify
population subgroups (using genetic, phenotypic, behavorial or
lifestyle markers) for such studies. Also a larger population size may
be more informative as to the exact role of some of the mutations
studied. Apart from providing more informative genetic content, a
larger population size would also be helpful in statistically analyzing
clinical subgroups (with respect to symptoms or the presence or
absence of other covariates such as smoking, hypertension etc) which
we felt could not be adequately adjusted for while performing the
statistical analyses owing to the smaller sample size of the study. Such
adjustments and more might give better information on the gene–
environment interaction, and may also help in establishing important
clinical correlations. All in all, future studies might consider a large
cross-sectional multicentric study that is more detailed in the clinical
aspects.
Conflict of interest statement
No conflict of interest declared.
References
[1] Bushnell CD, Goldstein LB. Diagnostic testing for coagulopathies in patients with
ischemic stroke. Stroke 2000;31:3067–78.
[2] Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular
defect in factor VR506Q. J Biol Chem 1995;270:4053–7.
[3] Nicolaes GA, Tans G, Thomassen MC, Hemker HC, Pabinger I, Varadi K, Schwarz HP,
et al. Peptide bond cleavages and loss of functional activity during inactivation of
factor Va and factorVaR506Q by activated protein C. J Biol Chem 1995;270:21158–66.
[4] Varadi K, Rosing J, Tans G, Pabinger I, Keil B, Schwarz HP. Factor V enhances the
cofactor function of protein S in the APC-mediated inactivation of factor VIII:
influence of the factor VR506Q mutation. Thromb Haemost 1996;76:208–14.
[5] Thorelli E, Kaufman RJ, Dahlback B. Cleavage of Factor V at Arg 506 by activated
protein C and the expression of anticoagulant activity of factor V. Blood 1999;93:
2552–8.
[6] Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in
patients homozygous for factor V Leiden (activated protein C resistance). Blood
1995;85:1504–8.
[7] Williamson D, Brown K, Luddington R, Baglin C. Factor V Cambridge. A new
mutation (Arg306 to Thr) associated with resistance to activated protein C. Blood
1998;91:1140.
[8] Chan WP, Lee CK, Kwong YL, Lam CK, Liang R. A novel mutation of Arg306 of factor
V gene in Hong Kong Chinese. Blood 1998;91:1135.
[9] Bernardi F, Faioni EM, Castoldi E, Lunghi B, Castaman G, Sacchi E, Mannucci PM, et
al. A factor V genetic component differing from factor V R506Q contributes to the
activated protein C resistance phenotype. Blood 1997;90: 1552–7.
[10] Lunghi B, Iacoviello L, Gemmati D, Dilasio MG, Castoldi E, Pinotti M, Castaman G, et
al. Detection of new polymorphic markers in the factor V gene: association with
factor V levels in plasma. Thromb Haemost 1996;75:45–8.
[11] Pepe G, Rickards O, Vanegas OC, Brunelli T, Gori AM, Giusti B, Attanasio M, et al.
Prevalence of factor V Leiden mutation in non-European populations. Thromb
Haemost 1997;77:329–31.
[12] Cushman M. Inherited risk factors for venous thrombosis. Hematol Am Soc
Hematol Educ Program 2005:452–7.
[13] Noe DA, Murphy PA, Bell WR, Siegel JN. Acute-phase behavior of factor VIII
procoagulant and other acute-phase reactants in rabbits. Am J Physiol 1989;257:
R49–56.
[14] Tsai AW, Cushman M, Rosamond WD, Heckbert SR, Tracy RP, Aleksic N, Folsom AR.
Coagulation factors, inflammation markers, and venous thromboembolism: the
longitudinal investigation of thromboembolism etiology (LITE). Am J Med
2002;113:636–42.
[15] Christiansen SC, Cannegieter SC, Koster T, Vandenbroucke JP, Rosendaal FR.
Thrombophilia, clinical factors, and recurrent venous thrombotic events. JAMA
May 18 2005;293:2352–61.
[16] Biswas A, Bajaj J, Ranjan R, Meena A, Akhter MS, Yadav BK, Sharma V, et al. Factor V
Leiden: Is it the chief contributor to activated protein C resistance in Asian-Indian
patients with deep vein thrombosis? Clin Chim Acta 2008;392:21–4.
[17] Undas A, Williams EB, Butenas S, Orfeo T, Mann KG. Homocysteine inhibits
inactivation of factor Va by activated protein C. J Biol Chem 2001;276:4389–97.
[18] Graf LL, Welsh CH, Qamar Z, Marlar RA. Activated protein C resistance assay detects
thrombotic risk factors other than factor V Leidenq. Am J Clin Pathol
2003;119:52–60.
[19] Lecumberri R, Ceberio I, Montes R, López ML, Alberca I, Rocha E, et al. qEvaluation
of the factor V HR2 haplotype as a risk factor for ischemic cerebrovascular diseaseq.
Haematologica 2003;88:236–7.
[20] Duran R, Biner B, Demir M, Celtik C, Karasalihoğlu S. Factor V Leiden mutation and
other thrombophilia markers in childhood ischemic stroke. Clin Appl Thromb
Hemost 2005;11:83–8.
[21] Folsom AR, Rosamond WD, Shahar E, Cooper LS, Aleksic N, Nieto FJ, Rasmussen ML,
et al. Prospective study of markers of hemostatic function with risk of ischemic
stroke. The Atherosclerosis Risk in Communities (ARIC) Study Investigators.
Circulation 1999;100:736–42.
[22] O'Donnell J, Mumford AD, Manning RA, Laffan M. Elevation of FVIII: C in venous
thromboembolism is persistent and independent of the acute phase response.
Thromb Haemost 2000;83: 10–3.
[23] Henkens CM, Bom VJ, van der Meer J. Lowered APC-sensitivity ratio related to
increased factor VIII-clotting activity. Thromb Haemost 1995;74:1198–9.