Keywords
antiphospholipid antibodies - lupus coagulation inhibitor - prothrombin
Introduction
Lupus anticoagulant (LA) indicates the presence of a type of “antiphospholipid antibody”
that is frequently, but not always, associated with thromboembolic events. Only occasionally,
LA is present in an uncommon bleeding disorder, the LA hypoprothrombinemia syndrome
(LA-HPS).[1]
[2] Twenty-eight cases have been described between 1948 and 1994,[3] but the syndrome is rare and its prevalence is uncertain.[4] Here we describe a case of a patient with LA-HPS with an associated lupus cofactor
(LC) phenomenon. In 1959, a lupus patient with LA and hypoprothrombinemia was described
by Loeliger.[5] Interestingly, the mixing studies (patient plasma plus normal plasma) prolonged
instead of shortening the clotting time of patient's plasma. This phenomenon that
increased the inhibitor activity by a normal plasma component was called “lupus cofactor”
(LC).[6]
[7] Loeliger suggested that responsible for the (unknown) “cofactor” could be prothrombin
(PT), while others said that LC was driven by β2‐glycoprotein I (β2‐GPI).[8] In this report, we describe a patient with LA-HPS caused by circulating antibodies
against PT and prove that prothrombin is responsible for the observed LC phenomenon.
Materials and Methods
Coagulation and Immunological Studies
Venous blood was collected in 0.109M sodium citrate 9:1 and double centrifuged at
room temperature. Obtained plasma was stored at –80°C until use. All the coagulation
tests were performed using the appropriate reagents and the ACL TOP instrumentation
(Werfen Group, Milan, Italy). LA was detected according to the International Society
of Thrombosis and Haemostasis (ISTH) guidelines.[9] Diluted Russell viper venom time (dRVVT) and silica clotting time (SCT) were performed
in three steps (screening, mixing, and confirm) and expressed as ratio of coagulation
times of patient's plasma to pooled normal plasma (PNP) for all the procedures. To
diagnose the presence of LA avoiding the LC effect, the confirmatory test described
was also performed using the original plasma diluted 1:1 with PNP.
Solid phase assays for the detection of antiphospholipid (aPL) antibodies anticardiolipin
(aCL), aβ2-GPI, antiprothrombin (aPT), and antiphosphatidylserine/prothrombin (aPS/PT)
antibodies were performed as previously described[10] following the recommendations of a recent communication from Scientific and Standardization
Committee of the ISTH.[11]
Specific Factor Activity
Factor II, factor V, factor VII, and factor X immuno-depleted deficient plasmas (Werfen
Goup, Milan, Italy) were used in combination with prothrombin time reagents to determine
specific factor activity in citrated plasma. To evaluate a possible inhibitory effect
of antibodies present in the plasma on factor II activity, a Bethesda inhibition titration
was performed
Prothrombin Affinity Column
HighTrap 1 mL column (HiTrap NHS-activated HP, GE Healthcare, Uppsala, Sweden) was
washed with 1 mM ice-cold HCl to eliminate the preservative (isopropanol). Eight milligrams
of human prothrombin (Enzyme Research, South Bend, Indiana, United States) in 1 mL
of coupling buffer (0.2M NaHCO3, 0.5M NaCl, pH 8.3) was injected into the column.
After 30 minutes at room temperature, the column was washed six times with coupling
buffer and multiple washing alternating ethanolamine buffer (0.5M ethanolamine, 0.5M
NaCl pH 8.3) and acetate buffer (0.5M sodium acetate, 0.5M NaCl, pH 4.0) to deactivate
any excess group. The column was stored in Tris-buffered saline (20 mM Tris, 150 mM
NaCl, pH 7.4) until use. One milliliter of patient's plasma was poured into the column
and incubated for 1 hour at room temperature. The column was then washed 10 times
with 1 mL of Tris-buffered saline pH 7.4 and bound material eluted with glycine-HCl
buffer (0.1 M glycine, NaCL 0.5M, pH 2.8) and dialyzed against Tris-buffered saline
with pH 7.4.
