Quantitative measurement of APC-Resistant factor V clotting activity (FV-Leiden) and potential graduation of the associated thrombo-embolic risk
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Amiral Jean, et al. Medical Research Archives vol 9 issue 1. Medical Research Archives
RESEARCH ARTICLE
Quantitative measurement of APC-Resistant factor V clotting activity (FV-Leiden)
and potential graduation of the associated thrombo-embolic risk
Authors
Peyrafitte Marie1, Vissac Anne Marie1, Amiral Jean2
Affiliations
1.
HYPHEN BioMed research laboratory, 95130 Neuville sur Oise (France)
2.
Scientific Hemostasis, 95130 Franconville (France)
Corresponding author:
Email: jean.amiral@scientific-hemostasis.com
Abstract:
Coagulation Factor V (FV) is a key factor for regulating blood coagulation cascade, and it acts at
the crossroads of the intrinsic and extrinsic pathways. It shows a dual activity as the procoagulant
cofactor for Factor Xa in the prothrombinase complex, but it also supports an anticoagulant
activity in combination with TFPI and Protein S. Its rapid cleavage by Activated Protein C (APC)
complexed with Free Protein S (FPS), in presence of phospholipids and calcium, inhibits its
activity and limits the propagation of blood coagulation, keeping it to where it is beneficial. Rapid
inactivation of active FV by APC-FPS is essential for preventing the risk of thrombosis
development. In 1993, Dahlbäck and coworkers reported an inherited disorder characterized by
activated protein C resistance (APC-R) and associated to an increased occurrence of
thromboembolic events in affected families. In 1994 Bertina demonstrated that this diathesis
resulted from a Factor V mutation (R506Q), rendering this factor resistant to inactivation by APC.
This mutated Factor V was called Factor V Leiden (FV-L). APTT based assays and molecular
biology methods for detecting the mutation were developed, but these methods are only qualitative
and classify tested individuals as normals, heterozygous or homozygous for the coagulation defect.
Our group developed a quantitative assay for FV-L, which is described in this report, along with
its performances. This assay allows to quantitate specifically FV-L coagulant activity, and to
graduate its amount in heterozygous or homozygous patients. FV-L is absent in normal individuals
and present in homozygous or heterozygous patients, accounting respectively for 100 % or 50 %
of blood FV. Its amount is compared with FV clotting activity or antigenic concentration.
Measured FV-L activities overlap between heterozygous patients with high FV and homozygous
ones with low FV levels. This assay allows to better discriminate for the FV-L associated
thrombotic risk, which depends on the effective FV-L concentration rather than on patients’
genetic status. This expectation is supported by literature review, which shows that FV-L
concentrations correlate with presence of platelet released microparticles in patients carrying that
mutation.
Key words: Factor V Leiden; Quantitative assay; Activated Protein C; APC Resistance;
Thrombosis.
Copyright 2021 KEI Journals. All Rights ReservedAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 2 of 18
Introduction: thrombomodulin: its coagulant activity is
Factor V (FV) is a key cofactor, known since a transformed into an anticoagulant one; ii) PC is
long time as accelerin, and essential for the activated to APC, and in presence of Free
activity of the prothrombinase complex, Protein S (FPS), forms an APC-PS complex
involving activated Factor X (FXa), that inactivates thrombin activated Factor VIII
phospholipids and calcium, which generates (FVIIIa) and FV (FVa). These actions stop any
thrombin from prothrombin 1. During normal blood hemostasis activation pathway, which
physiology, in-vivo coagulation is a site- could occur in blood circulation away from the
targeted mechanism, which is initiated, initial activation site and they inactivate the
amplified, and propagated where its action is blood coagulation cascade when the beneficial
beneficial, and is then regulated at this site. In clot is formed 5, 6. This regulatory mechanism
pathology, coagulation can be activated in operates similarly in presence of diffuse blood
blood circulation by abnormal components activation, triggered by developing pathologies
induced by disease states, tissue injuries, (even occult) or by metabolic abnormalities. In
metabolism abnormalities, inflammation, or this disease context, the protective intra-
infection 2. When activated, coagulation vascular endothelial surface and the blood
process ends by inducing a clot formation, content are altered. For example, cancer,
which prevents from bleeding and allows traumatisms, atherosclerosis, diabetes,
healing in presence of vascular wounds, but it infections, autoimmune diseases, immun-
can be deleterious when it is provoked by complexes can activate hemostasis and
pathology. An efficient regulation process mobilize the blood regulatory system to
must control clot formation, to keep it at the prevent thrombus formation. Only when these
healing sites, and to prevent propagation of body defenses are overwhelmed because blood
coagulation pathways in blood circulation, activation exceeds body regulation capacity,
where it could provoke undesired disseminated thrombo-embolic diseases can develop 2.
