ANTIPHOSPHOLIPID ANTIBODY SYNDROME

DEPARTMENT OF BIOCHEMISTRY

By: Dr JYOTI

Antiphospholipid syndrome was described in full in the 1980s, after various previous reports of specific antibodies in people with systemic lupus erythematosus and thrombosis. The syndrome is sometimes referred to as "Hughes syndrome", after the rheumatologist Dr. Graham R.V. Hughes who worked at the Louise Coote Lupus Unit at St Thomas' Hospital in London and played a central role in the description of the condition.

History

"Hughes”

• In young,app healthy people---for LA, antiCL is 1-5%• Prevalence ↑ with age ,in elderly with chronic disease. • Mean age of onset-31 yrs,Low age 8 months• Risk of thrombosis is 0.5-30%• Women :men is 5:1• Females---arthritis,livedo,migraine• Males—MI,epilepsy,lower extremity arterial thrombosis• More common in african americans.• Without rheumatic disease at younger age and

with rh –older• Apl ab—30-40% in SLE---10% have APLS• Idiopathically—ACL-24%,LA-4%

Epidemology

Antiphospholipid syndrome or antiphospholipid antibody syndrome (APS or APLS or), often also Hughes syndrome, is an autoimmune, hypercoagulable state caused by antibodies against cell-membrane phospholipids that provokes blood clots (thrombosis) in both arteries and veins as well as pregnancy-related complications such as miscarriage, stillbirth, preterm delivery, or severe preeclampsia.

The syndrome occurs due to the autoimmune production of antibodies against phospholipid (aPL), a cell membrane substance. In particular, the disease is characterised by antibodies against cardiolipin (anti-cardiolipin antibodies) and β2 glycoprotein I.

Introduction

Risk factors for developing antiphospholipid syndrome include:•Primary APS

genetic marker HLA-DR7•Secondary APS

SLE or other autoimmune disordersGenetic markers: HLA-B8, HLA-DR2, HLA-DR3Race: Blacks, Hispanics, Asians, and Native Americans

Risk factors

Disruption of vascular endothelial lining allows exposure of blood to subendothelial connective tissue:

Primary hemostasis (seconds)- Platelet plug formation at site of injury- Stops bleeding from capillaries, small

arterioles and venules

Secondary hemostasis (minutes)- Fibrin formation by reactions of

the plasma coagulation system

Normal Hemostasis

Defects in primary hemostasis• Thrombocytopenia

Defects in secondary hemostasis

• Clotting factor deficiencies• Prethrombotic

(hypercoagulable) states

Bleeding and Thrombosis

• Inherited

• Acquired

Prethrombotic disorder

Inherited Disorder

• Anti-thrombin deficiency

• Deficiencies of protein C and S

• Resistance to activated protein C •( factor V Leiden mutation)

• Prothombin gene mutation• ( G20210A)

Homocystinemia

Aquired disorder

Conditions associated with a hypercoagulable state: - pregnancy and postpartum - major surgery - obesity and immobility - malignancy - congestive heart failure - nephrotic syndrome

Estrogen treatment

Antiphospholipid syndrome

Antiphospholipid syndrome is an autoimmune disease, in which

"antiphospholipid antibodies" :

1.anticardiolipin antibodies (ACA)2. lupus anticoagulant(LA)react against proteins that bind to anionic phospholipids on plasma membranes.

ACA– are directed against cardiolipinThey may be b2 gp1 dependent or independent (Independent—syphilis)

Mechanism

• B2 gp1-----apolipoprotein H• Bind with cardiolipin ab - thrombosis

• b2GPI a plasma protein with affinity for negatively charged phospholipids

• anti- b2GPI: are probably the major cause of APS

• Anticardiolipn abs recognize in most assays: b2 GPI

• Lupus Anticoagulant activity is caused by autoantibodies to: - b2 GPI - prothrombin

Beta 2 glycoprotien-1(b2gp1)

Anionic phospholipids

The exact cause is not known, but activation of the system of coagulation is evident.

