1. Hospital based prospective longitudinal
study of 231 patients of Antiphospholipid
Syndrome and exploring the role of AntiAnnexin A5, Plasminogen Activator
Inhibitor-1 (PAI-1) and platelet dysfunction
in its pathogenesis.
Departments of Medicine, Nephrology, Obstetrics and
Gynaecology, Biochemistry and Immuno-pathology Division.
Institute of Medical Sciences, Banaras Hindu University

2. INTRODUCTION
 Antiphospholipid syndrome (APS) was first described by
Graham R. V. Hughes in 1983 as a clinical syndrome
characterized
by
venous
and
arterial
thrombosis, recurrent pregnancy loss, neurological
disease and the presence of antiphospholipid
antibodies (aPLs).
 Primary APS: When this syndrome occur without evidence of
another identifiable autoimmune disorders.
 Secondary APS: When this syndrome occur in association with

3. The diagnosis of APS is confirmed in the presence of at least one clinical
and one laboratory criterion (modified Sapporo criteria, 2006)
Clinical Criteria1. Vascular thrombosis:
one or more venous, arterial, or small vessel thrombotic events in
any tissue or organ confirmed by imaging or histopathology .Thrombosis should be
present without any significant evidence of inflammation in the vessel wall. (superficial
vein thrombosis is not included in clinical criteria).
2. Pregnancy morbidity
 One or more unexplained deaths of a morphologically normal fetus at or beyond the
10th week of gestation.
 One or more premature births of a morphologically normal neonate before the 34th week
of gestation because of eclampsia, severe preeclampsia, or placental insufficiency.
 Three or more consecutive spontaneous abortions before the 10th week of gestation (in
the absence of parental chromosomal causes and maternal anatomic or hormonal
abnormality).
Miyakis S, Lockshin MD et al. 2006, J Thromb Haemo 4, 295-306

4. Laboratory Criteria1.
aCL-Abs (IgG and/or IgM) present in serum or plasma on 2 or more
occasions atleast 12 weeks apart (in medium or high titer, ie, >40 GPL [IgG
phospholipid] or MPL [IgM phospholipid] units or >99th
percentile, measured by a standardized enzyme-linked immunosorbent assay
[ELISA])
2.
LA (lupus anticoagulant) present in plasma on 2 or more occasions at least
12 weeks apart (measured according to the guidelines of the International
Society on Thrombosis and Haemostasis)
3.
Anti-b2GPI antibody (IgG and/or IgM) present in serum or plasma on 2 or
more occasions at least 12 weeks apart (in titer >99th percentile, measured by
a standardized ELISA).
The diagnosis of APS cannot be confirmed if less than 12 weeks (or more than
5 years) separate the time of the positive aPL-Ab test and the time of the clinical
manifestation.

5. Table : Clinical manifestations of APL
Organs/Systems Manifestations
Asymptomatic
15-20% develop clinical events over time
Venous Thrombosis
DVT, Budd-Chiari syndrome, Cortical vein/Sinus Thrombosis
Arterial Thrombosis
TIA & stroke (thrombo-embolism also), PVD, CAD, Renal,
Intestinal
Obstetrics
RSFL- early & late foetal losses, IUGR, Pre-term labor (<34
weeks)
Neurological
Migraine, Chorea, Seizure, Dementia, Cognitive Dysfunction,
Multiple Sclerosis like, Transverse myelitis, GBS, MNM,
Myasthenia Gravis
Cont……

6. Table : Clinical manifestations of APL
Organs/Systems
Manifestations
Cardiac
MI, Libman-Sacks endocarditis, Intra-cardiac thrombi
Pulmonary
Thrombo-embolism, Pulmonary hypertension,
Intra-alveolar hemorrhage, ARDS
Renal
Hypertension
Intestinal
Intestinal ischemia, Acute abdomen
Dermatological
Livedo-reticularis, Digital and cutaneous necrosis,
Gangrene,Pyoderma Gangrenosum
Hematological
Thrombocytopenia, Auto-immune hemolytic anemia
Catastrophic APS
Rapid thrombotic storm affecting multiple organ system