activation and fibrin formation, and then Efficient inactivation of FVIIIa, and FVa by
thrombosis can occur. Regulation of APC-PS-TM system is then essential for
hemostasis involves intact endothelium, serine maintaining blood fluidity, and its dysfunction
esterase inhibitors (such as Antithrombin and induces an increased risk of thrombosis. In
Heparin Cofactor II), the Protein C (PC)- practice, FVa has a higher survival time in
Protein S (PS)-Thrombomodulin (TM) system, blood circulation than FVIIIa, and its
which regulates and inhibits activated factors V prolonged survival was reported many years
(FVa) and VIII (FVIIIa), and fibrinolysis. The ago in patients with Lupus Anticoagulant (LA)
7
complementary action of these functions is of . If not readily controlled, FVa can
essence for keeping the procoagulant pathways disseminate activation of blood coagulation
at the right sites, and for a delayed dissolution pathways away from the beneficial clotting
of the clot once the healing function is site. FV has a complex implication in the
achieved. Abnormal concentration or activity coagulation cascade. Recent studies have
of these anticoagulant mechanisms favors shown that it has a dual role and can also be
occurrence of thromboembolic diseases 3, 4. FV anticoagulant 8-11. A small amount of FV is
plays then a key role in this process, being at truncated (FV short) and behaves as the PS and
the crossroads of blood coagulation pathways. Tissue Factor Pathway Inhibitor (TFPI)
cofactor for the inhibition of FXa 8. This
Regulation of FV/FVa clotting activity: additional function of FV, which is
The PC-PS-TM system has a double action: i) anticoagulant, will not be discussed further in
it captures thrombin in micro-circulation that report, which focuses on the procoagulant
through its binding to endothelium exposed potential of FV and its control. In contrast,
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 3 of 18
when FV carries the R506Q mutation 12, it and regulation of FVa is essential for a normal
becomes resistant to the action of APC-PS and coagulation homeostasis, and the APC-PS-TM
it has then a prolonged survival following system has a major role in this process 17.
activation of blood coagulation pathways, and
this increases the risk of developing thrombo- Factor V variations:
embolic diseases (TED). The FV concentrations present a high
distribution among normal individuals and
APC-Resistance and Factor V-Leiden: they can range from < 70% to > 150% 26, 27, 28.
Many years ago, the key role of APC to In patients with FV-L mutation, the same
inactivate FVa for preventing thrombosis was variation range is present: therefore,
already described 5, 13. A breakthrough homozygous FV-L patients can have FV-L
occurred in 1993, when Dahlbäck and concentrations from < 70% to > 150%, and
coworkers reported the elevated incidence of heterozygous patients from < 35% to > 75% 9.
familial thrombosis associated with APC There is some overlapping of FV
resistance (APC-R) in Sweden 14. Laboratory concentrations between heterozygous patients
testing for APC-R was performed by with high levels and homozygous patients with
measuring the Activated Partial low levels. Thrombosis risk results from the
Thromboplastin Time (APTT) in presence or APC-resistant FVa concentration, i.e. FV-L,
absence of APC, then calculating the clotting and not from the patient’s genetic status 26.
times (CT) ratios 14, 15. Inherited thrombosis Conventional APC-R clotting assays,
tendency, unprovoked, was associated with a performed with or without APC, are based on
decreased APC-R ratio 15. One year later, clotting time ratios, and allow an approximate
Bertina’s group in Leiden (NL) showed that classification of patients in heterozygous or
this defect is the consequence of a FV mutation homozygous 14, 29. Assay performance and
at position 506, where arginine is replaced by specificity was improved by introducing a
glutamine (noted R506Q), and the mutated dilution of tested plasma in FV deficient
factor was named Factor V Leiden (FV-L) 12. plasma 30. The molecular biology approach
This FV mutation is located on one of the FV gives a similar classification, which is definite,
cleaving sites by APC 6, 16-19. Thrombin and it is fully specific for the R506Q mutation
12, 15
activated FV-L has then an increased activity . The quantitative FV-L assay is expected
survival time in plasmas from patients with this to allow graduating FV-L levels and analyzing
R506Q mutation, and this can propagate their association with thrombotic diseases 26.
activation of coagulation pathways. APC
resistance and FV-L were rapidly identified as Measurement of FV-L and association with
the major hereditary diathesis associated with thrombosis:
an increased thrombotic risk 15, 20-23, although Thrombotic diseases associated with FV-L are
for patients with the R506Q mutation odds frequently reported as unprovoked, as they
ratios for occurrence of thrombosis are lower occur in the absence of any major inducing
than those associated with AT, PC or PS cause 21, 22, 31. In any case, the thrombotic risk
deficiencies 4, 23-26. Since these discoveries, of individuals carrying the FV-L mutation
testing for APC-R with clotting assays and for remains linked to the balance between
the FVa mutation with molecular biology, have coagulation triggers and body’s capacity to
become major parameters of the thrombosis control and regulate hemostasis 2. However,
check-up. Affected patients can be the presence of a low-level trigger, which has
homozygous (100 % of FV is mutated), and are no pathogenic effect on normal individuals,
then at the highest thrombotic risk 20, 25, or can induce thrombosis in patients with
heterozygous (50% of FV is mutated). Control decreased antithrombotic defenses, because an
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 4 of 18
anticoagulant protein is deficient or in presence thrombin and APC; APC was tested for the
of FV-L 2-4, 20, 25. The quantitative absence of trace amounts of FXa.
concentration of FV-L is then a very useful Factor V Deficient plasma was from Affinity
information for graduating the thrombotic risk Biologicals (Ancaster, Canada) and supplied
in affected patients 26. For this objective, we frozen, in bulk: the various coagulation factors
developed a quantitative assay for measuring (excepted FV) are within the normal range.