• Homeostatic regulation of blood coagulation is altered.

• 1.defect in cellular apoptosis- exposure of membrane phospholipids to the binding of various plasma proteins---b2gp1---complex—epitope---target for autoantibodies.

• 2.oxidized b2GP1---activates dendritic cells –autoantibodies are produced.

• 3.production of antibodies against prothrombin, proteinC, S annexins.

• 4.activation of platelets to enhance endothelial adherence.

• 5.activation of vascular endothelium—platelet and monocyte binding.• 6.ab against oxidized LDL — atherosclerosis.

Possible mechanism of aPL induced thrombosis

• Complement activation has been increasingly recognised as a possible significant role in the pathogenesis of APS.

Blood. Jan 15 2007;109(2):422-30. 

Nat Med. Nov 2004;10(11):1222-6.

The family of APL ab are heterogenous and the targets vary.

APS can be caused by –LA,ACA,B2GP1 or other antibodies.

There are distinct clinical ,laboratory and biochemical differences between the disorders mediated by the different antibodies.

ACL---risk of stroke—arterial thrombosis

LA-venous

TNF alpha –pregnancy loss

Coagulation pathway

Annexin ˅

The syndrome can be divided into primary (no underlying disease state) and secondary (in association with an underlying disease state) forms.

Anti-ApoH and a subset of anti-cardiolipin antibodies bind to ApoH, which in turn inhibits Protein C, a glycoprotein with regulatory function upon the common pathway of coagulation (by degradating activated factor V).

Clinically important antiphospholipid antibodies (those that arise as a result of the autoimmune process) are associated with thrombosis and vascular disease.

LAC antibodies bind to prothrombin , thus increasing its cleavage to thrombin, its active form .

In APS there are also antibodies binding to: Protein S, which is a co-factor of protein C. Thus, anti-protein S antibodies decrease protein C efficiency;

Annexin A5, which forms a shield around negatively-charged phospholipid molecules, thus reducing their availability for coagulation.

Thus, anti-annexin A5 antibodies increase phospholipid-dependent coagulation steps.

The Lupus anticoagulant antibodies are those that show the closest association with thrombosis, those that target β2glycoprotein 1 have a greater association with thrombosis

than those that target prothrombin.

Anticardiolipin antibodies are associated with thrombosis at moderate to high titres (>40 GPLU or MPLU).

Patients with both Lupus anticoagulant antibodies and moderate/high titre anticardiolipin antibodies show a greater risk of thrombosis than with one alone .

APL antibodies and NF-B

• Intracellular events in EC induced by aPL antibodies:– aPL induce activation of

NF-kB and correlates with EC activation in vitro and in vivo and with thrombosis in vivo.

• Espinola RG et al: J Thromb Haemost, 2003; 1: 843-848.• Dunoyer-Geindre S. et al. Thromb Haemost. 2002; 88:

851-857. • Bohgaki M, et al. Int Immunol. 2004; 16: 1632-1641.

pathogenesis

In pregnancy

is characterized by:• Arterial or Venous Thrombosis

• Thrombocytopenia

• Recurrent Fetal Loss

• Serum Anti-phospholipid antibodies (aPL)

Antiphospholipid Syndrome

1. Primary antiphospholipid syndrome APS occurs in the absence of any other related disease.

2. Secondary antiphospholipid syndromeAPS occuring in the context of other autoimmune diseases, such as systemic lupus erythematosus (SLE).

3. Catastrophic antiphospholipid syndrome In rare cases, APS leads to rapid organ failure due to generalised thrombosis; this is termed (CAPS) and is associated with a high risk of death.

