Pathophysiology of APS

Primary anti-phospholipid syndrome is an autoimmune disease, portrayed by obstetric

complications and vascular thrombosis in the presence of various antibodies including;
antiphospholipid (aPL) antibodies, anti¬β2glycoprotein I antibodies, anticardiolipin antibodies or
ACL, and lupus anticoagulant (LAC) (Erkan et al., 2011). APS is considered as the throbophilic
state denoted by venous and arterial thrombosis, presence of aPL and recurrent pregnancy
morbidity (Durrani et al., 2002). The renal involvement pose the high risk factor for thrombotic
events and renal artery stenosis. In the syndrome, the antibodies are directed against the
phospholipids that linked to cell membrane receptors, oxidized lipoprotein, mitochondria,
activated components of complement, and phospholipid-binding protein. Cells get activated
when antibodies attach to the complex antigens leading to triggering of complement cascade and
coagulation that results in thrombolytic events and morbidity in pregnancy-also termed as the
syndrome (Durrani et al., 2002). In most case the phospholipid-binding proteins involved in the
process are; β2-glycoprotein I, annexins II and V, cardiolipin and prothrombin (de Laat-Kremers
et al., 2022).

The distinctive feature of APS is the lupus anticoagulant which is not a single body but a family
of complex antibodies that work against the group of antigens consists of prothrombin and
glycoprotein attached to anionic phospholipid (Chahal et al., n.d.). In clinical experiments, these
antibodies compete with the clotting factors for the binding sites of the phospholipids and
prolong the clotting time but they are not responsible for the clinical bleeding. Rather, they are
thrombogenic and concentrate the prothrombin on phospholipid sites, resulting into thrombin
production (Erkan et al., 2011). While other antiphospholipid antibodies elevates the platelet
aggregation and adherence and reduces the endothelium’s clot-inhibitory properties. Those
antibodies that enhances the rate of lipid peroxidation by impairing the fibrinolytic activity and
decreasing the paraoxonase activity (Fiaccadori et al., 2021).

The pathophysiology of APS is complex as it is an syndrome in which multiple factors and

pathways involved in the disorder but still available many probable conceptual frameworks to
underpin the pathophysiological pathways of the disease (Greaves, 2009). Three most important
factors play the vital role in the pathophysiological pathways of the disease which are;
 1. Phospholipid binding-Protein
2. The antibodies and their Targets
3. Hemostatic Factors and Complement

(Greaves, 2009)

Role Phospholipid binding protein

Four proteins including; Annexins, β2-GPI, Cardiolipin, and Vimentin are play an important in
pathophysiology of primary APS(Lockshin, 1996).

Annexin consists of four monotonous domains and the molecule contains seventy amino acids
and each chain involved in Ca2þmediated binding to the phospholipids having negative
charge(Malaviya, 2003). Assembly of plasminogen activator and plasminogen is mediated on
cell membrane by by Annexin II that results into tissue based fibrinolysis. On the endothelial
surface of cell, 21 β2-GPI attached to annexin ll. The people with primary APS, β2-GPI–
annexin II network may trigger the formation of anti-β2- GPI antibody(Malaviya, 2003). The
cross linkage of β2-GPI with anti- β2-GPI antibodies and the attachments of this complex with
annexin ll results into endothelial cell activation(Oku et al., 2012). These particular antibodies
are thrombogenic in nature and perform two important functions; inhibition of plasmin surface
expression and stimulate the releasing of tissue factors. (Oku et al., 2012) The crystalline shield
in formed by annexin V over the revealed anionic phospholipids of cell membrane that prevent
the formation of complexes of active clotting factors. The shield is distorted by APA when
bound to G40-R43 epitope on zone I of β2-GPI(Rand, 2002).

β2-GPI is a 48-kDa plasma protein organized in five domains and containing 326 amino acid,
forming the circular structure as domain I interacts with domain V in plasma membrane(Rand,
2007). Lysine which has positive charged, binds to negatively charged phospholipid locatedon
domain V. Lysine is amino acid that contains positive charge and it attached phospholipids
having negative charge through domain giving a fishhook configuration and expose the epitopes
on domain I (Rand, 2007). Exposure of epitopes and oxidation of sulfhydryl groups of β2-GPI
are responsible for achieving the Immunogenicity (Tektonidou, 2018). Different domains of β2-
GPI become target of developing antibodies. This is due to formation of β2-GPI–antibody
networks that directly interact with factor V, weakening its activation by factor Xa(Weiler,
2008). With the formation of antibodies to the protein, the specific antithrombotic role of β2-
GPI is impeded. Moreover, the binding of complexes of β2-GPI–antibody with the cellular
receptors locating on endothelial cells, neutrophils, platelets and monocytes, which ultimately
results into activation of these cells and increasing their thrombogenicity(Tektonidou, 2018).
Cardiolipin which is an anionic phospholipid, act as a common for ACAs or antibodies that in
most cases cross-react with other phospholipids having negative charge. These phospholipids
play an important role while diagnosing the APS(Erkan et al., 2011).

Cardiolipin complexes have the clinical importance for the patient with APS. Vimentin which is
an phospholipid binding protein in the endothelial cell have the special affinity for the
cardiolipin. Anti-cardiolipin/vimentin antibodies triggers the phosphorylation of IL-1 related
kinase that lead to generation of NF-kB or nuclear factor-kappa B(Greaves, 2009).

The Antibodies and their Target

The Antibodies and Target antibodies strike with cellular receptors, cells, and hemostatic
proteins in two ways; either by forming the phospholipid composite binding proteins or alone. In
the APS, the neo-epitopes are exposed provoking the APA on the apoptotic cell surface
(Kaburaki, 1999).