FV-L clotting activity. We report the assay Cephalin was extracted from rabbits’ brain,
principle, its validation and performances, and supplied by Pelfreez (Rogers, Arkansas, USA)
the FV-L concentrations in normals, and qualified upon receipt. All coagulation
heterozygous or homozygous patients. In few reagents used (Owren Koller or imidazole
cases, in presence of congenital or acquired FV buffer, calcium solution, FV coagulation
deficiencies the FV concentration is reduced. reagent, Zymutest Factor V for FV antigen)
Then, comparison of FV-L levels with the were from HYPHEN BioMed (Neuville sur
conventional FV clotting activity or the FV Oise, France).
antigenic content is necessary for confirming Coagulation instruments used were semi-
the patient status. automated (ST4 from Stago, Asnières, France;
KC 10 from Amelung, Lemgo, Germany;
Development of a quantitative assay for FV- MC10 from Merlin Medical, Lemgo,
L: Germany), or automated (STA-R from Stago,
Material and methods: Asnières, France; CS-2100i or CS-5100 from
Plasma samples: normal human citrated Sysmex, Kobe, Japan; ACL-Top from IL-
plasmas were obtained from healthy blood Werfen, Le Pré Saint Gervais, France).
donors collected at Etablissement Français du Quantitative clotting assay for FV-L: the assay
Sang (EFS), Pontoise, France, or from principle is depicted on figure 1, and performed
Precision Biologics Inc. (PBI), Halifax, at 37°C. In a first step, tested plasma or
Canada; citrated plasma pouches or apheresis calibrator (100 µL), diluted 1:20 in Owren
plasma pouches were obtained either from EFS Koller or imidazole buffer, is added to a
Normandie, Rouen, France, or from Biomex, clotting mixture (100 µL) containing human
Heidelberg, Germany; citrated plasmas from APC (about 15 µg/ml), purified Prothrombin,
patients carrying homozygous or heterozygous Fibrinogen, and purified PS, all at a constant
FV-L mutation, duly characterized with concentration close to that in normal human
molecular biology, were kindly provided by Dr plasma and in excess, and a heparin
Leroy-Matheron from Henri Mondor Hospital, neutralizing substance (Polybren). Then, a
Créteil, France, as part of a collaborative study purified mixture (50 µL) of human factor Xa
(some plasmas were from patients and phospholipids is added, and coagulation is
anticoagulated with Vitamin K antagonists); triggered with 100 µL of 0.025 M calcium
alternatively, they were selected from the chloride. The clotting time is recorded. A
plasma pouches supplied by EFS or Biomex, calibration curve is obtained by mixing
and screened for the presence of FV-L with the plasmas from patients with the FV-L mutation
FV-L clotting assay. and normal plasma, all with a known FV
Purified proteins and enzymes: highly purified concentration. In heterozygous patients, 50%
human fibrinogen, prothrombin (FII), factor X of total FV is FV-L, and all is FV-L (i.e. 100%)
(FX), PS, factor Xa (FXa), and APC were all in homozygous. Measured clotting time (CT,
from Hyphen BioMed (Neuville sur Oise, sec) is inversely proportional to the FV-L
France); factor X (FX) was tested for the concentration, whilst normal factor V is not
absence of FXa, and of trace amounts of measured (it is fully inactivated by APC-PS
complex). This assay is available as
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 5 of 18 HEMOCLOT™ Quanti-VL (HYPHEN for establishing the ratio. In addition, the result BioMed). A single clotting test is required, is quantitative, whilst it is only qualitative for conversely to the APC-R method, which APC-R assay. involves 2 clotting tests, with or without APC, Figure1: (A) Assay principle for the FV-L quantitative clotting assay, performed in presence of a constant concentration of Activated Protein C (APC). Normal FV is inactivated by APC in presence of PS, phospholipids, and Calcium, whilst FV-L is resistant to inactivation. There is an inverse relationship between the clotting time (CT) measured and the FV-L concentration. CT shortens when FV-L concentration increases. The dose-response curve obtained between CT and FV-L concentration is shown in (B). The linearity range is presented in (C). (A) (B) (C) Other laboratory materials used: Plasma (Potters Bar, UK), COATEST™ APC™ WHO coagulation factors’ International Resistance V was used on ACL TOP® Standard and SSC/ISTH Secondary instrument (both from IL-Werfen, Le Pré Saint Coagulation Standard were from NIBSC Gervais, France), as per manufacturer’s Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra
Amiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 6 of 18
recommendations: for the lot and the inverse relationship between the concentration
instrument application used, the normal range of FV-L and the corresponding clotting time is
cut-off value was for a ratio > 2.39. obtained (Figure 1B).