Types :

• Venous thromboembolism:

Clinical presentation of APS

Arterial Occlusion: Stroke and TIAs are the most common

Pulmonary Embolism

Deep Vein Thrombosis

Recurrent fetal loss • In women with recurrent miscarriage due to APS

fetal loss rate: as high as 90%

• antiphospholipid abs are associated with: - placental insufficiency - early preeclamapsia - IUGR- intrauterine growth restriction

• Pregnancy morbidity

aPL associated manifestations (individual diagnosis)

• Thrombocytopenia (occurs in up to 50%)

• Cardiac valve disease• Livedo reticularis• Nephropathy ( late

manifestation)

Sydney revsion of Sapporo criteria 2006

Livedo reticularis with necrotic finger tips in Antiphospholipid syndrome

• Transverse myelitis• Migraine• Chorea• Leg ulcers• UBOs (white matter lesions) on brain MRI

Possible Clinical presentation of APSnot included in criteria

• Infection:

- Syphilis, TB, Q-fever, Spotted Fever, Klebsiella, HCV, Leprosy,HIV.

- The abs are usually transient, not b2 GPI dependent

• Malignancy: Lymphoma, paraproteinemia

• Drug induced:

phenothiazines, procainamide, quinidine, phenytoin, hydralazine

Antiphospholipid antibody in other diseases

Common auto immune diseases ass with APL ab are

• 1.SLE-25-50%• 2.sjogren’s –42%• 3.RA-33%• 4.AITP-30%• 5.AIHA-unknown• 6.MCD-22%• 7.behcet-20%

Laboratory diagnosis

• Spontaneous venous thromboembolism

• Recurrent VT, even in presence of other risk factors

• Stroke or peripheral arterial occlusive event at < 50 yrs

• In all SLE patients

• In women with > 3 consecutive pregnancy losses

loss of morphologically normal fetus at II-III trimester early severe preeclampsia severe placental insufficiency

low prevalence in general obstetric population (< 2% ): screening not warranted

Indication

• APA---IgG,IgA,IgM• SEVERAL antibodies are recognised• Recently—antibodies against annexin V,protein C

• IgM acl---HEMOLYTIC ANEMIA.• IgG ACL –thrombosis• False positive test result for syphilis• ACL—membrane phospholipids• LA-plasma coagulation molecules• Elongates APTT,Kaolin clotting time,dilute russells viper venom time.

Lab Diagnosis

• Advantages– Overwhelming majority of APS patients are anti cardiolipin positive– Test can be performed reproducibly.– Clinicians and laboratories generally familiar with units of

measurement.

• Disadvantages– Relatively nonspecific (particularly low positive, IgM positive).– Intra-laboratory and Inter-laboratory variability. – Problems with false positive results: aCL positive in a wide variety of

infectious diseases and in non-APS related autoimmune diseases.

Anticardiolipin test

Solid phase assays usually anti-Cardiolipin abs

• IgG aCL levels below 21.4 = probability of thrombosis 0.07

• IgG aCL levels >21.4 and < 65.0 GPL = probability of thrombosis 0.20

• IgG aCL levels >65.1 GPL units = probability of thrombosis 0.75

Predictive value of IgG aCL for thrombosis in patients with SLE

• Perform coagulation screen to detect prolongation in phospholipid dependent coagulation assay (usually use: APTT)

• If APTT is prolonged: Mix with normal plasma

- If due to factor deficiency: corrected - If due to inhibitor (antibody) not corrected

• Confirm inhibitor is phospholipid dependent : corrected by mixing with platelets or phospholipids

• Perform second test: KCT or DRVVT

Lupus Anticoagulant TestCoagulation Assay

• APTT: - variability in reagents result in

inconsistent sensitivity. - acute phase reaction and pregnancy may shorten APTT and

mask a weak LAC A normal APTT does not exclude LAC

• KCT- Kaolin clotting time more sensitive to presence of anti-II

• DRVVT- Dilute Russell’s viper venom time more sensitive to presence of b2 GPI

• TTI - Tissue thromboplastin inhibition test

No LAC shows 100% specificity and sensitivity because aPLs are heterogeneous. More than 1 test system is needed

Test for LAC

Diluted Russells viper venom test

Kaolin clotting test

• Based on observation that antiphospholipid antibodies cross-react with negatively charged phospholipids but syphilis and other infectious diseases sera largely limited to cardiolipin binding (no cross-reactivity)

• Construction of a kit with negatively charged phospholipids might eliminate non-specific binding.

