Hypercoagulability
and Thrombosis
Maria Domenica Cappellini
Erika Poggiali
University of Milan and
Policlinico Foundation IRCCS
Milan, Italy
Clinical challenges in NTDT
• Iron overload
• Hypercoagulability
Hypercoagulability
Why are we so concerned?
Epidemiology of thromboembolism
in thalassaemia patients
TI
TM
n (%)
n (%)
Zurlo et al., 1989
–
4/159 (2.5)
Michaeli et al., 1992
–
4/100 (4)
3/5 (60)
3/5 (60)
12/74 (16.2)
14/421 (3.3)
Borgna Pignatti et al., 1998
5/52 (9.6)
27/683 (4.0)
*
*
*
Cappellini et al., 2000
24/83 (29)
–
*
*
*
–
8/720 (1.1)
Reference
Aessopos J et al., 1997
Moratelli et al., 1998
Borgna Pignatti et al., 2004
TI = β-thalassaemia intermedia; TM = β-thalassaemia major;
VT = venous thrombosis; PE = pulmonary embolism; AT = arterial thrombosis; N/A = not available.
Type of thrombosis
VT
PE
AT
Stroke
*
*
N/A
*
*
*
N/A
*
N/A
Taher A, et al. Blood Rev 2008;22:283-92.
Taher A, et al. Thromb Haemost. 2006;96:488-91.
Thromboembolic events
• Patients (N = 8,860)
– 6,670 with TM
– 2,190 with TI
– 61 (0.9%) with TM
– 85 (3.9%) with TI
Risk factors for developing thrombosis
in TI were
age (> 20 years)
previous thrombotic event
family history
splenectomy
66
28
Stroke
Type of event
• 146 (1.65%) thrombotic events
48
Venous
9
23
DVT
39
8
PE
12
11
PVT
19
0
STP
TM (n = 61)
8
Others
12
0
20
TI (n = 85)
30
40
60
Thromboembolic events (%)
DVT = deep vein thrombosis; PE = pulmonary embolism;
PVT = portal vein thrombosis; STP = superficial thrombophlebitis.
Taher A, et al. Thromb Haemost. 2006;96:488-91.
80
Can we redefine
thalassaemia as
a hypercoagulable state?
Pathophysiology of Thalassaemia
Extravascular haemolysis + Ineffective erythropoiesis
Release into the peripheral circulation of
damaged RBCs and erythroid precursors
Pulmonary hypertension (PHT)
and thromboembolic events (TEE)
Pathophysiology of thrombosis in NTDT
• Cellular factors
– Platelet activation
– Pathology and alteration in red blood cells
– Endothelial cells and peripheral blood activation
(microparticles)
•
•
•
•
Nitric oxide
Splenectomy
Inherited and acquired coagulation defects
Other factors
Cappellini MD, et al. Ann N Y Acad Sci 2010;1202:231-6.
Peripheral
blood elements
• Expression of endothelial
adhesion molecules and tissue
factor on endothelial cells (ELAM1, ICAM-1, vWF, VCAM-1)
• Formation of microparticles
Platelets
• Increased platelet aggregation
• Increased expression of
activation markers
• Presence of platelet morphologic
abnormalities
Nitric oxide
• Hallmark of haemolysis
•↓ Levels leading to vasoconstriction
Hypercoagulability
Other factors
• Cardiac dysfunction
• Hepatic dysfunction
• Endocrine dysfunction
RBCs
• Formation of reactive oxygen
species
• Expression of negatively
charged phospholipids
• Enhanced cohesiveness and
aggregability
Thrombophilia
• No role for prothrombotic
mutations
• Decreased levels of antithrombin III,
protein C, and protein S
• Anti-phospholipid antibodies
Splenectomy
• High platelet counts and
hyperactivity
• High levels of negatively charged
RBCs
Cappellini MD, et al. Ann N Y Acad Sci 2010;1202:231-6.
Platelet activation
↑ expression of CD62P
(P-selectin)
↑ expression of CD63
Chronic platelet
activation
↓ platelet survival due to
enhanced consumption
↑ platelet aggregation
Winichagoon P, et al. Asian J Trop Med Public Health 1981;12:556–60.
Del Principe D, et al. Br J Hematol 1993;84:111–7.
