1. Pathogenesis of APS
Ritasman Baisya
21.2.20

2. Structure of talk
• Introduction
• Etiology – genetic and environment influence
• APL- pathogenic antibody
• Pathogenesis of vascular APS
• Pathogenesis of obstetric APS
• Pathogenesis based therapeutic targets

3. Introduction
• Antiphospholipid syndrome (APS) is an autoimmune vasculopathy mediated by
autoantibodies directed against phospholipid- binding proteins.
• Two main clinical variants of the syndrome: vascular and obstetric APS
• Although most patients with APS have both vascular and obstetric
manifestations, isolated vascular or obstetric variants exist.
• The clinical spectrum of APS is becoming more complex as additional symptoms are
attributed to the syndrome

5. Schreiber, K., Sciascia, S., de Groot, P. et al. Antiphospholipid syndrome. Nat Rev Dis Primers 4, 17103 (2018).
Clinical manifestations

6. Non criteria manifestations
• Thrombocytopenia , AIHA
• Nephropathy
• Livedo reticularis, skin ulcers, cutaneous necrosis
• cardiac valve disease
• Neurological manifestations such as chorea, epilepsy, cognitive dysfunction
and transverse myelopathy
• Diffuse pulmonary haemorrhage

7. Etiology

8. Autoimmune Highlights (2011) 2:35–52
GENETIC

9. Environmental
• Infections - perhaps to be the most prominent environmental trigger for aPL
production and APS development
• CMV, parvovirus B19, HIV , Hepatitis B and C viruses, HTLV and VZV, tetanus -
reported associations, more in CAPS
• Molecular mimicry – potential mechanism
• Others - selectively activating or destroying unique lymphocyte subsets,
directing cytokine/ chemokine release or exposing cryptic autoantigens during
cell necrosis and/or apoptosis

10. Molecular mimicry in APS

11. 1. Vaccination, drug therapy and certain malignancies ….
2. Chlorpromazine, amoxicillin, phenytoin, chlorothiazide, propranolol, oral
contraceptives, quinine, alpha-interferon and infliximab have been associated
with the presence of aPL .
3. Presence of aPL in solid and haematological malignancy is documented –ether
against tumour antigen or neoantigen formed due to drug therapy
Noninfectious environmental triggers

12. aPL – pathogenic antibody
• aPL are a heterogenous group of antibodies (Abs) that react with a myriad of
phospholipids (PLs), PL-protein complexes & PL-binding proteins
• The origin of these pathogenic autoantibodies still remains a mystery
• Complex interaction of the antibodies with multiple environmental factors and
genetics increase susceptibility to the disease
• aPL mediate several procoagulant mechanisms that can explain their
thrombogenic effect in animal models & epidemiological association with APS
in clinical studies

13. Beta 2 GP1 Ab – the main culprit
• Β2- glycoprotein I (β2GPI)- dependent autoantibodies seem to be the main
pathogenic subpopulation of aPL .
• β2 GPI & prothrombin - accounts for more than 90% of the antibody-binding
activity in APS patients
• The main antigenic target of these antibodies is recognized to be plasminogen
activator (tPA), phosphatidylserine (PS), plasmin, annexin2, activated protein C
(APC), thrombin, antithrombin III (AT-III) and annexin V

14. Β2- glycoprotein I (β2GPI)- structure
• It consists of 326 amino acids
• Arranged in 5 highly homologous complement-control protein domains ( D I-N
terminal to D V- C terminal )
• D I - represents the main antigenic target for autoantibodies
• D5 –highly flexible with hook like configuration , binds to PL cell membrane
• It can adopt two different conformations: a circular inactive conformation in
plasma, maintained by interaction between the first and fifth domain and an
‘activated’ open conformation (Agar et al, 2010)

15. Sorice, M., Misasi, R. Different domains of β2-glycoprotein I play a role in autoimmune pathogenesis. Cell
Mol Immunol (2018).

16. Bill Giannakopoulos, Steven A. Krilis, The Pathogenesis of the Antiphospholipid Syndrome. N Engl J Med 2013;
368:1033-1044
Post translational redox modification

17. Radic M and Pattanaik D (2018) Cellular and Molecular Mechanisms of Anti-Phospholipid
Syndrome. Front. Immunol. 9:969

18. Pathogenesis of vascular APS

19. Vascular thrombosis
• Disruption of fluid phase coagulation –
• Interference with natural anticoagulants , activation of anticoagulation protein
C
• Interference with annexin A5
• Inhibition of fibrinolysis
• Disruption of coagulation cascade cell functions -
• Endothelial cell perturbation , platelet activation , tissue factor expression
• Complement activation

20. Trigger of thrombosis – two hit hypothesis
• first hit - anti-PL antibody induces a thrombophilic state
• second hit – environmental factors (such as infection), inflammatory factors (such
as concomitant connective tissue diseases) or other nonimmunological
procoagulant factors (such as oestrogen containing contraceptives, surgery and
immobility)
• “first hit” injury disrupts the endothelium, and a “second hit” potentiates
thrombus formation

21. Meroni, P.L., Borghi, M.O., Grossi, C. et al. Obstetric and vascular antiphospholipid syndrome: same antibodies
but different diseases?. Nat Rev Rheumatol 14, 433–440 (2018).

