Lupus Anticoagulant

Lupus Anticoagulant
By:Jennifer Kirkland (Lambe)

Antiphospholipid
Antibody Syndrome
* Antibodies to phospholipids or plasma proteins bound to phospholipids
o Lupus anticoagulant antibodies
o Anticardiolipin antibodies
o Anti-ß2- glycoprotein I antibodies
* Other antibodies: prothrombin, annexin V, phosphatidylserine, phosphatidylinositol
o These antibodies are not standardized for clinical use and their clinical utility is not well characterized

Lupus anticoagulant
* Lupus anticoagulant
o Describes a group of antibodies which react with cardiolipins, other phospholipids, ß2-glycoprotein I, or proteins other than ß2-glycoprotein I
-AND-
o possess “lupus anticoagulant” activity

What is lupus anticoagulant activity?

* Ability to interfere with coagulation testing (in particular, the tests which are phospholipid dependent) leading to prolonged values
* Despite the “anticoagulant effect” in vitro, these antibodies actually cause coagulation in vivo, in the form of arterial and venous thromboses

Lupus anticoagulant:
Actually a Misnomer
* Associated with clotting, not anticoagulation
* More than one antibody is associated with lupus anticoagulant activity
* Only about 50% of individuals with a lupus anticoagulant meet the American College of Rheumatology criteria for the classification of lupus (SLE)

Definitions
* Cardiolipin= mitochondrial phospholipid
o Causes a biologic false positive test for syphilis
* ß2-glycoprotein I -(not a phospholipid but a plasma phospholipid binding protein)
o In early 1990s, discovery that some anticardiolipin antibodies require the presence of ß2-glycoprotein I in order to bind to cardiolipin
o Patients with SLE or the antiphospholipid syndrome require ß2-glycoprotein I in order to bind to cardiolipin
o Most ß2-glycoprotein I-dependent anticardiolipin antibodies recognize ß2-glycoprotein I equally well whether bound to cardiolipin or bound to other anionic phospholipids

Additional info on LAs
* Anticardiolipin antibodies and Anti-ß2- glycoprotein I antibodies may not possess lupus anticoagulant properties
* Specificity of anticardiolipin antibodies for antiphospholipid syndrome increases with titer and is higher for the IgG than for the IgM isotope
* There is no definitive association between specific clinical manifestations and particular subgroups of antiphospholipid antibodies


Effects of antiphospholipid antibodies on coagulation
* Actually has opposing effects on coagulation
Procoagulant Effects
* Inhibits activated protein C pathway
* Up-regulates TF pathway
* Inhibits antithrombin III activity
* Disrupts annexin V shield on membranes
* Inhibits anticoagulant activity of ß2-glycoprotein I
* Inhibits fibrinolysis
* Activates endothelial cells
* Activates and degranulates neutrophils
* Enhances expression of adhesion moleculres by endothelial cells and adherence of neutrophils and leukocytes to endothelial cells
* Potentiates platelet activation
* Enhances platelet aggregation
* Enhanced binding of ß2-glycoprotein I to membranes
* Enhanced binding of prothrombin to membranes
Anticoagulant Effect
* Inhibits activation of factor IX
* Inhibits activation of factor X
* Inhibits activation of prothrombin to thrombin
o “Microenvironment of cell membranes in vivo may promote greater inhibition of anticoagulant pathways and therefore thrombosis.”
o Ultimately, we don’t really know the mechanism by which thrombosis is promoted over anticoagulation

Criteria for detection of lupus anticoagulant antibodies
* Lupus anticoagulant
1. Must prolong coagulation in at least one phospholipid-dependent coagulation assay with the use of platelet poor plasma
+ Extrinsic (dPT)
+ Intrinsic (aPTT, dilute aPTT, KCT, colloidal silica clotting time)
+ Final common pathway (dRVVT, Taipan venom time, Textarin and Ecarin time)

2. Failure to correct the prolonged coagulation time by mixing the patient’s plasma with normal plasma (1:1)
3. Correction of the prolonged coagulation time after addition of excess phospholipid or platelets that have been frozen and then thawed (they release phospholipids)
4. Rule out other coagulopathies with the use of specific factor assays if the confirmatory test is negative or if a specific factor inhibitor is suspected

To rule out a lupus anticoagulant antibody
* Two or more assays that are sensitive to these antibodies must be negative (one should be based on low phospholipid concentration and they should evaluate distinct portions of the coagulation cascade)

Diagnosis of antiphospholipid antibody syndrome
* Clinical Criteria
o Vascular thrombosis (Venous or arterial: blood vessels, brain, kidneys, lung GI tract, placenta etc)
o 1 or more deaths of normal fetuses at or after 10th week of gestation,or 1 or more premature births at or before the 34th week of gestation; or 3 or more unexplained consecutive spontaneous abortions before the 10th week of gestation

* Laboratory criteria
o Anticardiolipin antibodies
+ Anticardiolipin IgG or IgM antibodies present at moderate or high levels in the blood on two or more occasions at least 6 weeks apart
o Lupus anticoagulant antibodies
+ LA detected in the blood on 2 or more occasions at least 6 weeks apart (?12 weeks)

Antiphospholipid syndrome
* Primary
o No other evidence of another autoimmune disease
* Secondary
o Associated with autoimmune or other diseases, most commonly SLE
* Sneddon’s syndrome: clinical triad of stroke, livedo reticularis, and hypertension may represent undiagnosed antiphospholipid syndrome.


