Antiphospholipid Syndrome (APS ) Michigan State University Houria Suzy Hassouna No relevant conflicts of interest to declare

We are born with what some psychologists call an “explanatory drive.” You give a baby a strange object or something that doesn’t make sense and she will become instantly absorbed; using all her abilities — taste, smell, force — to figure out how it fits in with the world. The Question-Driven Life By DAVID BROOKS Rift Valley, KenyaDAVID BROOKS Hassouna June 9, 2012

Hassouna June Diagnosis and management of bleeding and thrombotic diseases Professors with “ Explanatory Drive” Shinichiro Uchiyama (not in photo Akitada Ichinose ( not in photo) John Penner Tadanori Kawada Jin Shiomura

Antiphospholipid Syndrome ( APS) Diagnostic Challenge Primary APS is an acquired thrombophilia (primary auto-immune disorder, not associated with another autoimmune disorder) Secondary APS Is the expression of APS in conjunction with other autoimmune disorders Treatment Options Many specialties of involved physicians includes: hematology, rheumatology, obstetrics, neurology, cardiology and nephrology among others. Do different backgrounds influence the choice of treatment? Hassouna June 9,

Antiphospholipid Syndrome ( APS) 1963 Edward John Walter Bowie, MD Mayo Medical School identified APS as an acquired thrombophilia 1984 Graham Robert Vivian Hughes MD FRCP. St Thomas Hospital UK identified APS as a generalized autoimmune disorder hassouna

Evolving history of APS From prolonged clotting times that do not correct in mixing of SLE patient plasma To the thromboembolic, venous and arterial events, obstetric complications and phospholipid antibodies that presently define APS Hassouna June 9, 2012

Several investigators reported prolonged clotting times that do not correct in mixing of SLE patient plasma with normal plasma. They named the substance Circulating Anticoagulant, Acquired Inhibitor, Lupus Anticoagulant Conley CL Harman RC. A hemorrhagic disorder caused by circulating anticoagulants in patients with disseminated lupus erythymatosus. J Clin Invest 1952; 31: Lechner K Acquired inhibitors in auto and isoimmune diseases. Hemostasis 1974 ; 3:65-69 Feinstein DI, Rappaport SI. Acquired inhibitors of blood coagulation In Sapet TN, Eds; Progress in Hemostasis and Thrombosis, 1rst ed. Grune and Stratton New York, NY: 1982 ;6: Hassouna June 9, 2012

APS is considered a hemorrhagic disease 1963 is linked to thrombosis LA is identified Circulating anticoagulants in SLE are named lupus anticoagulants ( LA) because they prolong the clotting times in phospholipid dependent assays. LA believed to cause clinical hemorrhagic disease 1963 LA are reported by Bowie et al to be associated with thrombosis in SLE patients LA are identified by Harris et al to be antibodies that react with cardiolipin Lupus anticoagulants react with purified cardiolipin and they are identified as anticardiolipin antibodies by RIA Hassouna June 9, 2012

In 1963, Dr Bowie reported several cases of thrombosis in SLE despite circulating anticoagulants. This was the first report of an acquired thrombophilia. Thrombophilia is an inherited or acquired clinical phenotype that manifests in selected individuals as a greater risk to develop thrombosis in response to an ENDOGENOUS stimulus than the general population. APS is an acquired thrombophilia, ENDOGENOUS stimulus is IMMUNE AUTO-ANTIBODIES. Bowie EJW, Thompson JH,, Pascuzzi CA, Owen CA. Thrombosis in systemic lupus erythematosus despite circulating anticoagulants. J Lab Clin Med 1963; 62: Hassouna June 9, 20129

Hassouna September 7,

Hassouna June 9, 2012 Gala dinner Penner Blood Coagulation Conference held in honor of Walter Bowie. Barbara Alving, NHLBI Walter and Trudy Bowie, Dr and Mrs. David Ginsburg, Howard Hughes Institute U of M John Penner. 11

In 1984, a group of rheumatologists led by Graham Hughes found that in their sizeable collection of SLE patients the Lupus anticoagulant occurred with some frequency and was associated with thrombosis and thrombocytopenia and this raised the possibility that patients with the lupus anticoagulant and anti-cardiolipin antibodies might be a part of a larger body of patients with systemic auto-immune diseases Hassouna September 7,

background who we are what we do overview symptoms diagnosis treatment research prognosis patient support membership publications patient FAQs research conferences your options membership fundraising shop activities news events research publicity Hassouna February 2,

Criteria that define APS Clinical symptoms obstetric complications, arterial and venous thrombosis. Patient is at high risk if one of the clinical symptoms is not confirmed. APS is confirmed by a specific biological marker Lupus anticoagulant Differential diagnosis Primary or secondary APS Treatment targets the cause of the disease. Hassouna June 9, 2012