Interferences: substances used for testing assay Calibration is performed by mixing a pool of
interferences were spiked in citrated plasmas, plasmas from heterozygous FV-L patients
comparatively to a control sample added with (which are available in a rather abundant way,
the same volume of the corresponding dilution whilst homozygous patients are rare) with a
buffer. These products were: WHO normal plasma. This pool of heterozygous FV-
International Standards from NIBSC (Potters L patients (> 15 individual plasmas) has a FV-
Bar, UK) for Unfractionated Heparin (UFH L concentration corresponding to 50% of the
07/328) and Low Molecular Weight Heparin total FV clotting activity, which can be exactly
(LMWH 11/176); Rivaroxaban powder from measured by reference to the international
Bayer Pharma (Germany); Apixaban powder WHO International Standard for plasma FV.
from BMS (Princeton, NJ, USA); and When this pool is used diluted 1:20, its FV-L
Dabigatran powder (active form) from concentration is close to 50%, and it is exactly
Boehringer Ingelheim (Ingelheim, Germany). measured. The 1:10 dilution of this pool allows
Hemoglobin and bilirubin were from Sysmex obtaining a concentration twofold higher, close
(Japan), and Intralipids were from Sigma (St to 100 % of FV-L, and exactly defined. The
Louis, Mo, USA). other FV-L concentrations used for calibration
are obtained by diluting the FV-L plasma pool
Results: with a normal plasma, then 1:20 in the assay
Quantitative FV-L assay design: dilution buffer. In practice, a FV-L
The quantitative FV-L assay is performed at concentration close to 25% (2 volumes normal
37°C by incubating first 100 µL of the 1:20 plasma and 2 volumes FV-L plasma pool) and
diluted tested plasma (in imidazole, or Owren another one close to 10 % (4 volumes normal
Koller’s dilution buffer, or physiological plasma and 1 volume FV-L plasma pool) are
saline) first with 100 µL of a mixture of prepared. The 0% FV-L concentration is
fibrinogen, FII, PS, APC, and polybren (to obtained with a normal citrated plasma pool.
neutralize heparin), for exactly 1 minute at The standard calibration curve generated on the
37°C; then 50 µL of a FXa-phospholipids CS-5100 instrument is presented on figure 1B.
solution are added, and incubated for another The clotting time is measured for the tested
minute exactly at 37°C, and finally, 100 µL of plasma, and the FV-L concentration is directly
0.025 M CaCl2 are introduced and clotting deduced from the calibration curve, or directly
time is recorded. Fibrinogen, FII, and PS are at calculated by the instrument. In normals, FV-L
a constant concentration and in excess, close to concentration is always expected < 5% and it is
that present in normal human plasma, and APC frequently undetectable. The basic clotting
is used at about 15 µg/ml. FXa is adjusted to time for normal plasmas is always higher than
produce a clotting time of about 30 seconds for that corresponding to a FV-L concentration of
100% FV-L. The assay can be performed on 5% and it can be variable from plasma to
any laboratory instrument, semi-automated or plasma depending on the specimen
automated. We used successfully that method characteristics and possible presence of
with KC 10, MC 10, ST4, STA-R, and CS- activated factors. As FV-L is absent in normal
5100 instruments. Clotting times obtained are plasmas clotting time can be very long and
dependent on the clot detection mode of the correspond to unmeasurable FV-L
instrument, optical or mechanical, and they can concentrations. Therefore, to keep a sufficient
present some variations depending on the margin, a cut-off value of FV-L < 10 % in
instrument type used. A Log-Lin linear and normals is defined, although in our experience
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 7 of 18 all normals have a FV-L < 5%, and assay is concentration. This ratio is always < 0.10 for often unclottable. In practice, using automated normal individuals (and usually < 0.05), as no instruments, the setting needs to be adjusted to FV-L is present; it is of about 0.50 for 5%, as below this cut-off value it is heterozygous patients (50 % of FV is in the FV- unnecessary to record, all samples being L form) and of about 1.00 for homozygous without FV-L. With the assay conditions patients (all FV is in the FV-L form). reported, normal FV is totally inhibited for Comparative studies of FV-L, FV clotting concentrations > 200 %, and there is then no activity and FV-Antigen are presented later in impact of samples with a high factor V this article. concentration. Assay linearity: Assay interferences: The assay linearity was checked by serially These interferences were tested by spiking diluting plasmas from homozygous patients concentrated solutions of UFH, LMWH, with a high FV-L concentration with a normal rivaroxaban, apixaban, dabigatran, bilirubin, plasma pool, as shown on figure 1C. The hemoglobin, or intra-lipids in normal or FV-L measuring range obtained is then from < 5% to patients, and by testing plasmas with LA, > 100 % FV-L. No matrix effect is noted coagulation factor deficiencies or from vitamin whether the assayed plasma is diluted in assay K antagonist treated patients. No interferences dilution buffer or in 1:20 diluted normal were observed for UFH or LMWH up to 2 plasma. IU/mL in assayed samples, or for Dabigatran FV-L distribution: up to 200 ng/mL. Rivaroxaban and apixaban The incidence of the R506Q mutation is highly (but also other anti-FXa DOACs, not tested in linked to the ethnic group tested. It is mainly that study) showed an interference from distributed in Caucasians with a prevalence > concentrations from 50 ng/mL and above and 12% in Sweden, in Greece, and some North- can generate underestimated FV-L Africa or Middle East countries, but < 5% in concentrations in tested patients. This is Mediterranean European countries, and with expected as the clotting reaction is triggered by intermediary values in other European FXa in presence of phospholipids and calcium. countries, as referenced in discussion. This No interference was noted when testing a few mutation is absent in Africans, native patients with Anti-Phospholipid-Antibodies Americans, and Asians. In America, its (APA and LA: 5 with normal FV and 2 with distribution is closely linked to immigration FV-L mutation) or coagulation factor history. As an example, we tested 557 deficiencies, nor in VKA treated patients, individual plasma pouches from EFS without or with the FV-L mutation. No Normandy (France) and found 13 donors with interference of hemoglobin (up to 1,000 FV-L concentrations corresponding to a mg/dL), bilirubin (up to 60 mg/dL) or intra- heterozygous mutation (i.e. an occurrence of lipids (up to 1,000 mg/dL) was detected. 2.3 %), and 324 pouches from Biomex Limiting factor: (Heidelberg, Germany) and found 16 donors The assay limiting factor concerns the very rare with a FV-L concentration in line with a cases of patients carrying the R506Q mutation heterozygous mutation (i.e. an occurrence of and with an acquired or congenital FV 4.9%). This does not represent however deficiency. If FV concentration is low (≤ 10%), population statistics, because carriers of the tested FV-L patients can erroneously be R506Q mutation are usually known and this classed as normals. This pitfall can be can impact their participation to blood overcome by measuring FV clotting activity donation; plasma pouches from APC-R and calculating the ratio between FV-L and patients can also be selected by blood banks for FV: Clotting activity, or FV-L and FV: Antigen specific uses. FV-L concentrations ranged Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra
Amiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 8 of 18
from 24 to 71% for EFS Normandy and from ranges between heterozygotes with high
22 to 53 % for Biomex. These concentrations FV/FV-L and homozygotes with low FV-L.
