72 Annals of the Rheumatic Diseases 1994; 53: 72-79 REVIEW Ann Rheum Dis: first published as 10.1136/ard.53.1.72 on 1 January 1994. Downloaded from Thrombin in inflammation and healing: relevance to rheumatoid arthritis Ruth Morris, Paul G Winyard, David R Blake, Christopher J Morris Thrombin is classically recognised as factor IIa lymphocytes, neutrophils, and monocytes. 1 of the coagulation cascade, where it catalyses This 'prothrombinase' complex then catalyses the conversion of fibrinogen into fibrin and so cleavage ofprothrombin, with the resulting loss aids in the formation of a blood clot. Active of a 32 kD fragment and formation of active thrombin is generated from prothrombin at the thrombin. cell surface of platelets, lymphocytes, mono- As with other stages in the coagulation cytes, neutrophils and endothelial cells.' It cascade, thrombin generation is controlled by belongs to the serine protease enzyme group, several feedback loops which exert both members of which share a common catalytic positive and negative influences on the amount mechanism and also the triad of amino acids of thrombin formed. For example thrombin at their active site (aspartate-serine-histidine) binds to thrombomodulin, a glycoprotein from which their name derives. Thrombin has found in the endothelial cell membrane and the several forms, the largest and most active of membranes of other cell types including which is 39 kD ct-thrombin. It is able to synovial macrophages.5 6 This binding alters catalyse the cleavage of a wide range ofproteins the thrombin molecule so that it no longer including itself. catalyses fibrinogen cleavage, but instead Recently it has been found that thrombin becomes an efficient activator of protein C, a causes a number of pro-inflammatory and serine protease which inactivates Factor Va.7 In mitogenic effects, many of which appear to this way thrombin is able to curtail its own take place after thrombin cleavage of a generation (fig 1). Raised levels ofthrombomo- G-protein coupled transmembrane receptor. dulin have recently been reported in RA These in vitro observations suggest that synovial fluid,8 suggesting that thrombomo- thrombin may be important in the healing dulin may be important in the control of process, acting as a mediator of inflammation thrombin during inflammatory joint disease. and initiating repair through the stimulation of cell growth. It has also been linked with the http://ard.bmj.com/ development of vascular disease, notably ENDOGENOUS THROMBIN INHIBITORS atherosclerosis.2 However, the multiple actions Uncontrolled thrombin action is prevented by of thrombin implicate this enzyme in the members of the serine protease inhibitor pathogenesis of any disorder where there is (serpin) superfamily,9 which embraces a large sustained vascular injury and cellular number of regulatory plasma proteins such as infiltration. plasminogen activator inhibitor-I (PAI-1) and In particular thrombin may be important ot2-antiplasmin. Serpins display sequence on September 25, 2021 by guest. Protected copyright. during the progression of rheumatoid arthritis homology and share common structural (RA), a chronic inflammatory disease marked features, forming stable 1:1 complexes with by increased vascular permeability, synovial serine protease enzymes. They possess a proliferation, and an abnormal angiogenic reactive centre as part of an external response. Extravascular coagulation is also polypeptide loop, and within this reactive seen in RA, with fibrin deposition being a well centre is a bond that can be cleaved by the documented feature of inflamed synovial target enzyme. Particular serpins tend to be tissue. Advances in immunohistochemical more efficient as inhibitors of certain serine techniques have made it possible to demon- proteases, but none are entirely specific. Inflammation strate the existence of intact coagulation Antithrombin III (AT-III) is the main Research Group, pathways within the rheumatoid synovium.34 circulating inhibitor of thrombin, although London Hospital This review describes current knowledge about other serpins may also contribute. It complexes Medical Coliege, how thrombin works to London, United promote successful with thrombin through its reactive arginine- Kingdom healing, and outlines the pathological serine bond,'01' and can also inhibit Factors R Morris relevance of prolonged and inappropriate IXa, Xa, XIa and XIIa. However, thrombin P G Winyard thrombin in RA. D R Blake activity appears able to bind to the subendothelial C J Morris extracellular matrix without loss of function Correspondence to: and, once bound, is protected from inhibition Ms R Morris, Generation and regulation ofthrombin by plasma AT-III.12 It is therefore not Inflammation Research Group, Arthritis