23. Second hit related changes by APL
1. Upregulate tissue factor, adhesion molecule expression on endothelial cells
and blood monocytes
2. Cytokine and chemokine release
3. Potentiates platelet aggregation
4. interfere with plasma components of the coagulation cascade, by inhibiting
anticoagulant activity, by affecting fibrinolysis, and by displacing the binding of
the natural anticoagulant annexin A5 to anionic structures.

24. Final signaling pathway

27. Platelets
• Binding of aPL Abs to platelets is dependent on both the presence of dimeric
b2GPI and the exposure of anionic phospholipids, especially phospatidylserine
(PS)
• aPL Abs enhance the expression of GPIIb/IIIa, a major fibrinogen receptor
• aPL induced TXB2 production in platelets through the activation of p38 mitogen
activated protein kinase (MAPK) and subsequent phosphorylation of cytosolic
phospholipase A2

28. Vascular endothelial and monocyte
• Role for vascular endothelial cell and monocyte activation in aPL- mediated
thrombogenesis has been described.
• VCAM-1 , ICAM-1 and E-selectin – expression
• aPL- induced activation and TF upregulation in both endothelial cells and
monocytes has been shown to play a key role

29. Annexin – as anticoagulant shield
• Annexin A5 binds to phosphatidylserine surfaces, forming a shield that inhibits
the formation of procoagulant complexes
• In vitro study has shown that domain I of anti B2 GP1 complex can disrupt the
shield, exposing procoagulant phosphatidylserine and hence predisposing to
thrombosis

30. Anticoagulation & fibrinolytic systems
• Interference with natural anticoagulant and fibrinolytic systems also plays a role in
thrombus formation in APS patients
• Elevated levels of coagulation factor XI confer a predisposition to venous
thrombosis and stroke
• On vessel injury leading to exposure of phosphatidylserine, tissue factor becomes
de-encrypted and activated, enabling it to bind factor VIIa, which leads to activation
of factor IX and factor X – extrinsic pathway
• Inhibition of APC , antithrombin 3

31. Innate immunity and NETS
• Neutrophils contribute in a unique and relevant manner to the development of APS
• APS patient neutrophils are prone to spontaneous NET release , and thrombi
incorporate NET-derived materials
• PSGL-1, a neutrophil protein that mediates adhesion to endothelia, as an important
regulator of the prothrombotic functions of neutrophils
• Upregulation of the expression of TLR7 and TLR8 in plasmacytoid dendritic cells and
monocytes, respectively, as well as their translocation from the endoplasmic
reticulum to the endosome, sensitizing the cells to TLR7 and TLR8 ligand

32. summary of mechanisms of vascular thrombosis
1. Increased oxidative stress – post translation modification
2. Impaired function of eNOS
3. Activation of APL- beta 2 GP1
4. Increased expression of tissue factor
5. Increased activated factor xa
6. Antibody mediated activation of complement
7. Disruption of annexin shield
8. TLR signaling
9. BAFF stimulation

33. Pathogenesis in obstetric APS

34. Controversies
• The exact mechanisms underlying the obstetric manifestations of APS
are still unclear
• Whether thrombosis is a major contributor to pregnancy morbidity is
still debated
• Defective placentation is considered to be the major cause of
pregnancy morbidity
• Inflammation contributes to fetal loss

35. Fetal loss mechanism
• Placental tissue thrombosis and infarction
• Acute inflammation
• Inhibition of syncytium-trophoblast differentiation
• Induction of decidual cell inflammatory phenotype
• Complement activation
• Embryo and/or placental apoptosis

36. Potential pathogenic mechanisms mediated by β2gPI- dependent aPl in the placenta.

37. Placental thrombosis and infarction
• Intra placental thrombosis, with impairment of maternal–fetal blood exchange -
initially suggested to be the main pathogenic mechanism of fetal loss.
• aPL antibodies can disrupt the anticoagulant annexin A5 shield on trophoblast
and endothelial cell monolayers – procoagulant state