are only indicative, as they were measured on Precision:
apheresis plasma pouches, frozen for a variable Intra-assay, and inter-assays reproducibility (5-
time and thawed just before use. Plasma days, 2 runs per day, 3 repetitions per run)
preparation, storage conditions and duration evaluated on CS-5100 yielded coefficients of
can impact the FV-L concentration measured, variation (CV%) below 3% as reported on
as some loss of FV activity can occur. Table 1. The FV-L measurement with that
Nevertheless, this shows that FV-L plasma quantitative assay is accurate and reliable.
distribution is wide, with some overlapping of
Table 1: Intra- and inter-assay reproducibility obtained for the FV-L quantitative clotting assay on
the CS-5100 automated coagulation instrument, with 2 plasmas at different FV-L concentrations.
Intra assay Inter assays
Plasma Mean CV% Mean CV%
n n
% %
Level 1 40 10.8 2.9 30 11.1 2.8
Level 2 40 43.1 2.0 30 44.7 2.4
Measurements on patients with heterozygous Comparison with the APC-R ratio and
or homozygous FV-L mutations: molecular biology:
Plasma samples from heterozygous patients Comparison of FV-L concentrations with the
(n=61, including 20 patients treated with VKA) APC-R ratio (COATEST™ APC™ Resistance
had a FV-L concentration ranging from 24 to V) on 53 normal citrated plasmas (from
76 % with a mean FV-L concentration of 49 % Precision Biologics, or from EFS) and 63
when untreated, and of 53 % when VKA plasmas from patients with APC-R, yielded a
treated; this concentration ranged from 73 to 100% agreement (Table 2). In this comparison
118 % in FV-L homozygous patients, with a study, the 53 normal plasmas from Precision
mean value of 92%. There is a clear Biologics were confirmed as normals (FV-L
discrimination of patients carrying the R506Q was 2.90, for a reported cut-off
2. However, there is a slight overlapping of ratio of 2.39). The 63 APC-R plasmas were
FV-L concentrations between the heterozygous identified as positive for FV-L, and all
group with high FV concentration and the measured with a FV-L concentration > 25%,
homozygous one with low FV concentration and up to 64% with HEMOCLOT™ Quanti V-
L kit, whilst all had an APC-R ratio < 2.30 with
the Coatest® APC-R kit (Table 2).
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 9 of 18
Figure 2: FV-L concentrations in normals and various groups of patients (untreated or VKA treated
heterozygous, or homozygous) carrying the R506Q (FV-L) mutation.
125
100
% Factor V-L
75
50
25
0
Normals Heterozygous Heterozygous Homozygous
Untreated VKA treatment
Table 2: Comparison study of HEMOCLOT™ Quanti V-L on Sysmex CS-5100 versus
COATEST™ APC™ Resistance V on ACL TOP® (qualitative assay, based on the APTT assay
performed with or without APC and calculating the CT ratio). Study was performed on 116 citrated
plasmas tested with the COATEST™ APC™ Resistance V qualitative method (cut-off for normal
range for the lot and application used: 2.39) or the quantitative Hemoclot™ Quanti VL assay. There
is a full consistence between both assays.
TM
HEMOCLOT Quanti.