Immunofixation
Plasma immunofixation was performed using antibodies anti-immunoglobulin G (IgG),
anti-IgA, anti-IgM, anti-kappa (free and bound light chains), and anti-lambda (free
and bound light chains) provided by Sebia (Bagno a Ripoli, Florence, Italy), according
to the instructions of the assay (Hydragel 2 IF-BJ [HR]). Immunofixation on 10x immunoaffinity
purified material was performed with the same technique used for plasma sample.
Coagulation Tests Using Affinity Purified IgMλ or Human Prothrombin
LA activity of affinity purified immunoglobulin M lambda (IgMλ) aPS/PT was evaluated
by dRVVT and SCT. Fifty microliters of PNP were combined with 100 µL of purified material
or Tris-buffered saline with pH 7.4. Following incubation of the mixtures for 30 seconds
at 37°C, 50 µL of dRVVT or SCT reagents were added and time recorded. To check for
the LC effect of prothrombin in dRVVT, 10 µL of a solution of human prothrombin to
get a final concentration of 18, 75, and 150 µg/mL were added to patient's plasma.
Ten microliters of Tris-buffered saline served as control.
Clinical Summary
On May 2018, a 74-year-old male Caucasian patient, who had never bled abnormally,
was admitted to hospital for an episode of severe hematuria. The patient was not on
anticoagulant therapy. In the preceding days, he had a similar episode that spontaneously
resolved. Laboratory tests for liver and renal function as well as blood cell count
were normal. Remaining blood examinations were unremarkable except for a prolonged
prothrombin time and activated partial thromboplastin time (aPTT). Protein electrophoresis
and immune-fixation revealed a monoclonal component IgMλ. The patient was affected
by Double-Hit large B Cell Lymphoma stage IVB, a condition resistant to chemotherapy
and with poor prognosis. An abdominal computed tomography scan revealed a metastatic
invasion of the liver and right kidney. Large para-aortic and mediastinal lymph nodes
as well as free fluid in the abdomen were evident. Diagnosis was confirmed by liver
biopsy. His performance status was ECOG (Eastern Cooperative Oncology Group), that
is, capable of only limited self-care, confined to bed 50% or more of waking hours.
After a cycle of chemotherapy, the patients presented an acute renal failure and underwent
five hemodialysis sessions, while there was a progressive clinical deterioration in
the patient's condition. He died after 2 months from the admission to the hospital.
Results
Diagnosis of LA-HPS
As shown in [Table 1], the increased prothrombin time ratio was partially corrected in mixing studies,
while the high aPTT ratio further increased. Testing for LA was remarkable as both
dRVVT and SCT ratio further increased in mixing studies (LC). When patient's plasma
diluted 1:1 (thus providing the cofactor present in normal plasma) was initially used
as an original plasma the ratio decreased in mixing studies (no more LC phenomenon).
Confirming tests proved the diagnosis of LA (significant reduction in coagulation
times). Thrombin time was normal. Testing for single coagulation factors showed normal
factor V (90%), VII (70%), and X (75%), while prothrombin level was 4%. A low inhibitory
effect on factor II was found: 0.8 Bethesda U/mL.
Table 1
Patient's coagulation and immunological studies at hospital admission
|
Coagulation studies[a]
|
|
Test
|
Normal values
|
Ratio
|
Mixing
|
Confirm
|
|
Prothrombin time
|
<1.2
|
2.34
|
1.56
|
–
|
|
aPTT
|
<1.16
|
1.50
|
2.06
|
–
|
|
dRVVT
|
<1.2
|
2.94
|
5.23
|
2.64
|
|
SCT
|
<1.2
|
2.30
|
3.00
|
2.25
|
|
Modified dRVVT[b]
|
<1.2
|
4.34
|
3.92
|
1.77
|
|
Modified SCT[b]
|
<1.2
|
2.60
|
2.45
|
1.54
|
|
Immunological studies
|
|
Test
|
IgG/IgM
Normal values
|
IgG
|
IgM
|
|
aCL (GPL/MPL units/mL)
|
<10/< 8
|
13
|
94
|
|
aβ2-GPI (U/mL)
|
<13/< 7
|
6
|
47
|
|
aPT (U/mL)
|
<14/< 7
|
6
|
56
|
|
aPS/PT (U/mL)
|
<30
|
117
|
14,400
|
Abbreviations: aβ2-GPI, anti-β2-glycoprotein I antibodies; aCL, anticardiolipin antibodies;
aPTT, activated partial thromboplastin time; aPS/PT, antiphosphatidylserine/prothrombin
antibodies; dRVVT, diluted Russell viper venom time; IgG, immunoglobulin G; SCT, silica
clotting time.