Fig 2 Clinical manifestations of antiphospholipid syndrome. Cohen D et al. BMJ 2010;340:bmj.c2541 ©2010 by British Medical Journal Publishing Group Hassouna June 9, 2012

In 1963 Walter Bowie reported several cases of thrombosis in SLE despite ‘Lupus Anticoagulants” ( circulating anticoagulants) Lupus anticoagulant generates a prolonged clotting time in patient plasma that does not correct when mixed in equal volume with pooled normal plasma. The identity of the lupus anticoagulant remained a mystery until 1983 ( 20 years) Hassouna June 9, 2012

Two mechanisms are identified in the induction of autoimmune Antiphospholipid antibodies (a PL). Apoptotic phospholipid beta 2 glycoprotein-1 complexes. Phospholipids released from injured cells bound to beta 2 glycoprotein 1 Infection related antiphospholipid antibodies. From experiments in mice, antiphospholipid antibodies are produced by binding of beta2- glycoprotein 1 to phospholipid viral or bacterial proteins by molecular mimicry June 9,

In 1983 Lupus anticoagulant was identified by the anticardiolipin test to be autoimmune antiphospholipid antibodies Introduction of the anticardiolipin test identified the lupus anticoagulant as auto-antibodies directed at cardiolipin, a phospholipid in inner mitochondrial membranes Harris EN, Gharavi AE, Boey ML. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983; 2: Hassouna September 7,

Cardiolipin is a phospholipid on the inner membrane of mitochondria first identified in bovine heart. Anticardiolipin antibodies ( lupus anticoagulant) represent an autoimmune process Hassouna June 9, 2012

Autoimmune anti-phospholipid antibodies are generated by apoptotic phospholipid protein complexes in mitochondria and other cellular components Increased apoptosis as observed in blood of SLE patients, followed by enhanced phosphatidyl serine exposure provides molecular signal to macrophages and other antigen presenting cells leading to autoimmune antiphospholipid antibodies Hassouna June 9, 2012

Antiphospholipid antibodies The most significant target antigen is the Beta 2 glycoprotein-1 In 1990 Monica Galli and her group in Bergamo, Italy reported that the true antigenic targets of antiphospholipid antibodies are not the phospholipids but a plasma protein beta 2 glycoprotein-1 bound to an anionic surface. Galli M,Comfurius P, Massen C et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein co-factor. Lancet 1990;334: Hassouna June 9, 2012

Beta-2 glycoprotein 1, also known as apolipoprotein H is an anticoagulant plasma protein belonging to the complement control protein family, and it is present in plasma at a concentration of approximately micrograms per milliliter It is the major brain anticoagulant Hassouna June 9, 2012

Beta 2 glycoprotein 1 prevents clotting factor assembly on phospholipid membranes and clotting activation Beta-2 glycoprotein 1 anticoagulant action is by interaction with anionic phospholipids via a lysine rich region in the extra 20 amino acid C-terminal loop of the fifth domain that prevents thrombin amplification by the prothrombinase activity Hassouna June 9, 2012

Beta-2 glycoprotein 1 prevents ADP induced platelet aggregation via interaction with Apo ER 2 platelet receptor ( anticoagulant action) auto antibody against beta2-glycoprotein 1 prevents dimerization and this interaction with Apo ER 2 enhances platelet procoagulant activity Hassouna June 9,

Antiphospholipid Syndrome has 3 components: Lupus anticoagulant is a term applied to a prolonged clotting time in a phospholipid dependent clotting test Antiphospholipid antibodies is a general term applied to antibodies generated by an autoimmune or an infectious process An acquired thrombophilia ( arterial and venous thrombosis, recurrent pregnancy loss, thrombocytopenia) caused by autoimmune or infectious processes that generate antibodies harmful to endothelium and platelets. Hassouna June 9,

Antibodies to bacteria or viruses recognize β2GPI phospholipid binding site Evidence in animal models that antibodies binding to β2GPI 15 amino acid sequence GDKV a major β2GPI phospholipid binding site is induced by molecular mimicry. Immunization of BALB/c mice with Hemophilus influenza Neisseria gonorrhoeae Tetanus toxoid Resulted in high titer antibody to β2GPI 15 amino acid sequence GDKV a major β2GPI phospholipid binding site synthesized by Gharavi et al. Hassouna June 9, 2012

Computer generated model of autoimmune anti-phospholipid antibody UK4 UK 4 is directed at β2GPI 15 amino acid sequence GDKV a major β2GPI phospholipid binding site synthesized by Gharavi et al. Beta 2 glycoprotein 1 Purple wire UK-4 white. Contact in light chain Yellow Kumar S, Nagl SCB, Kalsi JK, Ravirajan CT et al. Beta 2-glycoprotiein specificity of human antiphospholipid antibody resides on the light chain Molecualr immunology 42: Hassouna June 9,