V-L
(CS-5100)
FV-L + Normal Total
TM
COATEST FV-L + 63 0 63
TM
APC Resistance
Normal 0 53 53
V
(ACL TOP) Total 63 53 116
We also tested 21 citrated healthy normal a current VKA therapy, and 18 homozygous).
plasmas (French Blood Bank, EFS), and 79 They were all duly characterized with
citrated plasmas from patients carrying the FV- molecular biology. FV-L was measured with
L, R506Q mutation, from Créteil Hospital (of HEMOCLOT™ Quanti VL, on the Stago STA-
which 61 were heterozygous, including 20 with R system. Normals were all measured with a
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 10 of 18
FV-L < 5%. An excellent consistency of FV-L EFS, with a FV-L < 5% were further analyzed
concentrations with molecular biology status for Factor V clotting activity and FV: Antigen
was obtained. In patients, the FV-L (Figures 3 and 4). FV-L concentration for
concentration ranged respectively from 24 to normal plasmas is usually expected < 5%. The
76% (mean value of 50.3%) for heterozygotes, expected FV-L concentration in plasmas from
and from 73 to 118% (mean value of 92.0%) patients with the R506Q mutation is for
for homozygotes (Figure 3). A clear heterozygotes usually between 25 and 75%,
discrimination was thus obtained between and for homozygotes > 75%.
plasmas from patients carrying the R506Q FV-L to FV: clotting activity or to FV: Antigen
mutation and normals. Furthermore, the ratios were between 0.4 and 1.0 for
discrimination was globally acceptable (but not heterozygous plasma samples, and > 1.0 for
perfect) between heterozygotes and homozygous samples (Table 3 and Figure 4),
homozygotes when using directly the FV-L including those with less than 75% FV-L
concentration. In presence of doubts, concentration and a similar Factor V clotting
establishing the FV-L/FV activity or FV-L/FV: activity presented in Figure 3. These ratios are
Antigen ratios can help in confirming the expected to be of about 0.5 for heterozygotes
classification as explained below. and 1.00 for homozygotes. The higher ratios
obtained for some patients are due to the use of
Comparison of FV-L with FV: clotting activity plasma samples stored frozen for a long time,
and FV: Antigen: and for which some loss of FV activity
occurred, whilst FV-L is more resistant. These
Seventy-three plasma samples from patients FV-L/FV: Antigen ratios are closer to the
with the FV-L mutation (38 heterozygous, expected ones, as FV: Antigen is better
untreated, 19 heterozygous, with VKA therapy, preserved during storage.
16 homozygous) and 7 normal samples from
Figure 3: Comparison of FV-L concentration with FV Clotting Activity (left panel) or with FV
Antigen concentration (right panel) for normals, FV-L heterozygous patients untreated, or VKA
treated, or homozygous patients.
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 11 of 18
Table 3: Mean values and ranges of FV-L (%), FV:Clotting (%), FV:Antigen (%), and their
respective ratios measured on citrated plasmas form normal individuals, heterozygous untreated or
VKA treated and homozygous patients carrying the R506Q mutation.
Assays and measures
FV-L/FV: Clotting FV-L/FV: Antigen
Samples FV-L (%) FV: Clotting (%) FV: Antigen (%) ratio ratio
Normals (n=7)Amiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 12 of 18
phospholipids. In addition, APC-PS can cleave the measurement of other anticoagulant
FV to other sites, at amino acids 306 and 609 factors, such as Antithrombin, PC, FPS, and
for a complete inhibition of FVa. However, in eventually Plasminogen 3, 4. Whilst AT, PC,
presence of the R506Q mutation, APC-PS FPS deficiencies contribute to about 15% of
cannot cleave FV at this position 506. Cleavage total unprovoked thrombosis, especially in
of the other FV sites is then delayed, and this younger patients, APC-R and the FV-L
induces the long-term survival of FVa-L in mutation are involved in about 60 % of these
blood circulation. In vitro investigations have events 24. Introduction of the APC-R clotting
shown that in defined conditions normal FVa is assay, performed with or without APC, or its
fully inhibited after 5 min incubation with subsequent improved versions, using a dilution
APC-PS-Phospholipids and Ca++, but 50% to in FV deficient plasma, have contributed to
70% FVa-L is still present after 10 minutes characterize most of the concerned patients,
incubation, and still > 10% after 1 hour 16. FVa- and to investigate affected families 14, 30. With
L is then inactivated with a 10-fold lower rate the discovery of the R506Q mutation, as the
than normal FVa 16, 17. cause for APC-R, molecular biology tools were
The FV-L / R506Q mutation is rather abundant developed, and it became possible to
in Caucasians (about 5%), with 2 types of allele genetically characterize the concerned
distribution: a low one in western and individuals 12. The molecular biology tool for
Mediterranean Europe (< 2.8%); and a high the R506Q mutation appeared to be more
one on a band in North and Central Europe, accurate and reliable, and became rapidly the
from Scandinavia to Greece, with a pocket of gold standard for that diagnosis, even if it was
low allele distribution in Balkans 21, 37-39. This first of limited implementation in laboratories,
mutation is also present in North Africa and and if it required a more complex laboratory
some Middle East countries, such as Lebanon. methodology than performing the combination
In North and South America, its presence of 2 clotting assays (with or without APC), and
reflects the immigration history. In countries calculating the clotting time ratios.
where the R506Q mutation is present, most of Furthermore, the very first APTT based APC-
the unprovoked spontaneous familial R assay had many interferences of
thrombosis are associated with this defect. anticoagulant therapies, presence of
There is an odds ratio of 6 to 7 for occurrence coagulation inhibitors, coagulation factor
of thrombosis in heterozygous patients, and deficiencies or LA.
