Native- Human Plasma
Autoantibodies directed to negatively charged phospholipids, determined as anti-cardiolipin and/or lupus anticoagulant (LA), have been detected in the serum of patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). APS is characterised by high incidences of venous and/or arterial thrombosis, recurrent spontaneous abortions and thrombocytopenia. It is now known that serum cofactors are required for the binding of phospholipids by autoantibodies in the sera of patients with APS.
ß2-glycoprotein 1 was the first target protein identified and autoantibodies directed against this protein are known to be strongly associated with thrombosis. More recently autoantibodies to prothrombin have been implicated in LA activity. The detection of anti-prothrombin antibodies is now considered to be confirmatory for LA and to have a very high specificity for the diagnosis of APS.
Prothrombin (also known as coagulation factor II or F2) is a vitamin K-dependent glycoprotein that is physiologically activated by the prothrombinase complex (activated factor X, factor V, calcium and phospholipids) via a series of proteolytic cleavages to yield α-thrombin. As well as triggering fibrinogen polymerisation into fibrin, α-thrombin also acts on factors V, VIII, and XIII as well as protein C, platelets and endothelial cells.
The amino acid sequence of prothrombin determined by cloning and sequencing its cDNA reveals a 622 amino acid protein of about 70 kDa. Mature prothrombin that circulates in blood consists of a single 65 kDa polypeptide chain of 579 amino acids due to the removal of signal and propeptide sequences at the N-terminal. The fact that the protein migrates on SDS-electrophoresis with a much larger apparent molecular weight (95 kDa) suggests that it is extensively glycosylated. The protein also exhibits a cluster of 10 γ-carboxyglutamic residues, referred to as the GLA domain, which is essential for the calcium-dependent binding of phospholipid to prothrombin.
As well as the α-thrombin domain there are also 2 kringle domains that are believed to interact with factor V during the assembly of the prothrombinase complex. Anti-prothrombin autoantibodies appear to be conformation-dependent and do not react with prothrombin in Western blot.
Systemic Lupus Erythematosus
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