The endothelium is responsible for releasing the various factors that inhibit the thrombosis, but
in APA this activity is compromised severely (Brusch, 2016). The hydrolyses of arachidonic acid
is impaired through inhibition of phospholipase A2 activity, leading to reduction in release and
production prostacyclin, which is a potential vasodilator and platelets aggregation inhibitor
(Brusch, 2016). The binding of β2-GPI interferes with the VWF-dependent platelet aggregation
and adhesion. While neutralizing the β2-GPI through anti-β2-GPI antibodies leads to elevation of
VWF levels by 1.5 times (Vandevelde & Devreese, 2022). The patient with primary APA, the
clot-inhibitory characteristics are reduced and platelet adhesion properties of endothelium
increased. This is due to that fact that endothelial nitric oxide synthase or (eNOS) is stopped and
leukocyte adhesion is increased when β2-GPI attached to apoER2 at the cell membrane of
endothelium. In many cases of primary APS, the platelet thrombocytopenia is observed.
(Vandevelde & Devreese, 2022)
The hemostatic factors
Premature atherosclerosis is another important feature of APS. In such case, the
low density lipoproteins attach to β2-GPI at domain V and display themselves as the target
for anti-β2-GPI and APA (Forastiero, 2014). These antibodies reduce the overall activity of a
particular enzyme, named paraoxonase that hinder the LDL’ oxidation. Uptake of oxidized LDL
by macrophages increased and bounded antibodies with LDL and cardiolipin are found in
atherosclerotic lesions (Forastiero, 2014).

Non-muscle type of myosin II regulatory light chain is phosphorylated by Anti-B2-GPI

antibodies, which is essential for expression of mRNA tissue factor and releasing the micro
particles of endothelial cells. In almost 67% of individuals suffering from APS, antibodies
against the factor VII / VIIa are reported while about 33.9% of the patient contain the antibodies
against the factor Xa (Erkan et al., 2011). There is upregulation of protein family of disulfide
isomerase that is capable of decreasing the disulfide bond factor XI. The reduced factor IX
basically activated to another factor XIa which is seen in high proportion in APS patients (Galli,
2000).

The aPL found interfering with the natural existing anticoagulant protein including C, and S
protein. Moreover, aPL also interfere with the fibrinolytic system resulting in breakage of fibrin
(Green, 2021).
Protein C:
In APS, the anticoagulant activity is stopped as aPL inhibit the protein C activation and its
ability to inactivate the factors VIII and V (Miesbach et al., 2005).
Antithrombin activity
The antithrombin (AT) activity is reduced by aPL by inhibiting the binding of heparin necessary
for the complete activation of AT. Apl with AT activity may result into further reducing the
thrombin inactivation while at the same time the antibodies which are activated against the factor
IX and X may also create interference with negative regulation (Léránt et al., 2005).

Laboratory Essays
Diagnoses of the APS is achieved through different clinical assessments including; medical
history, laboratory examination, physical examination, and antiphospholipid antibodies (aPL )
(Tripodi et al., 2011) . Here the laboratory assays will be discussed.
Laboratory evaluation is divided into two categorized;

1. Routine laboratory testing

2. Antiphospholipid antibody testing

Routine Lab testing
Complete blood cell count:
It is performed to check whether the patient is thrombocytopenic or not as thrombocytopenia
may be found in the APS patients (Tripodi et al., 2011).
Baseline coagulation testing
Activated partial thromboplastin time and prothrombin time are paramount prior to commencing
the anticoagulation, particularly in the case when used for monitoring. In lupus anticoagulant
testing, aPTT is utilized (Tripodi et al., 2011).
Urinalysis and Serum creatinine level analyses
  A serum urinalysis with urine sediment and creatinine analyses used to identify the involvement
of kidney in APS. Wrong findings may also depicts a alternative or concomitant diagnosis (eg,
SLE). Patients with microscopic hematuria, abnormal kidney function, active urinary sediment
and proteinuria, will need further assessment.
If the results suggesting SLE should also go through the proper clinical evaluation for SLE
(Chahal et al., n.d.).
Antiphospholipid antibody testing

Specific antiphospholipid antibody testing

Suspected APS patient undergoes through two immunoassays and a functional coagulation assay.
1. Anti-beta2 glycoprotein I / anti-beta2GPI antibodies; IgM and IgG by ELISA.
2. Anticardiolipin antibodies (aCL); IgM and IgG by enzyme-linked immunosorbent assay
(ELISA) (de Laat-Kremers et al., 2022).
LA functional coagulation assay
It is basically a three-step procedure:
 For hemostasis a screening test is performed which depends upon prolonged
phospholipid. Most common used screening tests are; aPTT and (dRVVT)
 When the patient plasma is mixed with normal plasma the prolonged screening test can’t
be corrected. This suggests the presence of inhibitor not the deficiency of coagulation
factor.
 When excess phospholipid is added, the prolonged coagulation test is corrected or
shortens which manifests its dependency on phospholipid (de Laat-Kremers et al., 2022)

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 Diagnostic criteria for APS
aPL –Associated demonstration

Vascular thrombosis
Thrombocytopenia
Confirmed episodes of venous,
arterial or small vessel Renal manifestation
thrombosis

Suspected APS

Non-aPL
aPL criteria
criteria

aCL Anti-B2 GPI PT/aPS

LgA,
LA Other
lgM/lgG lgM/ lgG aCL ,anti- IgG/ factors
B2 GPI IgM/IgA

Positive Negative
Negative Positive

Repeating
the test > 12
weeks No APS Repeating
the test > 12
Weeks
Positive Negative

Positive Negative

Increased risk No APS

No APS
APS of thrombosis
Possible
diagnosed
APS