Epidemiology
* Antiphospholipid antibodies are found among young, apparently healthy control subjects at a prevalence of 1 to 5% for both anticardiolipin antibodies and lupus anticoagulant antibodies
o Meta-analysis
+ LA= 11.1 Odds ratio for venous thrombosis compared with 3.21 with anticardiolipin Ab
o Multivariant analysis
+ Odds ratio for venous and arterial thromboembolism is 4.4 with LA and 1.2 with anticardiolipin
* Prevalence increases with age

Prevalence of LAs in patients with SLE
* Anticardiolipin antibodies= 12-30%
* Lupus anticoagulant antibodies= 15-34%
* B2glycoprotein I antibodies=20%
o Antiphospholipid syndrome may develop in 50 to 70% of patients with both SLE and antiphospholipid antibodies after 20 years of follow-up
o Up to 30% of patients with SLE and anticardiolipin antibodies lacked any clinical evidence of the antiphospholipid syndrome over an average follow-up of seven years

Prospective study
* In a recent prospective study involving individuals with antiphospholipid antibodies, the incidence of thrombosis per year was:
o 1% in individuals with no history of thrombosis
o 4% in patients with systemic lupus erythematosus
o 5.5% in patients with a history of thrombosis
o 6% in individuals with high titer IgG anticardiolipin antibody (>40 units).

Functional Assays of Lupus Anticoagulants
* aPTT
o Some manufacturers offer aPTT reagent which contains a low amount of phospholipid, therefore it is more sensitive for lupus anticoagulant
o Conditions causing acute phase reactants associated with increased fibrinogen and factor VIII, may shorten the aPTT and mask a weak LA
* Prothrombin Time:
o patients with LA will have a normal PT unless they are receiving oral anticoagulants or they develop an inhibitor to prothrombin (PT reagents contain more phospholipids than PTT reagents)

* DRVVT (screening)
o Activates factor X which in the presence of PL, calcium, and factor V activates prothrombin, leading to the formation of a fibrin clot
o Dilution of the venom yields a clotting time in which concentration of the PL reagent is the rate limiting step (there is low amount of phospholipids)
o Inhibition by LA leads to prolongation
o After positive screen, perform the mixing study- if does not correct then:
* DRVVT (confirm)
o Adds a higher amount of phospholipids to neutralize the lupus anticoagulant
o Ratio is derived from the screen clotting time divided by the confirmatory clotting time
o If ratio exceeds the established cutoff, then lupus anticoagulant is in the specimen

Tissue thromboplastin inhibition test (TTI)
o Modified PT assay
o Thromboplastin, which is rich in phospholipid, can be diluted so that its concentration becomes the rate limiting step
o Inhibition of prothrombinase by a LA will cause prolongation of the PT assay
o Due to the various PL and its concentration in the reagent, the test varies in its sensitivity and specificity

STACLOT LA: Hexagonal II Phase Phospholipid Assay
* Two part aPTT screening assay for LA
* Patient’s plasma is mixed with buffer (screening test) or hexagonal phase phosphatidyl ethanolamine (confirmatory test) to neutralize any lupus anticoagulant present
* Mixtures are incubated with normal plasma to correct any coagulation factor deficiency
* Measure aPTT in both mixtures
* If specimen contains LA, the aPTT of the confirmatory test will be significantly shorter than that of the screening test

Staclot-LA
* Phospholipid antibody positive= difference in the clotting times between the two tubes is greater than + 8.0 seconds.
* The aPTT reagent in this assay contains a heparin inhibitor which makes the test system insensitive to heparin levels up to 2.0 U/mL.
* False positive results may occur in patients with high titer Factor VIII inhibitors

Summary
* Lupus anticoagulant causes thrombosis
* Lupus anticoagulant is a group of antibodies that bind to phospholipids or phospholipid binding proteins
* Due to the heterogeneity of the phospholipid antibody, there is no single test that is confirmatory for all phospholipid dependent antibodies.

References

* Kaolin clotting time
o Sensitive for LA when no additional PL is used
o LA is identified when the KCT fails to correct after the addition of even large amounts of plasma
o Problems with the KCT, owing to the particular nature of kaolin, is that it is unsuitable for some photo-optical devices, which makes full automation difficult
* Taipan (Oxysuranus scutellatus) venom activates prothrombin in the presence of PL and Ca2+
* Textarin (Pseudonaja textiles) acts similarly but requires the presence of factor V
* Specificity of both of the above tests can be improved by mixing tests and/or confirmation with the use of ecarin, an enzyme purified from the venom of Echis carinatus, in conjunction with the Textarin test

Lupus Anticoagulant.ppt