this odds ratio is up to 80 for homozygous 20. A variant method was introduced later and is
Thrombotic disease is often venous, but it can based on the use of activators extracted from
also be arterial 22-24. Following a first snake venom for activating FX and FV. This
thromboembolic event in individuals carrying assay also uses the ratio of clotting times
the FV-L mutation, the recurrence risk is high, performed with or without APC 40. Specificity
and an anticoagulant prophylactic therapy is and accuracy of APC-R assays were improved
usually instaured. In the absence of thrombosis, by testing patient’s plasma diluted in FV
the need for a preventive anticoagulation is deficient plasma 30. In current practice, the
discussed 25. APC-R clotting assays allow classifying tested
APC-R, FV-L and risk for thrombosis: individuals as normals, heterozygous or
Diagnosis of individuals carrying the R506Q homozygous, depending on the ratio obtained
29, 41
mutation is then very important and useful for . With the former versions of APC-R
understanding the diathesis favoring the clotting assays, normals had a ratio > 2.00,
development of thrombotic diseases. It is yet of heterozygous between 1.50 and 2.00, and
higher usefulness for investigating patients homozygous < 1.50, although the cut-off
with unprovoked thrombotic events, along with values need to be adjusted and validated in
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 13 of 18 each laboratory. In addition, there is some 1:20 diluted tested sample is mixed with a overlapping between ratios obtained for clotting factors’ mixture including human homozygous and heterozygous. Furthermore, fibrinogen, human FII, human PS, and human some heterozygous patients can generate ratios APC, all extracted from a pool of human close to the cut-off value of the normal range, citrated plasma pouches, and highly purified. or slightly above, i.e. 2.00 in the example cited. Of importance is the absence of activated Normals can also have ratios close to this cut- factors in these protein preparations. off value or slightly below. There is then a Concentrations are constant and in excess and significant number of unconclusive assays. correspond to those present in a normal plasma These variations are explained by the pool, excepted for APC, which is used at the characteristics of the tested plasmas or their high concentration of 15 µg/ml, which allows eventual activation grade, occurring during a fast and complete inhibition of normal FV. plasma preparation and storage. The rate of Clotting is initiated by a mixture of FXa and inaccurate diagnosis is then not negligeable. phospholipids, then Ca++. In these conditions, Using the Coatest® APC-R kit with the ACL- all normal FV, including plasma Top instrument, the cut-off value for the concentrations up to 200 %, is readily normal range was in our hands of 2.39. FV-L inactivated, whilst FV-L is resistant to that and APC-R assays are well documented in inactivation. The clotting time obtained is then recent reports 29, 41. Patients tested with the a direct relationship of the FV-L concentration qualitative assays, using either molecular in tested sample. This method is accurate, biology or the APC-R method, are classed in 3 reliable, and insensitive to heparin or VKA groups: normal, heterozygous, or homozygous, therapy, coagulation factor deficiencies, or and not according to the actual FVa-L presence of antiphospholipid antibodies and concentration present. The understanding of LA. Direct Oral anticoagulants targeted to mechanisms favoring development of FXa, can impact the assay, by prolonging the thrombotic events in patients with the R506Q clotting time. mutation shows that the risk is directly The assay limitations concern those samples dependent on the FV and FV-L concentration, with a very low FV concentration, as present in and to the amount of FVa-L resistant to the the rare FV congenital or acquired deficiencies APC-PS inactivation, and not on the genetic (severe hepatic diseases). However, there is status 26. FV and FV-L concentrations present only a risk of false diagnosis, if FV is < 20%, important variations in individuals 9, 26, 27. i.e. a FV-L < 10 % if tested patients are Therefore, FVa-L concentrations can present heterozygous. This very rare risk can be high variations among patients heterozygous or controlled by testing the FV clotting activity or homozygous for the R506Q mutation, and the FV: Antigen and comparing them with the these concentrations are expected to be directly FV-L concentration. The ratio obtained is < associated with the thrombotic risk. Measuring 0.05 in normals, of about 0.50 in heterozygous, the real concentration of FV-L can allow to and of about 1.00 in homozygous patients. individualize the risk analysis for Some concern can occur when testing is thromboembolic events, in affected patients. performed on citrated plasma samples, stored Laboratory usefulness of the quantitative FV- frozen for a long time, as this is frequently the L assay: case when performing clinical or research In this article, we describe a quantitative assay, studies. FV is a labile factor, especially if Hemoclot™ Quanti V-L, for the measurement samples have been slightly activated during of FV-L clotting activity. The assay is designed plasma preparation, processing, and storage. with purified factors, which allows an excellent FV activities are then decreased, but in a much standardization of assay conditions. First, the lesser extend for FV: Antigen. This can impact Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra
Amiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 14 of 18
the FV and FV-L concentrations reported, homozygous. We can anticipate that the
especially when testing patients’ libraries thrombotic risk is then very similar for those
progressively constituted over time. When patients in the overlapping zone, whether
performed on fresh samples, or freshly frozen heterozygous or homozygous, as they share a
and thawed plasmas, the method offers its best same level of FV-L.
performances, with clear FV-L to FV: Clotting At this time, we had no opportunity to
Activity or to FV: Antigen ratios < 0.10 (and in investigate the association between FV-L
practice < 0.05) for normals, of about 0.50 for concentrations with the occurrence of
heterozygous and of about 1.00 for thrombosis in affected patients, when adjusted
homozygous. for all the other thrombotic risk factors and
In a first study, we had the opportunity to physiological variables. This study would be of
compare the FV-L concentrations obtained high value, and support the individualization of
with the Hemoclot™ Quanti V-L assay, to the the thrombotic risk associated to the R506Q
APC-R ratios measured with the Coatest® mutation, with the actual concentration of FV-
APC-R method. With the lot and the ACL-Top L. However, a published study totally supports
instrument application used, the cut-off value this concept by using a subrogate endpoint.
between normals and FV-L patients was of Presence of micro-particles (MPs), now
2.39, i.e. normals had a ratio > 2.39, and globally named extracellular vesicles (EVs), is
patients with the R506Q mutation had ratios < closely associated to thromboembolic risk.