38. Inflammation
• Repeated intraperitoneal injections of large amounts of human IgG with aPL
activity to pregnant naive mice induces considerable placental inflammatory
damage that results in fetal resorption and growth retardation
• Immunohistochemistry of decidua showed deposition of human IgG and mouse
complement, neutrophil infiltration and local TNF secretion

39. • C5a acts through the upregulated expression of TF on neutrophils infiltrating
placental tissues.
• Mice deficient in chemokine-binding protein D6 (chemokine-binding protein 2), a
placental receptor that recognizes the majority of inflammatory CC chemokines
and targets them for degradation, are more susceptible to fetal loss
Inflammation ( contd )

40. Defective placentation – fetal side
aPL ( β2GPI-dependent antibodies) bind to human trophoblasts and affect several
cell functions in vitro, inducing –
1. Cell injury and apoptosis
2. Inhibition of proliferation and syncytia formation
3. Decreased production of human chorionic gonadotrophin
4. Defective secretion of growth factors and impaired invasiveness.

41. Defective placentation –maternal side
A. aPL also cause abnormalities at the maternal side ( decidua ) of the placenta.
B. Impaired endometrial differentiation & reduced expression of complement
decay-accelerating factor (CD55) were found on endometrial biopsies.
C. These alterations before conception might compromise implantation and
predispose to complement-mediated pregnancy failure
D. In addition, β2GPI-dependent aPL are able to react with human stromal
decidual cells in vitro, inducing a proinflammatory phenotype

42. Willis, R., Pierangeli, S.S. Pathophysiology of the antiphospholipid antibody syndrome. Autoimmun Highlights 2, 35–52
(2011)

43. Schreiber, K., Sciascia, S., de Groot, P. et al. Antiphospholipid
syndrome. Nat Rev Dis Primers 4, 17103 (2018).

44. Pathogenesis of non-criteria features
• Renal involvement in APS, although involving thrombosis in glomeruli and larger
vessels, endothelial cell dysfunction induced by aPL is likely to play a major role
• An association exists among cognitive dysfunction, livedo reticularis and white
matter MRI lesions suggesting a microangiopathic mechanism
• Cognitive dysfunction in APS may be due to aPL binding to CNS cells causing
permeabilization and depolarization
• Contribution of aPL to atherosclerotic plaque formation through endothelial cell
and monocyte activation

45. Future Therapeutic target based on pathogenesis
Bill Giannakopoulos, Steven A. Krilis, The Pathogenesis of the Antiphospholipid Syndrome. N Engl J Med 2013; 368:1033-
1044
NAC
Statin
HCQS
Synthetic domain
of BGP1
Coenzyme Q10
PDI -inh
Xa-inh
eculizumab
HCQS
belimumab

46. Take home message
1. Pathogenesis of APS differs in vascular and obstetric variant
2. Vascular event is mediated by thrombosis – by two hit model
3. Obstetric event has more abnormal placentation and inflammatory changes
4. Anti Beta 2 gp1 Ab – plays a major role in pathogenesis
5. Studies are still ongoing for evaluation of exact mechanism in different manifestations –same
antibodies but different diseases ?
6. Future therapeutic target may have a promising role
7. More study is needed to explore the exact pathogenesis , source of APL antibodies , true
physiological role of beta2gp1 and epitope specificity

47. References
1. Bill Giannakopoulos, Steven A. Krilis, The Pathogenesis of the Antiphospholipid Syndrome. N Engl J Med
2013; 368:1033-1044
2. Radic M and Pattanaik D (2018) Cellular and Molecular Mechanisms of Anti-Phospholipid Syndrome. Front.
Immunol. 9:969
3. Meroni, P., Borghi, M., Raschi, E. et al. Pathogenesis of antiphospholipid syndrome: understanding the
antibodies. Nat Rev Rheumatol 7, 330–339 (2011)
4. Sorice, M., Misasi, R. Different domains of β2-glycoprotein I play a role in autoimmune pathogenesis. Cell Mol
Immunol (2018).
5. Meroni PL, Borghi MO, Grossi C, Chighizola CB, Durigutto P, Tedesco F. Obstetric and vascular
antiphospholipid syndrome: Same antibodies but different diseases? Nature Reviews Rheumatology. 2018 Jul
1;14(7):433-440
6. Willis, R., Pierangeli, S.S. Pathophysiology of the antiphospholipid antibody syndrome. Autoimmun
Highlights 2, 35–52 (2011)
7. Schreiber, K., Sciascia, S., de Groot, P. et al. Antiphospholipid syndrome. Nat Rev Dis Primers 4, 17103 (2018).