The coagulation system in synovial fluid from inflamed joints is strongly activated, resulting in the deposition of fibrin; these fibrin deposits are called rice bodies (1–4). Joint lavage removes the rice bodies and alleviates the pain.
Coagulation activation requires not only (activated) coagulation proteins and calcium ions, but also a procoagulant surface to which coagulation proteins can bind to assemble tenase and prothrombinase complexes. Membranes exposing negatively charged phospholipids, such as phosphatidylserine (PS), act as such a procoagulant surface. PS is not exposed on resting cells, but during cell activation or apoptosis it appears in the outer leaflet of the membrane. In vitro, platelets are known to release microparticles that expose PS and bind coagulation factors V, VIII, IX, and XI and/or their activated forms (5–8). Monocytes, endothelial cells, and erythrocytes also release PS-exposing microparticles upon appropriate activation, which supports coagulation in vitro (9–11). In vivo, microparticles can be found in the systemic circulation; their numbers and cellular origin are dependent on the state of health of the individual. Compared with healthy control subjects, patients at risk of thromboembolic complications have elevated numbers of platelet microparticles in their venous blood (12–19), and we and other investigators have recently demonstrated the presence of microparticles of nonplatelet origin in the circulation of healthy subjects as well as patients with various clinical conditions (17, 20–22).
Synovial fluid also contains cell-derived microparticles. These microparticles contain relatively high concentrations of lysophospholipids such as lysophosphatidylcholine and lysophosphatidylethanolamine (23). Most likely, the presence of the lysophospholipids is due to secretory phospholipase A2 (sPLA2), a phospholipid-hydrolyzing enzyme found in elevated concentrations in synovial fluid (24). The cellular origin of the microparticles in synovial fluid has not been reported, and the breakdown of the phospholipids to their lyso compounds is likely to inhibit their possible thrombin-generating capacity. The aims of the present study were to characterize the cellular origin of microparticles from synovial fluid and to study their procoagulant properties and, thus, find a possible explanation for the hypercoagulation occurring in synovial fluid of inflamed joints.
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- PATIENTS AND METHODS
The findings of the present study demonstrate that synovial fluid from the inflamed joints of RA patients as well as non-RA patients contains cell-derived microparticles of mainly granulocyte (CD66b, CD66e) and monocyte/macrophage (CD14) origin and, to a lesser extent, CD4+ and CD8+ T cells, B cells (CD20), and erythrocytes (glycophorin A). Synovial microparticles from the 6 RA patients tested and from 4 of the 7 non-RA patients tested evoked thrombin generation via the tissue factor/factor VII–initiating pathway. A subpopulation of synovial microparticles exposed tissue factor, and we found colocalization of tissue factor and the CD66e antigen (i.e., granulocyte origin of the microparticles) on part of this subpopulation. This suggests a role of granulocyte-derived microparticles in local hypercoagulation. The origin of the majority of the tissue factor–exposing microparticles, however, could not be established.
Tissue factor antigen was demonstrated by flow cytometry only in the presence of a threshold concentration of the detergent Triton X-100. In previous studies, especially on microparticles from the pericardial cavity of patients undergoing cardiac surgery with cardiopulmonary bypass, we had noticed discrepancies between the tissue factor–mediated thrombin-generating activity and the apparent absence of the tissue factor antigen. On cells, the presence of cryptic tissue factor is a well-known phenomenon, where activity rather than antigen expression appears cryptic (31). Upon incubation with detergent, the tissue factor exposed on the cell surface becomes active; this has been explained by either changes in membrane phospholipid distribution, conformational changes in the tissue factor molecule, or the multimerization of tissue factor molecules (32–34). In contrast to cells, however, incubation of the microparticles with Triton X-100 completely abolished their ability to induce thrombin generation (data not shown).
The discrepancy between the presence of tissue factor in thrombin-generation experiments and the lack of detection by flow cytometry in the absence of Triton X-100 may simply be due to a lower detection limit of biologic versus antigen assays, involving only a subclass of active tissue factor molecules. Our present findings of granulocyte-derived microparticles bearing tissue factor may support the reports by other investigators that granulocytes are capable of producing tissue factor (35). However, we cannot exclude a possible transfer of tissue factor from other cells (e.g., monocytes) to granulocytes or their microparticles, similar to the transfer between leukocytes and platelets (36). The occurrence of microparticles of granulocyte origin in synovial fluid was confirmed by measuring concentrations of elastase (a protease secreted by activated granulocytes) and by the use of both CD66b and CD66e as granulocyte markers. We therefore propose that the microparticles originated from granulocytes.