11. Table: Clinical Manifestations of 179 pts of
primary APS
CLINICAL MANIFESTATIONS
A. Venous Thrombosis
No. of Patients
Percentage %
( 33 )
( 18.4)
- Deep vein thrombosis (DVT)
19
10.6
- Cortical vein &/or sinus thrombosis
11
6.1
- Budd-Chiari Syndrome
3
1.8
(24)
(13.4)
- Peripheral arterial thrombosis
12
6.7
-Intracranial arterial thrombosis
9
5
- Visceral arterial thrombosis
3
1.7
(140)
(78.2)
- Recurrent early pregnancy losses
32
17.8
- Late pregnancy losses
97
54.2
- Premature birth/ Still birth
28
15.6
- Preeclampsia/ Eclampsia
13
7.3
B. Arterial Thrombosis
C. Adverse Pregnancy Outcome

12. Continued
CLINICAL MANIFESTATIONS
No. of Patients
Percentage
D. Neurological involvement
- Psychosis
3
1.7
- Seizure
2
1.1
2
1.1
3
1.7
- Thrombocytopenia
75
41.9
- Coombs positive hemolytic anemia
4
2.2
- Digital gangrene
7
3.9
- Livedo reticularis
6
3.3
- Chronic leg ulcer
2
1.1
E. Cardiac involvement
- Myocardial Infarction
F. Respiratory involvement
- Pulmonary Hypertension
G. Hematologic manifestations
H. Skin manifestations

19. Table: Immunological Parameters in 179 pts of
primary APS
Immunological tests
No. of patients
Percentage
- IgG
141
78.8
- IgM
32
17.9
- Both
29
16.2
B. Lupus Anti-coagulant
99
55.3
C. Both aCL and LA
64
35.7
A. Anti-Cardiolipin Ab (aCL) : mod-high

25. Table :Treatment of APL
Clinical
condition
Treatment
Asymptomic
No treatment; short term antticoagulant if required
Venous thrombosis
Long-term anticoagulant (Heparin & warfarin); INR 2 to 3
Arterial thrombosis
L ong-term anticoagulant; INR 2 to 3 vs 3.1 to 4
+ low-dose antiplatelet (controversial)
Pregnancy morbidty
Low-dose anticoagulant (Heparin 10 to 20,000 u/day or LMWH
1u/kg/day) + Low-dose antiplatelet
If having throbotic event : As above
Catestrophic APS
Anticoagulant (INR 3 to4) + corticosterod
IV Ig/ Plasmapheresis
Others (Fibrinolytic/Cyclophosphmide/Prostacyclin etc)

26. Pathogenesis of APS
It appears to be multifactorial and multifaceted and is an area of
intense research in recent time.
1. Endothelial cell activation leading to increased expression of
2.
3.
4.
5.
6.
adhesion molecules and TF.
Displacement of Annexin A5 from endothelial cells.
Platelet activation
Activation of complement system
Inhibition of naturally occurring anticoagulants (Protein C,S
and AT-3)
Impaired fibrinolysis
Exact pathogenesis still remains inconclusive.

28. Annexin A-V weight 36kd)
a glycoprotein (molecular

 Ability to bind to negatively charged phospholipids with high
affinity in a calcium-dependent manner.
 Forms a protective anticoagulant shield on vascular endothelial
cells
 Antiß2- GPI Abs/ ß2-GPI complex disturb the shield and
predispose to placental thrombosis and fetal loss.

31. Baseline data of antiphospholipid syndrome (APS) patients
CLASS
IFICA
TIOn
Mean Sex
+/-S.D. (M/F)
age
(years
)
Thrombosis(n
=48)
Venous Arteri
no.(%) al no.
(%)
Primar
y APS
(n=86
Bad obstetric history
(n=64)
(Early
Pr.loss)
(<10 wk
%)
ACLA
IgG>2
0 GPL
LA no.
(%)
(Late
Others
pr. loss No. %
(>10wk
%)
27 +/4.33
6/80
19
(22.05)
11
13
(12.79) (15.11)
33
(38.37)
7
(10.93)
77
45
(89.53) (52.32)
Second 28.5
ary
+/-6.9
APS
(n=26
3/23
9
(34.61)
9
4
(34.61) (15.38)
5
(19.23)
2
(7.69)
23
14
(88.46) (53.84)
Total
(n=112)
9/103
28
20
(25.00) (17.85)
17
(15.17)
9
(8.03)
100
59
(89.28) (52.67)
27.75
+/-
20
(17.85)

32. Estimation of Anti – annexin A-V
 Antibody levels were measured by quantitative
enzyme-linked immunosorbent assay (ELISA),
using the Zymutest kit

33. Results

36. p<.001
p<.001
p<.001

39. CONCLUSION
 The present study revealed significantly higher
positivity rates and significantly raised levels of aANX
IgG in primary as well as secondary APS patients as
compared to healthy controls (P < 0.001).

