from patients with rheumatoid arthritis Inactivation of antithrombin III in synovial fluid and

Objective—To investigate the thrombin inhibitory capacity of antithrombin III in the inflamed human joint. Methods—Thrombin inhibitory capacity was measured, using a kinetic spectophotometric method, in matched plasma and synovial fluid samples of patients with rheumatoid arthritis (n=22) and osteoarthritis (n=16), together with normal control plasma samples (n=13). In the same samples, the concentration of antithrombin III was also determined by the method of radial immunodiVusion. The combination of these measurements allowed the calculation of the specific thrombin inhibitory capacity of these samples. Results—An increased concentration of antithrombin III in rheumatoid compared with osteoarthritic synovial fluid was noted (p<0.05). However, there was a significant depression in the specific activity of antithrombin III in rheumatoid synovial fluid when compared with matched plasma samples (p<0.001) or with osteoarthritic synovial fluid (p<0.05). Conclusion—In rheumatoid synovial fluid the thrombin inhibitory capacity of antithrombin III is disproportionately depressed relative to the concentration of antithrombin III, indicating the inactivation of antithrombin III in the rheumatoid joint. (Ann Rheum Dis 1998;57:162–165) Thrombin is a multifunctional enzyme that plays a central part in haemostasis and coagulation. It cleaves fibrinogen to form the fibrin clot and can activate factors V, VIII, XIII, and protein C and is a potent stimulator of platelet aggregation. Aside from these functions, thrombin has pro-inflammatory properties with a number of eVects on the vascular endothelium, including stimulation of neutrophil adhesion to the vessel wall and of prostacyclin release through upregulation of arachidonic acid. Thrombin also acts as a mitogen for synovial cells and there is increased thrombin receptor expression by cells within the rheumatoid synovium. Fibrin deposition is a prominent finding within the rheumatoid synovium and hyperfibrinogenaemia is associated with rheumatoid arthritis (RA). The most important physiological inhibitor of thrombin is antithrombin III (AT III). This single chain glycoprotein of molecular weight 58 000 is synthesised primarily in the liver and belongs to the serpin (serine proteinase inhibitor) superfamily. Serpins display sequence homology, share common structural features, and form a 1:1 complex with their cognate proteinase. A common structural feature of serpins is an exposed polypeptide loop that contains the reactive centre involved in proteinase inhibition. In the case of AT III, this is centred on Arg 393. The rate of thrombin inhibition by AT III is greatly enhanced by the presence of glycosaminoglycans, such as heparin. In vitro, serpins are susceptible to the inactivation of their proteinase inhibitory activity by proteolytic attack at an exposed loop of the polypeptide chain (which contains the reactive centre) and/or oxidative inactivation involving reactive oxygen/nitrogen species. Both metalloproteinases and reactive oxygen species are thought to contribute to biomolecular damage within the RA joint. Previous studies have shown a decreased concentration of AT III in RA synovial fluid compared with RA plasma but the concentration of AT III protein has not been related to the thrombin inhibitory capacity of these fluids to obtain the specific activity of synovial fluid AT III. In vitro, ATIII is proteolytically inactivated by metalloproteinases and by neutrophil elastase. We therefore hypothesised that in RA, AT III may be inactivated, thus potentiating the pro-inflammatory actions of thrombin. Method Matched samples of plasma and synovial fluid were obtained from 38 patients. The 22 patients (16 female, 6 male) with RA and mean age of 64 years (range 45–84) satisfied the 1987 ARA revised criteria. The 16 patients (9 female, 7 male) with osteoarthritis (OA) of the knee and mean age of 71 years (range 54–81) were diagnosed on combined clinical and radiological grounds, which included pain on joint use and the presence of joint space narrowing with osteophytosis in at least one tibiofemoral compartment of the knee joint in all cases. Plasma was also obtained from 13 healthy subjects (7 female, 6 male) with a mean age of 36 years (range 21–53). All samples were collected in EDTA, centrifuged at 1500 g for 10 minutes, and immediately frozen at −70°C until analysis. All of the patients with RA were receiving disease modifying drugs or non-steroidal antiinflammatory drugs, or both, while most of the OA patients were receiving either no drug or paracetamol. No patients were receiving corticosteroids, nor had any received Ann Rheum Dis 1998;57:162–165 162 Department of Rheumatology, Royal Hampshire County Hospital, Winchester HW Jones N L Cox Inflammation Research Group, St Bartholomew’s and the Royal London School of Medicine and Dentistry, London R Bailey Z Zang D R Blake C J Morris P G Winyard Department of Rheumatology, Christchurch Hospital, Christchurch K A Dunne