Steven A. Krilis

The Pathogenesis of the Antiphospholipid Syndrome

The antiphospholipid syndrome is a prothrombotic disorder associated with autoantibodies. It is associated with obstetrical complications (mainly spontaneous abortion) as well as venous and arterial thrombotic risks. Insights into disease mechanisms have led to new therapies.

Evidence-based recommendations for the prevention and long-term management of thrombosis in antiphospholipid antibody-positive patients: Report of a Task Force at the 13th International Congress on Antiphospholipid Antibodies:

The antiphospholipid syndrome (APS) is defined by the presence of thrombosis and/or pregnancy morbidity in combination with the persistent presence of circulating antiphospholipid antibodies: lupus anticoagulant, anticardiolipin antibodies and/or anti-β2-glycoprotein I antibodies in medium to high titers. The management of thrombosis in patients with APS is a subject of controversy. This set of recommendations is the result of an effort to produce guidelines for therapy within a group of specialist physicians in Cardiology, Neurology, Hematology, Rheumatology and Internal Medicine, with a clinical and research focus on APS.

A Phospholipid-β2-Glycoprotein I Complex Is an Antigen for Anticardiolipin Antibodies Occurring in Autoimmune Disease But Not with Infection

Anticardiolipin antibodies (aCL) purified from patients with autoimmune disease have recently been shown to interact with a phospholipid-binding plasma protein, β2-glycoprotein I (β2-GPI). The aim of this study was to determine whether aCL purified from patients with infection also interact with β2 -GPI. aCL purified from 23 patients with malaria, infectious mononucleosis, tuberculosis, hepatitis A or syphilis did not require the presence of β2-GPI to bind cardiolipin (CL). In contrast, aCL were purified from 11 out of 12 patients with autoimmune disease that bound CL only in the presence of β2-GPI. Thrombotic complications appear to be associated with aCL occurring in autoimmune disease but not with aCL associated with infections. We postulate that this increased risk of thrombosis in the autoimmune group may be due to the presence of aCL that bind CL in association with β2-GPI, a plasma protein with anticoagulant activity.

Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics

Autoantibodies to phospholipid antigens can be characterized using solid phase immunoassays to detect anticardiolipin antibodies (ACA) or in phospholipid‐dependent clotting tests where lupus anticoagulant (LA) activity can be demonstrated. It has not been established whether each activity is due to the same or separate antibody subgroups. Plasma from two patients with high levels of both activities were used for purification of ACA and LA using sequential ion‐exchange, gel‐filtration, and anti‐Ig affinity chromatography. Plasma could be separated into fractions containing each activity in the absence of the other. In these fractions, antibodies responsible for LA activity do not bind to isolated phospholipids in solid phase immunoassays, and conversely antibodies binding in these assays (ACA) do not possess LA activity, suggesting LA are directed against a more complex lipid epitope. In addition, in one patient ACA was of IgG isotype only, whilst LA was due to IgG and IgM isotypes. In this patient, the IgG‐ACA was heterogenous with three peaks clearly separated on ion‐exchange chromatography. Affinity purified antiphospholipid antibodies have been previously prepared from a number of patients using a phospha‐tidyl‐serine column and antibodies purified in this manner possess ACA but not LA activity. Taken together, these data indicate that tests for ACA and LA define separable subgroups of phospholipid binding antibodies, thus explaining the discordance often seen between the two activities.

How we diagnose the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is an acquired thrombophilia, characterized by the occurrence of venous and arterial events. This article examines the laboratory and key clinical aspects of APS. Particular focus is given to anti-beta 2-glycoprotein I (beta(2)GPI) antibodies in view of their recent inclusion in the APS classification criteria. The clinical utility of using the beta(2)GPI enzyme-linked immunosorbent assay, in conjunction with the established lupus anticoagulant assays and cardiolipin enzyme-linked immunosorbent assay, for diagnosing and risk stratifying patients suspected of having APS is discussed. The relative importance of the various assays in diagnosing obstetric APS (early and late gestation miscarriages) is explored. The implications of recent epidemiologic findings for possibly understanding the underlying pathophysiologic mechanisms of obstetric APS are highlighted. Insights into which patients with obstetric APS may be at most risk of thrombotic complications are presented.

Multifocal cutaneous mucormycosis complicating polymicrobial wound infections in a tsunami survivor from Sri Lanka

A man injured in the tsunami of Dec 26, 2004, returned to Sydney for management of his soft-tissue injuries. Despite broad-spectrum antibiotics, surgical wound debridement, and vigilant wound care, his condition worsened. Muscle and fat necrosis developed in a previously debrided thigh wound, and necrotising lesions arose from previous abrasions. Histological analysis showed mucormycosis in three non-contiguous sites, and Apophysomyces elegans was isolated from excised wound tissue. Wound infections, both bacterial and fungal, will undoubtedly add to the morbidity and mortality already recorded in tsunami-affected areas. Other cases [correction] of cutaneous mucormycosis might develop in survivors, but this disease can be difficult to diagnose and even harder to treat, particularly in those remaining in affected regions.

Identification of a new member of the tryptase family of mouse and human mast cell proteases which possesses a novel COOH-terminal hydrophobic extension.