As stated above, synovial microparticles generated thrombin via factor VII. In previous studies, we demonstrated that microparticles isolated from the pericardial cavity of patients undergoing cardiopulmonary bypass (22) and from the plasma of a patient with meningococcal sepsis and disseminated intravascular coagulation (17) also triggered thrombin generation via factor VII and tissue factor. In those studies, anti–tissue factor and/or anti–factor VII antibodies merely delayed the onset of thrombin generation by 5–10 minutes, whereas in the present study, thrombin generation was permanently blocked. This may indicate that thrombin generation by these synovial fluid microparticles occurs continually through de novo activation of the coagulation system via tissue factor/factor VIIa, whereas in other conditions, other microparticle populations propagate thrombin generation, for instance, via their negatively charged phospholipid surface, and thus have the extra ability to form tenase and prothrombinase complexes.
We recently showed that microparticles derived from the plasma of healthy subjects slowly generated a modest quantity of thrombin (27). This thrombin generation was independent from tissue factor/factor VII and was only partially sensitive to inhibition by factor XII (10%) or factor XI (40%). Since combinations of antibodies also failed to inhibit more than 40% of thrombin generation, we concluded that thrombin generation by microparticles is, at least in part, independent from the common intrinsic and extrinsic initiation pathways of coagulation activation. Due to the (almost complete) absence of these microparticles in synovial fluid, this alternative route of initiation of thrombin generation may be lacking. Whether this route only amplifies thrombin generation, e.g., via factor VIIIa/IXa and Va that are possibly already present on the surface of microparticles, or whether this is a completely novel coagulation initiation pathway remains to be elucidated. However, we cannot exclude the involvement of elastase, cathepsins, (macrophage) fgl-2 prothrombinase, or (monocyte) Mac-1 integrin in the initiation of coagulation under these conditions.
The high thrombin-generating capacity of synovial microparticles may contribute to local hypercoagulation and account for the fibrin depositions called rice bodies. Actually, this procoagulant nature was not anticipated, because previous studies showed that sPLA2, which is present in high concentrations in synovial fluid, inhibits coagulation (23, 24, 37, 38). In line with such an effect of sPLA2 was the lower exposure of PS by synovial microparticles, as reflected by the reduced binding of annexin V, most likely due to the breakdown of PS to lyso-PS. Despite the reduced binding of annexin V, the synovial microparticles still efficiently supported thrombin generation. Thus, the presence of lysophospholipids apparently does not inhibit thrombin generation, and the amount of intact negatively charged phospholipids on the microparticles still appears to be sufficient to support coagulation.
At present, we have no explanation for the inability of 3 microparticle fractions (from 2 patients with undifferentiated arthritis and 1 patient with symmetric synovitis) to generate thrombin in normal plasma. The binding of annexin V to preparations of microparticles from these 3 patients was comparable to that of microparticle preparations from the other patients, indicating that the lack of thrombin-generating activity is not explained simply by an insufficient exposure of negatively charged phospholipids. However, preliminary experiments indicated that these microparticles expose sufficient negatively charged phospholipids to facilitate the propagation of thrombin generation.
Kaolin as an activator of factor XII requires the presence of negatively charged phospholipids, either microparticles or artificial phospholipid vesicles, to initiate and propagate thrombin generation. Addition of synovial microparticles from 1 of the 3 patients showing no thrombin generation was tested and did indeed propagate the kaolin-induced thrombin generation (data not shown). The absence of thrombin initiation by these microparticles could not be attributed to the absence of tissue factor on the microparticles, which was detectable by flow cytometry and was similar to microparticles from the other patients, which did generate thrombin. We hypothesized that the synovial fluid from this particular patient may contain one or more substances that interfere with thrombin initiation. Synovial microparticles from another patient lost more than 90% of their thrombin-generating capacity after preincubation in microparticle-free synovial fluid from this patient. Although these data are preliminary, they suggest that in some patients, one or more factors that hamper the initiation of thrombin generation by microparticles may be present in the synovial fluid.
Microparticles may also play a role in inflammation. Leukocyte-derived microparticles trigger endothelial cells to produce interleukin-6 (39, 40). P-selectin, which is present on platelet-derived microparticles, triggers monocytes to express not only tissue factor, but also chemokines (41, 42). Platelet-derived microparticles transport arachidonic acid to endothelial cells and induce the expression of cyclooxygenase 2 and the increased production of prostacyclin (43), enhance the interaction between monocytes and endothelial cells (44), and enhance leukocyte aggregation and their accumulation on the endothelium (45). Furthermore, thrombin, factor Xa, and fibrin degradation products are all potent inducers of the inflammatory response (46–50). Hence, the presence of microparticles in synovial fluid may facilitate not only coagulation, but also inflammation via direct and indirect mechanisms.
In summary, microparticles in the synovial fluid of arthritis patients differ in cellular origin from those found in plasma. In the majority of these patients, subpopulations of these microparticles expose tissue factor on their surface, enabling them to generate thrombin. Synovial microparticles may underlie the extensive activation of the coagulation system in the inflamed arthritic joint, leading to local fibrin deposition, which aggravates the condition in both RA and non-RA patients.