However, it did not differ significantly between
healthy controls and patients with SLE, RA, non-APS
thrombosis and non- APS pregnancy complications.

Furthermore, the level of aANX IgG was significantly
higher in subsets of APS patients having bad obstetric
history and/or thrombosis (P < 0.001).

41. Role Of Impaired Fibrinolysis In APS
 Relatively understudied, few recent reports suggest that
deficient fibrinolysis may contribute to hypercoagulable
state in APS.

42. Patients’ Characteristics

43. Estimation of PAI-1Ag
 Estimation of PAI-1Ag was done by quantitative ELISA,
using the Zymutest kit according to the manufacturer’s
instructions (Hyphen Biomed)..

44. Results

47. Statistical correlation of level of mean ± SEM PAI-1 in
healthy controls, SLE, RA, total APS, primary APS and
secondary APS

48. Statistical correlation of PAI-1 Ag (mean ±SEM)
with clinical parameters

49. Conclusion
 Level of PAI-1Ag was higher in APS patients in relation to
control groups.
 Significantly higher proportion of prim. and sec. APS
patients showed positivity in relation to healthy controls
(p=0.006 & 0.001 respectively).
 Mean±SEM levels of PAI-1Ag were significantly higher in APS
patients (p<0.001) but didn’t differ in 3 control groups.

52. Platelet function studies included :
 Freshly prepared 1.4 ml of citratephosphate-
dextrose anticoagulant (CPDA) was added to
blood.
 Platelet rich plasma (PRP) was separated from
the samples and were analysed within 3 hours.
The laboratory personnel were blinded to the
source of samples.

53.  The following platelet function studies were performed:
1. Platelet aggregation studies
2. Studies pertaining to platelet secretion of dense
granules, which comprised of :
• Total degranulation
• Platelet secretion of granules in relation to time
• Visualisation of platelet degranulation and alteration of its
morphology by phase contrast microscopy
3. Clot retraction studies by tube method
4. Western blot studies on clot retracted samples for
demonstration of activated proteomes

54. RESULTS
 A significant increase (P < 0.001) in the platelet aggregation in
APS patients as compared to healthy controls was noted.
 The subjects also showed a significant increase (P < 0.05) in the
platelet granule release as well as more degranulation (P < 0.001)
in relation to time at stored condition, which were well-visualized
under phase-contrast microscope.
 Sixty-five percent of APS patients showed lesser as well as delayed
clot retraction as compared to healthy controls, signifying that the
platelet clots are less retractile in APS patients.

56. Total degranulation of dense granule

58. Microscopic visualization of platelet
degranulation

59. Clot retraction studies

60. Western blot of clot retracted samples of control and
APS patients showing additional bands at 37 kDa of
anti-phosphotyrosine antibodies

61. CONCLUSION
 In-vitro studies on platelets from APS patients, revealed:
1. Significantly increased platelet aggregation.
2. Increased degranulation of platelets.
3. Poor clot retraction and
4. Additional band of 37kDa of anti-phosphotyrosine
antibodies in APS patients by Western Blot study
Thus, platelets seem to play an important role in formation of
occlusive clot in vessels in APS patients.

62. Pathogenic Mechanisms of APS
Endothelial Cells
Activation
Platelet
Activation
Expression of adhesion molecules and TF
Raised pro-inflammatory cytrokines
Antiannexin A5
antibodies
Anti-Phospholipid Syndome
Antiphospholipid antibodies
Impaired
Fibrinolysis
Inhibition of Natural
Anti-coagulants
Activation of
complement
system

63. LEARNING POINTS: Anti Phospholipid Syndrome (APS) is a leading cause of venous and/or arterial
thrombosis as well as adverse pregnancy outcomes such as early (<10 weeks)
and late pregnancy losses, preterm labor, IUGR, still birth and toxemia of
pregnancy.
 High index of clinical suspicion is necessary as clinical presentation varies
widely.
 When clinically suspected, laboratory confirmation should be done by IgG
anticardiolipin antibody (ACLA), Lupus Anticoagulant (LA) and β2GP-1. All 3
tests should be done because in a given patient any one of them may be
positive.
 Pathogenesis of APS appears to be multi-factorial and complex and is an area of
ongoing research and investigation. Studies establishes the role of antiannexinA5, plasminogen activator inhibitor 1 (PAI-1) and hyperactivity of
platelets in the pathogenesis of APS.

64. THANK YOU