a Ratio is obtained dividing patient's coagulation time (PT) in seconds by that of
pooled normal plasma (PNP); Mixing is the coagulation time of the 1:1 divided by that
of PNP; Confirm is the ratio obtained between patient to PNP coagulation times performed
with high aPL concentration.
b Before testing, patient's plasma was diluted 1:1 with normal plasma to avoid the
Lupus Cofactor phenomenon and false negative confirming test.
LA, hypoprothrombinemia, and bleeding led to the recognition of LA-HPS.
Solid phase assays for aPL antibodies showed moderate-to-high titer of IgM aCL, IgM
aβ2-GPI, and IgM aPT. Surprisingly, patient's plasma contained high titer of IgG and
extremely high titer of IgM aPS/PT (14400 U/mL). The value of 14,400 U/mL in aPS/PT
enzyme-linked immunosorbent assay (ELISA) was obtained diluting patient's plasma to
an extent (1:10,000) to allow optical density to fall into the reference curve.
Immuno-Affinity Purification of Antiprothrombin Antibodies
As shown in [Fig. 1], the material recovered from prothrombin affinity column was a monoclonal IgMλ (34
µg/mL) that showed a marked positivity in IgM aPS/PT ELISA (440 U/mL) and negligible
positivity in IgM antiprothrombin ELISA (14 U/mL). Spiking PNP with the eluate containing
IgMλ anti PS/PT resulted in marked LA activity in both dRVVT and SCT (12.2 seconds
prolongation and 25.5 seconds prolongation compared with buffer, respectively).
Fig. 1 Immunofixation of patient's plasma showing a monoclonal immunoglobulin M lambda (IgMλ)
(A) and its immunoaffinity purification using a prothrombin affinity column (B, arrowheads). Purified material yielded a marked positivity in anti-phosphatidylserine/prothrombin
enzyme-linked immunosorbent assay (C) and possessed lupus anticoagulant activity as shown by diluted Russell viper venom
time (dRVVT) and silica clotting time (SCT) (D).
Prothrombin and the Lupus Cofactor Phenomenon
To ascertain whether prothrombin was responsible for the LC phenomenon, 10µL of human
prothrombin (18, 75, and 150 µg/mL final concentration) was added to patient plasma
and results are shown in [Fig. 2]. A marked prolongation of coagulation time in a prothrombin concentration manner
with respect to buffer was observed in dRVVT.
Fig. 2 Diluted Russell viper venom time (dRVVT) of patient's plasma without addition of
human prothrombin (buffer) and after addition of human prothrombin in increasing amounts
(18, 75, 150 µg/mL final concentration in patient's plasma).
Discussion
LA-HPS is a rare acquired disorder caused by prothrombin antibodies. The disease is
most common in children and more prevalent in women. Systemic lupus erythematosus
and viral infections are the most frequent associated conditions. A recent review
of the literature[12] reported that in only 3 of 87 cases the disease was associated with lymphoma. The
suspicion of LA-HPS derives from the coexistence of bleeding events with a prolonged
aPTT and prothrombin time in combination with a LA. The diagnosis is confirmed by
the finding of a reduced prothrombin level. The patient described here showed a strong
LA and the LC phenomenon and a huge amount of IgM aPS/PT antibodies. The presence
of IgM aCL and IgM aβ2-GPI in the patient's aPL profile might be interpreted as nonspecific
binding (false positive results) due to the large amount of circulating IgMλ aPS/PT.