• Antigen composed of mixture of phospholipids instead of cardiolipin• Sensitivity of APS (greater than 90%)• More specific than anticardiolipin test and at least as specific (or

more) compared to anti-ß2GPI• Incorporation of an in-house positive control• Can be utilized for first line testing, and certainly important in

confirmation of APS

AphlELISAPrinciple

Patient Laboratory DataPT 20.6 sec aPTT 100.3

sec

TCT 8.8 sec DRVVT ‘No clot’

Factor VIII

‘Inhibitory’ Factor IX <1.6%

Factor XI <1.6% Bethesda titer

2.8 U

Platelets 120,000/ml

Factor X 68%

Additional Laboratory Data

Factor V (aPTT) “Inhibitory”

Factor V (PT) 115%

Factor II 38%

Fibrinogen 795.6 mg/dl

D-dimer >4.37 mcg FEU/ml

Repeat DRVVT (ratio) 3.23

DRVVT Confirm (ratio) 2.17

Alternative strategies to identify a lupus anticoagulant

• Platelet neutralization procedure (PNP; uses platelet membranes).

• Hexagonal phase phospholipid assay (StaClot LA; uses PE in a hexagonal phase conformation).

• Textarin/Ecarin clot time.• Factor V analysis by PT and aPTT-based assays.

What if LA,ACL are negative

• If patient experiencing thrombosis or recurrent miscarriages

• Order • Antibodies to b2 gp1• Ab to phosphatidyl serine,ethonalamine,glycerol,inositol• Annexin V• Phosphatidyl choline.

Imaging studies• For confirmation• USG• COLOR DOPPLER• CT SCAN• MRI• 2D ECHO

• Histology----

• non inflammatory bland thrombosis with no signs of perivascular inflammation or leukocytoclastic vasculitis.

• Lupus anticoagulant detected and confirmed.• Multiple factor deficiencies in aPTT pathway

reflect high-titer lupus anticoagulant.• Prolonged PT reflects mild factor II deficiency

and lupus anticoagulant effect.• Elevated D-dimer reflects recent thrombosis.• Elevated inhibitor titer due to lupus

anticoagulant.

Assessment of coagulation Assay

DIFFERENTIAL DIAGNOSIS

• Think of any other thrombophilic states before making a diagnosis of APLS.

• Malignancy• OCP• Homocysteinemia• Antithrombin 111 def• Protein C,S def• Factor V leiden mutation

MANAGEMENT

Incidental finding of antiphospholipid antibodies

• Anti-thrombotic therapy not usually indicated

• Low threshold for thromboprophylaxis at times of high risk

• Some suggest low dose Aspirin prophylaxis

• Reduce other risk factors for thrombosis

INR

• The ISI—1.0-2.0• INR—5 high chance of bleeding• 0.5--- clot formation• Normal range is 0.9-1.3• Warfarin ---2.0-3.0• Prosthetic valves—3.0-4.0

• Eliminate other risk factors, such as oral contraceptives, smoking, hypertension, or hyperlipidemia.

• Low-dose aspirin is used widely in this setting; however, the effectiveness of low-dose aspirin as primary prevention for APS remains unproven.

• Clopidogrel has anecdotally been reported to be helpful in persons with APS and may be useful in patients allergic to aspirin. • In patients with SLE, consider hydroxychloroquine, which may have intrinsic antithrombotic properties.

•Consider the use of statins, especially in patients with hyperlipidemia.

Prophylactic therapy

• Perform full anticoagulation with intravenous or subcutaneous heparin followed by warfarin therapy.

• Based on the most recent evidence, a reasonable target for the international normalized ratio (INR) is 2.0-3.0 for venous thrombosis and 3.0 for arterial thrombosis.

• Patients with recurrent thrombotic events, while well maintained on the above regimens, may require an INR of 3.0-4.0.

• For severe or refractory cases, a combination of warfarin and aspirin may be used. •Treatment for significant thrombotic events in patients with APS is generally lifelong.