Ruf A, et al. Br J Hematol 1997;98:51–6.
Eldor A, et al. Am J Hematol 1989;32:94–9.
Pathology and alteration in red blood cells
Globin
oxidation in
erythroid cells
(fastest in αglobin chains)
Reactive
oxygen species
Irondependant
oxidation of
membrane
proteins
Hemichromes
Toxic
nontransferrin
bound iron
Senescence
antigens
(phosphatidylserine)
Heme
disintegrates
Rigid,
deformed
RBCs
aggregate and
are
prematurely
removed
Bind to
membrane
band 3,
ankyrin and
spectrin
Endothelial cells and
peripheral blood activation
↑ELAM-1
↑ICAM-1
Vessel Obstruction
Haemolysis
↑VWF
Tissue Hypoxia
Tissue Death
↑E-selectin
↑VCAM-1
Butthep P, et al. Thromb Hemost 1995;74:1045–9.
Butthep P, et al. Southeast Asian J Trop Med Public Health 1997;28(Suppl. 3):141A–8A.
Hovav T, et al. Br J Hematol 1999;106:178–81.
Peripheral
blood elements
• Expression of endothelial
adhesion molecules and tissue
factor on endothelial cells (ELAM1, ICAM-1, vWF, VCAM-1)
• Formation of microparticles
Platelets
• Increased platelet aggregation
• Increased expression of
activation markers
• Presence of platelet morphologic
abnormalities
Nitric oxide
• Hallmark of haemolysis
•↓ levels leading to vasoconstriction
Hypercoagulability
Other factors
• Cardiac dysfunction
• Hepatic dysfunction
• Endocrine dysfunction
RBCs
• Formation of reactive oxygen
species
• Expression of negatively
charged phospholipids
• Enhanced cohesiveness and
aggregability
Thrombophilia
• No role for prothrombotic
mutations
• Decreased levels of antithrombin III,
protein C, and protein S
• Anti-phospholipid antibodies
Splenectomy
• High platelet counts and
hyperactivity
• High levels of negatively charged
RBCs
Cappellini MD, et al. Ann N Y Acad Sci 2010;1202:231-6.
The epidemiological data
and the clinical experience
OPTIMAL CARE study:
incidence and risk factors for thrombosis
Parameter
Age (years)
< 18
18–35
> 35
Male : Female
Splenectomized
Serum ferritin (µg/L)
< 1,000
1,000–2,500
> 2,500
Complications
Osteoporosis
EMH
Hypogonadism
Cholelithiasis
Thrombosis
Pulmonary hypertension
Abnormal liver function
Leg ulcers
Hypothyroidisim
Heart failure
Diabetes mellitus
Frequency
n (%)
172 (29.5 )
288 (49.3)
124 (21.2)
291 (49.8) : 293 (50.2)
325 (55.7)
376 (64.4)
179 (30.6)
29 (5)
134 (22.9)
124 (21.2)
101 (17.3)
100 (17.1)
82 (14)
64 (11)
57 (9.8)
46 (7.9)
33 (5.7)
25 (4.3)
10 (1.7)
EMH = extramedullary haematopoiesis; CI = confidence interval.
Complication Parameter
RR* 95% CI p value
Thrombosis
Age > 35 years
2.60
1.39-4.87
0.003
Hb ≥ 9 g/dL
0.41
0.23-0.71
0.001
Serum ferritin
≥ 1,000 µg/L
1.86
1.09-3.16
0.023
Splenectomy
6.59 3.09-14.05 < 0.001
Transfusion
0.28
0.16-0.48 < 0.001
*RR indicates adjusted relative risk.
Taher AT, et al. Blood. 2010;115:1886-92.
Frequency of thrombosis increases
with age in NTDT patients
● N = 120 treatment-naive* TI patients
11–20years
years
11-20
10 years
years
<<10
45
21–32 years
years
21-32
*
40.0
40
Frequency (%)
35
*
*
26.7
26.7
25
20.0
20
10
5
* = statistically significant trend
30.0*
33.3
30
15
> 32years
years
>32
16.7
6.7
*
20.0
20.0
*
16.7 16.7
16.7
13.3
13.3
13.3
6.7
3.3
3.3
10.0
6.7
3.3
0
23.3
23.3
10.0
6.7
3.3
0
10.0
13.3
10.0
6.7
3.3
20.0
16.7
13.3
10.0
6.7
3.3
0 0
3.3
0
0
*never received any treatment intervention (splenectomy, transfusion,
iron chelation therapy, or HbF-inducing agents).