We found that anti-PS/PT antibodies were monoclonal IgMλ with LA activity. There was
a large discrepancy between the presence of aPS/PT antibodies and those directed toward
plain prothrombin (aPT). Indeed, whereas several LA could bind to soluble human prothrombin,[13]
[14]
[15] adding PS/phosphatidylcholine vesicles enhanced the extent of the binding.[15] LA antibodies reacting with prothrombin have been postulated to recognize an epitope
that becomes exposed only after Ca2+-mediated binding of prothrombin to phospholipids.[14]
[16] How we could get the IgMλ anti-PS/PT from a column containing immobilized prothrombin
is difficult to comment. One simple explanation might be that the coupling of ligand
(prothrombin) through primary amines determines a conformational change of the protein
similar to that induced by PS and calcium ions. Another explanation is that the abundant
amount of prothrombin (8 mg) used for coupling might increase the local concentration
of the antigen without necessarily inducing a radical structural reengagement rearrangement
of the protein but increasing the affinity for aPS/PT antibodies. Antiprothrombin
antibodies are better identified by Kaolin clotting time, while dRVVT is preferentially
sensitive to aβ2-GPI antibodies.[17] In the described patient, both the dRVVT and SCT were equally prolonged suggesting
the presence of LA. The confirmatory test showed a modest shortening of coagulation
times (dRVVT ratio from 2.90 to 2.64 and SCT ratio from 2.30 to 2.25), a fact that
challenged the presence of a LA. Reporting a negative LA without performing mixing
tests may thus be misleading[18] and the mixing step is particularly important when a baseline factor deficiency
is also present. In fact, upon mixing patient with PNP 1:1, dRVVT and SCT ratios markedly
increased showing the presence of a LC. When providing the cofactor by mean of normal
plasma (patient plasma mixed 1:1 with normal plasma) in the confirmatory test, a clear
reduction in dRVVT and SCT ratios was diagnostic for LA (see the last two rows of
[Table 1]). Most likely, the confirmatory test did not work in the original plasma because
the very low prothrombin amount limited the rate of reaction despite the increase
in phospholipid surface. Prothrombin is a target of aPL antibodies as aPT antibodies
are detected in ∼50 to 90% of the patients.[19] On the other hand, aPS/PT antibodies are extremely frequent in patients with LA.[10]
The patient suffered from spontaneous bleeding because of very low prothrombin level
(4%) and this scenario defines a condition named LA-HPS. Although an association between
LA activity and acquired hypoprothrombinemia has been described many years earlier,[1]
[5] it was not until the early 1980s that the plasma of a patient with the acquired
LA-HPS was shown to contain non-neutralizing antiprothrombin antibodies capable of
binding to prothrombin in solution thus resulting in rapid clearance of the prothrombin–antibody
complex.[20] The very low inhibitory activity found on factor II underlies the presence of mainly
non-neutralizing antibodies. Treatment with corticosteroids rapidly increased prothrombin
level probably by interrupting the clearance of antigen–antibody complexes.[13]
In this study, we have clearly shown for the first time, to the best of our knowledge,
that prothrombin is responsible for the observed LC phenomenon. Thus, the insufficient
amount of protein impedes the full expression of antibodies inhibitory effect on PL-dependent
coagulation tests. Originally, the phenomenon was thought to be due to a patient's
plasma being deficient in an undefined cofactor that is essential for LA to exert
its anticoagulant effect. The cofactor has been proposed but not proven to be prothrombin
or β2‐GPI.[5]
[8] Although prothrombin is clearly the LC in the patient described here, we cannot
exclude that other cofactors may be responsible for the LC phenomenon. However, the
role of β2‐GPI as a cofactor can be excluded as the concentration of this protein
was normal in the patient's plasma (data not shown). In 1965, Yin and Gaston[6] postulated that the cofactor was a gamma globulin that should be present in correct
proportion for the anticoagulant to exert its maximal activity. Furthermore, on the
basis of chemical and physical properties, a detailed study by Rivard et al[7] concluded that the cofactor was a complement component with a molecular weight of
200 kD. In our patient, we obtained the LC effect by adding prothrombin to patient's
plasma; a minimal amount almost doubled the coagulation time in dRVVT. In conclusion,
although these findings are not generalizable, we have shown that in this patient
LA-HPS may be attributable to antibodies to aPS/PT and the LC phenomenon is caused
by prothrombin.