Thrombosis

• Mild to moderate- Platelets > 50,000: No treatment

• Severe- <50,000:

- corticosteroids - corticosteroid resistant cases:

HCQ , IVIG, Immunosuppressive drugs, Splenectomy

Thrombocytopenia

Management of aPL positive patients with adverse pregnancy history

• Poor obstetric history - the most important predictor

• The risk of fetal loss is related to aCL ab titer • Presence of aPL are a marker for a high risk

pregnancy

• Once APS is diagnosed, serial aPL testing is not useful

Current Recommendations

Pregnancy Fetal protection• Asymptomatic aPL no treatment• Single loss <10wks no treatment• Recurrent loss* <10wks prophylactic heparin +ASA up to 6-12 wks postpartum, ASA

after(?)

• Recurrent loss < 10 wks therapeutic heparin + ASA, + thrombosis warfarin postpartum

• Prior thrombosis therapeutic heparin + ASA warfarin postpartum

* Late fetal loss IUGR severe pre-eclampsia

Other therapies for aPL associated pregnancy loss

• Corticosteroids : - associated with significant maternal and fetal morbidity - ineffective

• Immunosuppression: azathioprine, plasmapheresis: numbers treated too small for conclusion

• IVIG: may be salvage therapy in women who fail on Heparin + Aspirin

Fetal Monitoring

• US monitoring of fetal growth and amniotic fluid every 4 weeks

• US monitoring of uteroplacental blood flow: uterine artery waveforms assessed at 20-24 wks

• If early diastolic notch seen: do 2 weekly growth scans due to high risk of IUGR

FOLLOW UP

• Frequent check ups• Adequate patient education• Avoidance of smoking• Strict control with anticoagulants.• In case of bleeding –hospital.

• Normal healthy life

With appropriate medication and lifestyle modifications, most individuals with primary antiphospholipid syndrome (APS) lead normal healthy lives.

However, subsets of patients continue to have thrombotic events despite aggressive therapies. In these patients and in patients with CAPS, the disease course can be devastating, often leading to significant morbidity or early death. A retrospective study suggested that hypertension or medium-to-high titers of IgG anticardiolipin antibody are risk factors for a first thrombotic event in asymptomatic patients with antiphospholipid (aPL) antibodies.[18] Primary prophylaxis against thrombosis appears to offer significant protection in such cases.

Prognosis

Patients with secondary APS carry a prognosis similar to that of patients with primary APS; in the former, however, morbidity and mortality may also be influenced by these patients' underlying autoimmune or rheumatic condition. In patients with SLE and APS, aPL antibodies have been associated with neuropsychiatric disease and have been recognized as a major predictor of irreversible organ damage. Women with aPL antibodies who experience recurrent miscarriages may have favorable prognoses in subsequent pregnancies if treated with aspirin and heparin.

Future Directions

• Can we predict which patients with antiphospholipid antibodies will develop thromboembolic complications?

• Is there an inherited predisposition to developing antiphospholipid antibody syndrome?

Genomic strategy

• Whole blood RNA prepared using PAXgene system from patients with APS and selected control populations.

• RNA extracted and validated.• Oligonucleotide arrays printed at the Duke

Microarray Facility, using the Operon Human Genome Oligo Set Version 3.0 (Operon, Huntsville, AL).

-- Potti, et al., Blood, 2006; 107: In press.

Discovery ModePreliminary data with patients and ‘controls’

Controls with VTE APS NormalaPLA

Up regulated Down regulated

-- Potti, et al., Blood, 2006; 107: 1391.

Family history

Asymptomatic daughter tests positive for a lupus anticoagulant.

Mother developed arterial thrombosis and thrombocytopenia prior to her death.

Familial Antiphospholipid Syndrome

• Family members of patients with APS have an increased incidence of autoimmune disorders.

• “Genetics of APS” is a clinical trial being developed by the Rare Thrombotic Diseases Clinical Research Consortium.

Thank you