PHT = pulmonary hypertension; HF = heart failure; ALF = abnormal liver function; DM = diabetes mellitus.
Taher A, et al. Br J Haematol 2010;150:486-9.
High prevalence of thromboembolic events,
particularly in splenectomized patients
Thrombin-generated (nM)
150
Splenectomized patient with TI
Non-splenectomized patient with TI
Non-splenectomized controls
Splenectomized controls
120
90
60
30
0
0
10
30
60
Time (s)
90
120
150
Thromboembolic events occurred in 24/83 (29%) transfusion-independent patients
with TI who had undergone splenectomy
Cappellini MD, et al. Br J Haematol. 2000;111:467-73.
OPTIMAL CARE study: patient stratification according
to splenectomy and TEE status
•
Three groups of patients identified
– Group I, splenectomized patients with a documented TEE (n = 73)
– Group II, age- and sex-matched splenectomized patients without TEE (n = 73)
– Group III, age- and sex-matched non-splenectomized patients without TEE (n = 73)
Type of thromboembolic event in
splenectomized TI patients (Group I)
n (%)
DVT
46 (63.0)
PE*
13 (17.8)
STP
12 (16.4)
PVT
11 (15.1)
Stroke
4 (5.5)
*All patients who had PE had confirmed DVT.
TEE = thromboembolic events; DVT = deep vein thrombosis; PE = pulmonary
embolism; STP = superficial thrombophlebitis; PVT = portal vein thrombosis.
Taher A, et al. J Thromb Haemost. 2010;8:2152-8.
OPTIMAL CARE study: patient stratification according
to splenectomy and TEE status (cont.)
Parameter
Group I
Splenectomized
with TEE
(n = 73)
Group II
Splenectomized
without TEE
(n = 73)
Group III
Nonsplenectomized
(n = 73)
p value
Mean age ± SD, years
Male : female
Mean Hb ± SD, g/dL
Mean HbF ± SD, %
Mean NRBC count ± SD, x106/L
Mean platelet count ± SD, x109/L
33.1 ± 11.7
33 : 40
9.0 ± 1.3
45.9 ± 28.0
436.5 ± 205.5
712.6 ± 192.5
33.3 ± 11.9
35 : 38
8.8 ± 1.2
54.4 ± 32.8
279.0 ± 105.2
506.3 ± 142.1
33.4 ± 13.1
34 : 39
8.7 ± 1.3
44.2 ± 27.2
239.5 ± 128.7
319.2 ± 122.0
0.991
0.946
0.174
0.429
< 0.001
< 0.001
PHT, n (%)
HF, n (%)
DM, n (%)
Abnormal liver function, n (%)
Family history of TEE
Thrombophilia, n (%)
Malignancy, n (%)
Transfused, n (%)
Anti-platelet or anticoagulant use, n (%)
Hydroxyurea use, n (%)
25 (34.2)
7 (9.6)
4 (5.5)
2 (2.7)
3 (4.7)
3 (4.7)
1 (1.4)
32 (43.8)
1 (1.4)
13 (17.8)
17 (23.3)
5 (6.8)
5 (6.8)
2 (2.7)
1 (1.4)
2 (2.7)
2 (2.7)
48 (65.8)
3 (4.1)
17 (23.3)
3 (4.1)
1 (1.4)
1 (1.4)
3 (4.1)
3 (4.7)
2 (2.7)
0 (0)
54 (74.0)
2 (2.7)
29 (27.4)
< 0.001
0.101
0.256
0.863
0.554
0.863
0.363
0.001
0.598
0.383
Hb = total haemoglobin; HbF = fetal haemoglobin; NRBC = nucleated red blood cell;
PHT = pulmonary hypertension; HF = heart failure; DM = diabetes mellitus.
Taher A, et al. J Thromb Haemost. 2010;8:2152-8.
