Patients with meningococcal sepsis generally suffer from disseminated intravascular coagulation (DIC). The aim of this study was to address whether these patients have elevated numbers of circulating microparticles that contribute to the development of DIC. Plasma samples from 5 survivors, 2 nonsurvivors, and 5 healthy volunteers were analyzed for the presence of microparticles by flow cytometry. Ongoing coagulation activation in vivo was quantified by enzyme-linked immunosorbent assay of plasma prothrombin fragment F1 + 2, and procoagulant properties of microparticles in vitro were estimated by thrombin-generation assay. On admission, all patients had increased numbers of microparticles originating from platelets or granulocytes when compared with controls (P = .004 and P = .008, respectively). Patients had elevated levels of F1 + 2 (P = .004), and their microparticles supported thrombin generation more strongly in vitro (P = .003) than those of controls. Plasma from the patient with the most fulminant disease course and severe DIC contained microparticles that expressed both CD14 and tissue factor, and these microparticles demonstrated extreme thrombin generation in vitro. We conclude that patients with meningococcal sepsis have elevated numbers of circulating microparticles that are procoagulant. These findings may suggest a novel therapeutic approach to combat clinical conditions with excessive coagulation activation.

To cite this article: Tesselaar MET, Romijn FPHTM, van der Linden IK, Prins FA, Bertina RM, Osanto S. Microparticle-associated tissue factor activity: a link between cancer and thrombosis? J Thromb Haemost 2007; 5: 520-7.Summary. Background: Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis. Patient/methods: We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects. Results: Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TFactivity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19-0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model. Conclusions: Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

Background: Cancer, in particular mucinous adenocarcinomas, is associated with an increased risk of venous thromboembolism (VTE). Tissue factor (TF), a transmembrane-receptor protein and initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF is associated with poor differentiation of tumors and poor prognosis. We initiated a study in cancer patients, to find out whether microparticles (MP) derived from malignant epithelial cells, may directly initiate blood coagulation locally or at distant sites via TF expressed on these MP and play a critical role in the development of the hypercoagulability Patients & Methods: MP were isolated from peripheral blood samples obtained from healthy subjects, and unselected cancer patients, i.e. patients with primary breast carcinoma (before and after surgery), with metastatic breast carcinoma and with pancreatic carcinoma. Tissue factor procoagulant activity associated with isolated MP was studied by factor VIIa dependent generation of factor Xa. MP were examined by flow cytrometric phenotyping, and electron and confocal immunofluorescence laser scanning microscopical examination. Results: Compared to 37 healthy sujects, significantly increased levels of MP-associated TF activity were found in patients with disseminated breast and pancreatic cancer. Pancreatic (n=5) and breast cancer patients (n=2) who presented with clinically manifest venous thrombosis had a 18-fold increase in MP-associated TF activity as compared to healthy subjects or 6 subjects with idiopathic VTE (P<0.003). In all individuals most of the circulating MP expressed the platelet antigen CD61. MP expressing the epithelial antigen MUC1 -most likely derived from malignant cells- were found in 65% of metastatic breast and 59% of advanced c.q. metastatic pancreatic patients. Co-expression of CD61 and MUC1 on MPs was shown by flow cytometry and confocal immunofluorescence microscopy. Conclusion: Highly elevated MP-associated TF activity significantly correlated with development of VTE and with the presence of circulating MUC1-positive MP, suggesting a decisive role in the pathogenesis of the prothrombotic state in cancer patients with disseminated mucinous carcinomas. Patients with a low level of TF-activity on MP that also lacked expression of mucin had a higher survival rate at 3–9 months follow-up than those with a high TF-activity and mucin present: the estimated risk of dying was about 0.42 (95% CI 0.19–0.94) for an individual with these 2 predictor variables present, adjusting for the other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model.

Summary Aim. We investigated the occurrence and thrombin generating mechanisms of circulating microparticles (MP) in patients with multiple organ dysfunction syndrome (MODS) and sepsis. Methods. MP, isolated from blood of patients (n = 9) and healthy controls (n = 14), were stained with cell-specific monoclonal antibodies (MoAbs) or anti-tissue factor (anti-TF) MoAb and annexin V, and analyzed by flow cytometry. To assess their thrombin-generating capacity, MP were reconstituted in normal plasma. The coagulation activation status in vivo was quantified by plasma prothrombin fragment F1+2- and thrombin-antithrombin (TAT) measurements. Results. Annexin V-positive MP in the patients originated predominantly from platelets (PMP), and to a lesser extent from erythrocytes, endothelial cells (EMP) and granulocytes (GMP). Compared to healthy controls, the numbers of annexin V-positive PMP and TF-exposing MP were decreased (p = <0.001 for both), EMP were decreased (E-selectin, p = 0.003) or found equal (CD144, p = 0.063), erythrocyte-derived MP were equal (p = 0.726), and GMP were increased (p = 0.008). GMP numbers correlated with plasma concentrations of elastase (r = 0.70, p = 0.036), but not with C-reactive-protein or interleukin-6 concentrations. Patient samples also contained reduced numbers of annexin V-negative PMP, and increased numbers of erythrocyte-derived MP and GMP (p = 0.005, p = 0.021 and p <0.001, respectively). Patient MP triggered thrombin formation, which was reduced compared to the healthy controls (p = 0.008) and strongly inhibited by an anti-factor XII MoAb (two patients), by anti-factor XI MoAb (eight patients) or by anti-TF MoAb (four patients). Concentrations of F1+2 and TAT were elevated (p = 0.005 and p = 0.001, respectively) and correlated inversely with the number of circulating MP (and r = –0.51, p = 0.013, and r = –0.65, p = 0.001, respectively) and their thrombin generation capacity (F1+2: r = –0.62, p = 0.013). Conclusions. In patients with MODS and sepsis relatively low numbers of MP are present that differ from controls in their cellular origin, numbers and coagulation activation mechanisms.

Objective. To determine the cellular origin of synovial microparticles, their procoagulant properties, and their relationship to local hypercoagulation.Methods. Microparticles in synovial fluid and plasma from patients with rheumatoid arthritis (RA; n ‫؍‬ 10) and patients with other forms of arthritis (non-RA; n ‫؍‬ 10) and in plasma from healthy subjects (n ‫؍‬ 20) were isolated by centrifugation. Microparticles were identified by flow cytometry. The ability of microparticles to support coagulation was determined in normal plasma. Concentrations of prothrombin fragment F 1؉2 (by enzyme-linked immunosorbent assay [ELISA]) and thrombin-antithrombin (TAT) complexes (by ELISA) were determined as estimates of the coagulation activation status in vivo.Results. Plasma from patients and healthy controls contained comparable numbers of microparticles, which originated from platelets and erythrocytes. Synovial microparticles from RA patients and non-RA patients originated mainly from monocytes and granulocytes; few originated from platelets and erythrocytes. Synovial microparticles bound less annexin V (which binds to negatively charged phospholipids) than did plasma microparticles, exposed tissue factor, and supported thrombin generation via factor VII. F 1؉2 (median 66 nM) and TAT complex (median 710 g/liter) concentrations were elevated in synovial fluid compared with plasma from the patients (1.6 nM and 7.0 g/liter, respectively) as well as the controls (1.0 nM and 2.9 g/liter, respectively). Conclusion. Synovial fluid contains high numbers of microparticles derived from leukocytes that are strongly coagulant via the factor VII-dependent pathway. We propose that these microparticles contribute to the local hypercoagulation and fibrin deposition in inflamed joints of patients with RA and other arthritic disorders.