Mapping of the tryptase locus on chromosome 17 revealed a novel gene 2.3 kilobase 3′ of the mouse mast cell protease (mMCP) 6 gene. This 3.7-kilobase gene encodes the first example of a protease in the tryptase family that contains a membrane-spanning segment located at its COOH terminus. Comparative structural studies indicated that the putative transmembrane tryptase (TMT) possesses a unique substrate-binding cleft. As assessed by RNA blot analyses, mTMT is expressed in mice in both strain- and tissue-dependent manners. Thus, different transcriptional and/or post-transcriptional mechanisms are used to control the expression of mTMT in vivo. Analysis of the corresponding tryptase locus in the human genome resulted in the isolation and characterization of the hTMT gene. ThehTMT transcript is expressed in numerous tissues and is also translated. Analysis of the tryptase family of genes in mice and humans now indicates that a primordial serine protease gene duplicated early and often during the evolution of mammals to generate a panel of homologous tryptases in each species that differ in their tissue expression, substrate specificities, and physical properties.

Cardiac disease in the antiphospholipid syndrome: recommendations for treatment. Committee consensus report:

The Committee reviewed cardiac involvement in the antiphospholipid antibody syndrome. The Committee’s recommendations are: Valve abnormalities: anticoagulation is recommended for symptomatic patients with valvulopathy. Prophylactic antiplatelet therapy may be appropriate for asymptomatic patients (recommended by 13/17 experts in an independent review). Committee members disagreed whether corticosteroid therapy is helpful, but agree that distinguishing among presumptive valvulitis (valve thickening on echocardiogram), valve deformity and vegetations is important, as treatment implications may differ. Occlusive arterial disease (angina, myocardial infarction): the Committee recommends aggressive treatment of all risk factors for atherosclerosis (hypertension, hypercholesterolaemia, smoking) and liberal use of folic acid, B vitamins and cholesterol-lowering drugs (preferably statins). Hydroxychloroquine for cardiac protection in APS patients may be considered. The Committee also recommends warfarin anticoagulationfor those who have suffered thrombosis in the absence of atherosclerosis, but recognizesthat developingdata may support the use of antiplateletagents instead. Intracardiac thrombi: the Committee recommends intensive warfarin anticoagulation, and consultation with cardiac surgeons when appropriate. Ventricular dysfunction: the Committee has no recommendations on this aspect of cardiac disease. Pulmonary hypertension: the Committee recommends intensive anticoagulationwith warfarin and clinical trials of bosentan, epoprostenol and other new agents.

Essential role for mast cell tryptase in acute experimental colitis

Patients with inflammatory bowel disease (IBD) have increased numbers of human tryptase-β (hTryptase-β)-positive mast cells (MCs) in the gastrointestinal tract. The amino acid sequence of mouse mast cell protease (mMCP)-6 is most similar to that of hTryptase-β. We therefore hypothesized that this mMCP, or the related tryptase mMCP-7, might have a prominent proinflammatory role in experimental colitis. The dextran sodium sulfate (DSS) and trinitrobenzene sulfonic acid (TNBS) colitis models were used to evaluate the differences between C57BL/6 (B6) mouse lines that differ in their expression of mMCP-6 and mMCP-7 with regard to weight loss, colon histopathology, and endoscopy scores. Microarray analyses were performed, and confirmatory real-time PCR, ELISA, and/or immunohistochemical analyses were carried out on a number of differentially expressed cytokines, chemokines, and matrix metalloproteinases (MMPs). The mMCP-6–null mice that had been exposed to DSS had significantly less weight loss as well as significantly lower pathology and endoscopy scores than similarly treated mMCP-6–expressing mice. This difference in colitis severity was confirmed endoscopically in the TNBS-treated mice. Evaluation of the distal colon segments revealed that numerous proinflammatory cytokines, chemokines that preferentially attract neutrophils, and MMPs that participate in the remodeling of the ECM were all markedly increased in the colons of DSS-treated WT mice relative to untreated WT mice and DSS-treated mMCP-6–null mice. Collectively, our data show that mMCP-6 (but not mMCP-7) is an essential MC-restricted mediator in chemically induced colitis and that this tryptase acts upstream of many of the factors implicated in IBD.

Beta 2-glycoprotein I.

an apparent molecular weight of 50 kD and is highly glycosylated . Schultze et all reported the carbohydrate content of !32GPI as being approximately 18% of the molecular weight. When tested in phosphate buffer at pH 7.4, P2GPI existed as 40% !3-sheet, 30% 0-tum and 30% random coil. They suggested that after removal of at least 90% of the carbohydrate (deglycosylation) from P2GPI, the primary structure of the polypeptide chain is maintained but the secondary structure alters with greater number of !3-turns, accompanied by reduction in random coil structures. P2GPI inhibits contact activation of the intrinsic coagulation pathway8, platelet prothrombinase activity~ and ADPinduced platelet aggregation. P2GPI can bind negatively charged macromolecular structures other than phospholipids, including DNA&dquo;, heparin1Z, platelets 13 and negatively charged phospholipid vesicles 14