2.39. In practice all tested patients carrying the MPs are the consequence and a contributing
R506Q mutation presented a ratio < 2.30 with cause of disease development and evolution.
the APC-R method, and the FV-L The major MPs source in blood circulation is
concentrations measured with Hemoclot™ released from platelets. In an elegant study,
Quanti VL had a mean value of 46.1 % and Lincz and coworkers demonstrated that the
were always > 25%. There was a full amount of FV-L correlated with the history of
consistency between both types of assays, and thrombosis and the concentration of MPs
the 63 patients tested for having FV-L exposing CD41a 26. This analysis supports the
presented all an APC-R ratio below 2.30, concept developed and presented in this article.
whilst all normals tested had a ratio > 2.39,
with FV-L concentrations < 0.05%. Conclusion:
In another study, FV-L clotting activity was This new quantitative assay allows measuring
measured on a library of patients carrying the any mutated FV resistant to the action of the
R506Q mutation, and kindly supplied by Dr APC-PS-Phospholipids-Ca++ complex. In
Leroy-Matheron from Henri Mondor most cases, it concerns FV-L, resulting from
University Hospital in Créteil (France). All the R506Q single mutation. This latter has been
these patients were duly characterized with selected during evolution, for its beneficial
molecular biology, and for being homozygous effects in protecting individuals against
or heterozygous. A subset of patients was bleeding, inflammation, fertility, and
under current VKA therapy. A group of normal pregnancy risks, at a time where blood loss was
plasmas was tested as controls. All patients one of the most frequent life-threatening events
42
presented a FV: Clotting Activity from 60% up . However, a few other FV mutations, which
to 155 %, in line with usually reported FV induce a prolonged survival of FVa, have been
distribution range 27. FV-L ranged from 25% reported 24, 34-36, 43. These mutations remain
up to 75 % in heterozygous, and from 74% up very rare, and we had no opportunity to
to 118% in homozygous patients, with a slight evaluate the behavior of these various mutated
overlapping between the high concentrations FV in that Hemoclot™ Quanti VL assay.
for heterozygous and the low ones for Nevertheless, the method presented in this
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 15 of 18
report, is not fully specific for FV-L itself, but APC-R: activated Protein C resistance
it measures any FVa resistant to APC cleavage APTT: Activated Partial Thromboplastin Time
and inactivation. There is then a high AT: Antithrombin
probability that other FV variants than FV-L Ca++: Calcium
are also detected, when they present a FV CT: clotting times
resistant activity, as it was reported in the DOACs: Direct Oral Anticoagulants
recent ISTH review article on assays available EFS: Etablissement Français du Sang (French
for testing the APC-R 29. As most of patients Blood Bank)
with FV-L mutations are well-documented, the EVs: extracellular vesicles
clotting assays practice is decreasing over time, FII: Prothrombin
and the systematic testing is now discussed 41. FV: Factor V
Molecular biology is then a definite diagnosis FVIIIa: activated Factor VIII
for patients with FV mutations. However, the FVa (FVa-L): activated Factor V (activated
introduction of the quantitative assay, Factor V Leiden).
restimulates the interest for FV-L laboratory FX (FXa): Factor X (activated Factor X)
assays by personalizing the thrombotic risk FPS: Free Protein S
associated with this mutated factor. Clinical FV-L: Factor V Leiden
studies, analyzing the risk between the LA: Lupus Anticoagulant
quantitative amount of FV-L and occurrence of LMWH: Low Molecular Weight Heparin
thrombosis, are required for documenting this MPs: micro-particles
association. NIBSC: National Institute for Biological
Standards and Controls
Acknowledgements: The authors are highly PBI: Precision Biologics Inc.
grateful to Dr C. LEROY-MATHERON, from PC: Protein C
University Hospital Henry MONDOR, at 94- PS: Protein S
CRETEIL (France) for her invaluable support SSC/ISTH: Secondary Coagulation Standard /
in providing plasmas from patients International Society for Thrombosis and
heterozygous or homozygous for the R506Q Hemostasis
mutation (FV-L), along with the molecular TED: Thrombo-Embolic Disease
biology results, which made this study TFPI: Tissue Factor Pathway Inhibitor
possible. TM: Thrombomodulin
UFH: Unfractionated Heparin
Abbreviations: VKA: Vitamin K antagonists
APA: Anti-Phospholipid-Antibodies WHO: World Health Organization
APC: Activated Protein C
Copyright 2020 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mraAmiral Jean, et al. Medical Research Archives vol 9 issue 1. January 2021 Page 16 of 18
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