Time-to-thrombosis (TTT) since splenectomy
• The median TTT following splenectomy was 8 years (range 1–33 years)
Taher A, et al. J Thromb Haemost. 2010;8:2152-8.
Time-to-thrombosis (TTT) since splenectomy
(cont.)
NRBC count
< 300 x 106/L
≥ 300 x 106/L
0.8
0.6
0.4
0.2
0
0
1
5 10 15 20 25 30 35 40
Duration since splenectomy (years)
Transfused
Yes
No
0.8
0.6
0.4
0.2
0
0
5 10 15 20 25 30 35 40
Duration since splenectomy (years)
Cumulative
thrombosis-free survival
1
Cumulative
thrombosis-free survival
Cumulative
thrombosis-free survival
Cumulative
thrombosis-free survival
Time to thrombosis
1
Platelet count
< 500 x 109/L
≥ 500 x 109/L
0.8
0.6
0.4
0.2
0
0
1
5 10 15 20 25 30 35 40
Duration since splenectomy (years)
Pulmonary hypertension
Yes
No
0.8
0.6
0.4
0.2
0
0
5 10 15 20 25 30 35 40
Duration since splenectomy (years)
Taher A, et al. J Thromb Haemost. 2010;8:2152-8.
Clinical recommendations for the
prevention of thromboembolic events
• A guarded approach to splenectomy in β-thalassaemia patients is
recommended unless strongly indicated
• In already-splenectomized NTDT patients, those at high risk of
thrombosis may be identified early by their high NRBC and platelet
counts, evidence of PHT, and transfusion naivety
– attention should also be paid to the aging NTDT patients
• Prospective clinical trials that evaluate the efficacy, safety, and costeffectiveness of transfusions and anti-platelet and anticoagulant
therapy in preventing thromboembolism are necessary
– aspirin for stroke prevention and lifelong anticoagulation treatment in patients
with a history of thrombotic events
Taher AT, et al. Br J Haematol. 2011;152:512-23.
OPTIMAL CARE study:
transfusion therapy reduces the risk of complications
Complication
Parameter
RR
95% CI
p value
EMH
Splenectomy
Transfusion
Hydroxyurea
Age > 35 years
Splenectomy
Transfusion
Hydroxyurea
Iron chelation
Transfusion
Age > 35 years
Hb ≥ 9 g/dL
Serum ferritin ≥ 1,000 µg/L
Splenectomy
Transfusion
Age > 35 years
Female
Splenectomy
Transfusion
Iron chelation
Serum ferritin ≥ 1,000 µg/L
0.44
0.06
0.52
2.59
4.11
0.33
0.42
0.53
0.06
2.60
0.41
1.86
6.59
0.28
2.76
1.96
5.19
0.36
0.30
1.74
0.26–0.73
0.03–0.09
0.30–0.91
1.08–6.19
1.99–8.47
0.18–0.58
0.20–0.90
0.29–0.95
0.02–0.17
1.39–4.87
0.23–0.71
1.09–3.16
3.09–14.05
0.16–0.48
1.56–4.87
1.18–3.25
2.72–9.90
0.21–0.62
0.18–0.51
1.00–3.02
0.001
< 0.001
0.022
0.032
< 0.001
< 0.001
0.025
0.032
< 0.001
0.003
0.001
0.023
< 0.001
< 0.001
< 0.001
0.010
< 0.001
< 0.001
< 0.001
0.049
Pulmonary hypertension
Heart failure
Thrombosis
Cholelithiasis
Abnormal liver function
n = 445 occasionally/regularly transfused patients (N = 584)
Taher AT, et al. Blood. 2010;115:1886-92.
OPTIMAL CARE study:
transfusion therapy reduces the risk of complications
(cont.)