Experimental APS is induced in naive mice by molecular mimicry Immunization with microbial particles and tetanus toxoid having sequence homology to β2GPI 15 amino acid sequence GDKV a major β2GPI phospholipid binding site synthesized by Gharavi et al. causes thrombocytopenia, fetal loss and prolonged APTT Hassouna June 9, 2012

Catastrophic APS Asherson Syndrome Lupus anticoagulant discriminates from DIC and TTP There is a small group of APS patients who present with occlusive events at multiple sites over a short period of time resulting in multi organ failure. The majority of catastrophic episodes are preceded by a precipitating events mainly infections Less than 1% of APS patients Hassouna June 9, 2012

Fig 1 Classic histological findings in a patient with catastrophic antiphospholipid syndrome. Lupus anticoagulant differentiates fulminant DIC from Catastrophic APS. Clinical symptoms are identical Cohen D et al. BMJ 2010;340:bmj.c2541 ©2010 by British Medical Journal Publishing Group Hassouna June 9, 2012

Guillermo RI,Crowther M, Branch WKamashta NA. Antiphospholipid Syndrome Lancet 2010: Of the different antibodies The lupus anticoagulant Is the strongest predictor of features related to antiphospholipid syndrome Hassouna June 9, 2012

Significance of the Lupus anticoagulant in the diagnosis of APS Hassouna June 9, 2012

Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the APS syndrome: a systemic review of the literature. Monica Galli, David Luciani, et al Blood 2003;101:

Clinical Utility of Laboratory tests used to identify APS antibodies and to diagnose the APS Galli M Semin Thromb Hemost 2008; 34: Lupus Anticoagulant is essentially a measure of the functional activity of subgroups of auto-immune antibodies directed against beta2-glycoprotein 1 Hassouna June 9, 2012

Identity of the Lupus anticoagulant depends on the capture antibody in the ELISA Enzyme labeled 1.Anticardiolipin antibodies directed against antigen cardiolipin bound to beta-2 glycoprotein 1 2.Antiphospholipid antibodies directed against phospholipid bound to beta2 glycoprotein 1 3.Anti beta2 glycoprotein -1 antibodies directed against antigen beta 2 glycoprotein-1 37

Special Coagulation Laboratory Recipe for APS diagnosis APTT clotting times must be prolonged at least twice control plasma clotting times (correlates with high antibody titer) APTT prolonged clotting times does not correct in mixing patient plasma with pooled normal plasma ELISA identifies beta2-glycoprotein 1 antigen and auto-antibodies. Testing is repeated as indicated. APS induced by viral or bacterial proteins disappears after infection is cured Hassouna June 9, 2012

Summary Antiphospholipid Syndrome has 3 components: Lupus anticoagulant is a term applied to a prolonged clotting time in a phospholipid dependent clotting test. Clotting times correlate with antibody titer Antiphospholipid antibodies is a general term applied to antibodies generated by an autoimmune or an infectious process An acquired thrombophilia ( arterial and venous thrombosis, recurrent pregnancy loss, thrombocytopenia) caused by autoimmune or infectious processes that generate antibodies harmful to endothelium and platelets. Hassouna September 7,

Summary Lupus Anticoagulant is the “ identifier” or “ biomarker” unique to the Antiphospholipid syndrome Auto-antibodies that prolong clotting times in phospholipid dependent clotting assays are termed “lupus anticoagulant” Hassouna June 9, 2012

Antiphospholipid Syndrome Clinical Phenotype Arterial and venous Thrombosis Obstetric complications Recurrent miscarriages Pre-eclampsia Intrauterine growth retardation Thrombocytopenia Hassouna June 9, 2012

Intensity and duration of anticoagulation therapy in antiphospholipid syndrome (APS) Les I,Ruiz-Irastorza G, Khamashata M.A. Semin Thromb Hemost 2012:38:

Hassouna June 9, 2012

Intensity and duration of anticoagulation therapy in antiphospholipid syndrome (APS) Les I,Ruiz-Irastorza G, Khamashata M.A. Semin Thromb Hemost 2012:38:

Hassouna June 9, 2012

Conclusion Diagnosis is absolutely necessary for the proper clinical management A patient with Lupus anticoagulant and without thrombosis or recurrent obstetric complications is at high risk for APS, and should be managed accordingly In our experience, it is possible to remove antibodies in APS induced by molecular mimicry after complete eradication of the bacteria by pheresis (plasma exchange). Success is indicated by APTT clotting times significantly shortened to ( almost baseline levels over 6 months Hassouna June 9, 2012

Bergamo was built by Emperor Justinian ( )

Monastery of St Catherine in the Sinai was built by the Emperor Justinian I ( ) at the site where Moses is supposed to have seen the burning bush