and THE PROTHROMBINASE COMPLEX surprising to find a second serpin, protease Rheumatism Council Thrombin is generated through the action of nexin-1 (PN-1), that directly controls Building, 25-29 Ashfield Street, London El 2AD, Factor Xa and Factor Va, which form a thrombin action at the cell surface. PN-1 is United Kingdom. complex together with phospholipids in the secreted by many cell types in vitro including Accepted for publication membranes of platelets, endothelial cells, human and mouse fibroblasts'3 '4 and bovine 15 September 1993 Thrombin in inflammation and healing. relevance to rheumatoid arthritis 73 complex Ann Rheum Dis: first published as 10.1136/ard.53.1.72 on 1 January 1994. Downloaded from PrtImb V Va v !i o- !2_ Protein C -. I. Thrombomodulin Prothrombin h-romI jIThrombin 1.lj Plasma Antithrombin III Heparin cofactor 11 Other inhibitors Cell surface http://ard.bmj.com/ Protease nexin-1 sr Receptor cleavage on September 25, 2021 by guest. Protected copyright. N C' Thrombin receptor Figure 1 Simplified diagram showing generation, action, and inhibition of thrombin. Solid arrows = activation mechanisms, dotted arrows = inhibitory mechanisms. aortic endothelial cells,'5 where it acts primarily presence of glycosaminoglycans such as as a thrombin inhibitor after cell surface heparin and heparan sulphate,'9 22 which are binding.'6 Thrombin inhibition by a further found in mast cells and on endothelial cell plasma serpin, heparin cofactor II (HC-II), has surfaces respectively. In addition, PAI-I also been demonstrated.'7 HC-II differs from AT- becomes an efficient thrombin inhibitor in the III and PN-1 in that its reactive bond contains presence of heparin.23 The involvement of leucine in preference to the usual arginine glycosaminoglycans in coagulation has recently residue.'8 The physiological significance of this been reviewed,24 and heparin itself is used inhibitor is not well understood. clinically as an anticoagulant because of its The rates of thrombin-inhibition by AT-III, ability to accelerate the inhibition of thrombin PN-1 and HC-II are greatly increased in the by antithrombin III. 74 Morrs, Winyard, Morris Inactivation of serpins in inflamed tissues upregulate expression of the adhesive proteins Serpins are susceptible to inactivation during P-selectin and intercellular adhesion molecule- the inflammatory process, owing to the 1 (ICAM-1) on the endothelial cell membrane, exposed location of their reactive centre on the where the enzyme may act both alone and also Ann Rheum Dis: first published as 10.1136/ard.53.1.72 on 1 January 1994. Downloaded from outside of the molecule. Reactive oxygen synergistically with the cytokines IL-1 and species from neutrophils readily oxidise a tumour necrosis factor-ox (TNF-ot;48 49). critical methionine residue, present in the P-selectin itselfhas recently been shown to play reactive centre of several serpins, for example, a major role during monocyte adhesion to the oxl-antitrypsin (ox,-AT;25). Moreover the rheumatoid synovial vasculature.50 presence of inflammatory mediators such as interleukin-1 (IL-1) stimulates the release of various cell-derived protease enzymes.26 MITOGENICITY AND HEALING Neutrophils produce elastase, a serine The importance of platelet derived growth protease, and connective tissue cells secrete factors (PDGF), the fibroblast growth factors metalloproteinases including collagenase and (FGF), and epidermal growth factor in the stromelysins. These enzymes degrade joint proliferation and healing has been recognised tissues. They can also inactivate serpins by for many years. Like these well-known proteolysis within the exposed loop region, and mitogens, thrombin stimulates proliferation in so may disturb the delicate balance between fibroblastic and other cell types, with the thrombin and its inhibitors. response occurring at low concentrations ofthe Such an event has already been enzyme. In vitro thrombin has been shown to demonstrated for neutrophil elastase, whose be mitogenic for fibroblasts, lymphocytes, associated serpin, ot,-AT, can be cleaved by endothelial cells, vascular smooth muscle cells stromelysin.27 28 The proportion of cleaved and monocytes.1-'5 This mitogenesis can be ol-AT in RA synovial fluid is higher than in stimulated by low levels of active thrombin, osteoarthritic synovial fluid or normal serum,29 and we have observed thrombin-stimulated and consequently RA synovial fluid has a proliferation of murine 3T3 fibroblasts in the reduced capacity for elastase inhibition.30 presence of only five NIH units of thrombin Elastase itself will cleave both AT-III and activity/ml culture medium. Fibroblasts are PN-1 in vitro, and after cleavage these serpins able to regulate such thrombin-induced are no longer effective as