Complication
Parameter
RR
95% CI
p value
Leg ulcers
Age > 35 years
2.09
1.05–4.16
0.036
Splenectomy
3.98
1.68–9.39
0.002
Transfusion
0.39
0.20–0.76
0.006
Hydroxyurea
0.10
0.02–0.43
0.002
• Transfusion
therapy
was
protective
for
thrombosis,
EMH,
PHT,
Hypothyroidism
Splenectomy
6.04
2.03–17.92
0.001
cholelithiasis and legHydroxyurea
ulcers
0.05
0.01–0.45
0.003
Osteoporosis
Age > 35 years
3.51
2.06–5.99
< 0.001
Female
1.97
1.19–3.27
0.009
• Transfusion therapy
was
associated
with
an
increased
risk
of
Splenectomy
4.73
2.72–8.24
< 0.001
overload-related endocrinopathy
Transfusion
3.10
1.64–5.85
< 0.001
Hydroxyurea
0.02
0.01–0.09
< 0.001
Iron chelation
0.40
0.24–0.68
0.001
Hypogonadism
Female
2.98
1.79–4.96
< 0.001
Serum ferritin ≥ 1,000 µg/L
2.63
1.59–4.36
< 0.001
Transfusion
16.13
4.85–52.63
< 0.001
Hydroxyurea
4.32
2.49–7.49
< 0.001
Iron chelation
2.51
1.48–4.26
0.001
HF,
iron
n = 445 occasionally/regularly transfused patients (N = 584).
Only significant associations presented.
Taher AT, et al. Blood. 2010;115:1886-92.
Indications of RBC transfusion in TI
•
•
•
•
Hb < 5 g/dL
Declining Hb level with progressive splenic enlargement (> 3 cm/year)*
Poor growth and/or development
Evidence of
–
–
–
–
–
–
bone deformities
clinically relevant tendency to thrombosis
leg ulcers
EMH
PHT
infections
• Prior to surgical procedures
• Pregnancy
Hb = heamoglobin.
* At least in periods of maximal growth and development.
Taher A. et al. Blood Reviews 26S (2012); S24-S27.
Initiating transfusion therapy
in NTDT patients
• The decision to initiate transfusion in these patients is very
difficult due to the heterogeneity of the disease
• There is no benefit in limiting the quantity or frequency of
transfusions once they have begun
• Starting transfusions after the third year of life has been
shown to increase the risk of alloimmunization
• Transfused patients with TI experience fewer TEE, PHT and
silent brain infarcts compared to transfusion-naïve patients,
due to the correction of the ineffective erythropoiesis and
resulting damaged RBCs with thrombogenic potential
Taher A. et al. Blood Reviews 26S (2012); S24-S27.
Summary
• Thromboembolic events are frequent in β-thalassaemia patients
– oxidative damage to RBCs, impacting their membrane properties, resulting in increased
aggregation and risk of thromboembolism
– risk of thromboembolism increases with age, and is influenced strongly by splenectomy
and transfusion navïety
• Splenectomy is associated with a high risk of thrombosis,
particularly in patients with high NRBC or platelet counts, who are
transfusion-naïve
• Transfusion therapy reduces the risk of thrombosis in NTDT patients
– transfusion iron intake inevitably increases the risk of iron overload, but the
benefit of transfusion therapy may greatly outweigh the cost and inconvenience
of iron chelation therapy
Summary
•
Despite various treatment options are available, no clear
guidelines exist: each patient must be assessed individually
and assigned a personalized thrombotic risk based on intrinsic
and extrinsic factors
•
Several studies are highlighting the roles of transfusion, iron
chelation therapy, and fetal haemoglobin induction
(hydroxycarbamide, HU) in the management of NTDT; thus
these approaches merit large prospective evaluation
• Another approach would be to correct the reactive oxygen
species-induced RBC membrane damage using antioxidants,
although this approach has not yet been verified in clinical
trials
Acknowledgments
I would like to thank you for the attention,
and all the Congenital Anemia Center Staff
for their support
Prof. M.D. Cappellini
Giovanna Graziadei
Irene Motta
Alessia Marcon
Ilaria Gandolfi
Laura Zanaboni
Marianna Giuditta
Elena Cassinerio
Marta Mazzoleni
Claudia Cesaretti
Silvio De Fazio
All the nursing staff
Alteration of the phospholipid “Flip-Flop” mechanism:
RBCs with negatively charged phospholipids
Adherence of RBCs to
endothelial cells is increased
Thrombus
formation
Thrombin generation
Fibrin/
platelets
Phosphatidylserine on
damaged or senescent
RBCs leads to
● Recognition by
phagocytes
● Removal from
circulation
● Apoptosis
Splenectomy favours persistence of these RBCs in the circulation
Courtesy of Dr A. Taher.