Amounts Of Tissue Factor Activity Research Articles

Transmission of Procoagulant Properties to Daughter Generations of Endothelial Cells upon Exposure to Cancer Cell-Derived Microparticles. a New Implication of Cancer Derived Microparticles in the Pathogenesis of Cancer Associated Thrombosis

Introduction & Objectives: Endothelium activation by cancer cell derived MPs (CaCe-dMP) is essential in pathogenesis of cancer associated thrombosis (CAT). Recent studies demonstrated that exposed endothelial cells to CaCe-dMP acquire a new procoagulant phenotype characterized by endocytosis of tissue factor (TF) and enhancement of thrombin generation.The aim of the present study was to investigate whereas the procoagulant properties acquired upon exposure of endothelial cells to CaCe-dMP are transferred to daughter generations of the endothelial cells.Materials & Methods: Pancreas adenocarcinoma cells (BXPC3) and human breast cancer cells (MCF7) were cultured in 75 cm2 culture flasks at 37˚C in a 100% humidified atmosphere with 5% CO2 and were used for experiments at a confluence of 70%. Microparticles released in conditioned medium from BXPC3 and MCF7 cells were isolated by differential centrifugation. The pellets were washed twice and re-suspended in 1 ml endothelium cell basal medium (EBM-2). Human umbilical vein endothelial cells (HUVEC) were cultured for 72 h in the presence or absence of CaCe-dMP (mother generation : C0). Subsequently, they were washed and re-cultivated over three cycles to get daughter cell generations (Daughter Generations : C1, C2, C3). Tissue factor activity (TFa) was assessed with a chromogenic assay in the absence or presence of cells in normal PPP. Procoagulant phospholipid-dependent clotting time (Proag-PPL): was measured with STA®Procoag-PPL assay. Thrombin generation was studied as described elsewhere using Calibrated Automated Thrombogram®. All assays were from Diagnostica Stago (Asnières, France).Results: The HUVEC and their microparticles manifested faint amounts of TFa (0.18±0.03 pM and 1.2±1.0 pM respectively). At steady state, HUVEC did not induce any detectable thrombin generation. Abundant TFa was showed by BXPC3-dMP (92±50 pM). The levels of TFa were significantly lower at MCF7-dMP (12.1±2.2 pM; p<0.05). Following exposure to BXPC3-dMP, the HUVEC abundantly displayed TFa as compared to non-exposed cells (25±3 pM versus 0.18±0.03 respectively; p<0.001) and showed significantly shorter Procoag-PPL® as compared to non-exposed cells (56±2 sec versus 102±10 sec. respectively ; p<0.001). HUVEC exposed to MCF7-dMP showed significantly increased TFa as compared to non-exposed cells (0.75±0.2 pM versus 0.18±0.03 respectively; p<0.001) and reduced the Procoag-PPL® clotting time as compared to non-exposed cells (65±3 sec versus 102±10 sec respectively; p<0.001). HUVEC exposed to CaCe-dMP significantly increased thrombin generation as compared to non-exposed HUVEC. Data of thrombin generation experiments are summarized in Table 1.Daughter HUVEC from mother cells exposed to CaCe-dMP exhibited significantly higher TFa as compared to daughter generations from HUVEC non-exposed to CaCe-dMPs. The high TFa was conserved in three generations of cells derived from those exposed to CaCe-dMPs (Figure 1). Daughter generations of HUVEC exposed to CaCe-dMP induced significantly higher thrombin generation as compared to that induced by daughter generations of non exposed HUVEC (Table 1). The TFa of daughter cells stemming from HUVEC exposed to BXPC3 was significantly higher as compared to that of daughter cells stemming from HUVEC exposed to MCF7 (Figure 1). The Procoag-PPL® did not significantly change in daughter HUVEC exposed to microparticles derived either from BXPC3 or MCF7.Conclusion: Upon exposure to CaCe-dMP, endothelial cells acquire a procoagulant phenotype marked by abundant TFa and enhanced thrombin generation potential. The procoagulant properties are transferred to the daughter generations of cells. These results support the concept that CaCe-dMPs lead to a procoagulant shift of endothelial cells which, upon exposure, present high TF activity and enhance thrombin generation. The procoagulant shift of endothelial cells can be transferred to next generations of endothelial cells and could be an additional mechanism - together with cancer-induced blood hypercoagulability - in the pathogenesis of cancer associated thrombosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of thromboxane-dependent platelet activation in venous thrombosis: Aspirin effects in mouse model

Recent trials suggest that Aspirin (ASA) reduces the incidence of venous thromboembolism in human. However, the molecular mechanisms underlying this effect are still unclear. In this study we assessed the effects of ASA in venous thrombosis mouse model induced by inferior vena cava (IVC) ligation and we investigated the mechanisms responsible for this effect.ASA (3mg/kg daily for 2 days) treatment decreased the thrombus size, the amounts of tissue factor activity in plasma microvesicles (TF-MP) and the levels of 2,3-dinor Thromboxane B2 (TXB-M) in urine compared to control mice. Interestingly, the thrombus size positively correlated with both TF-MP activity and TXB-M. In addition, positive correlation was observed between TF-MP activity and TXB-M. A reduced number of neutrophils and monocytes, and of TF-positive cells accompanied to a lower amount of fibrin and neutrophil extracellular traps (NETs) were also found in thrombi of ASA-treated mice. Similar results were obtained when mice were treated 24h before IVC ligation with SQ29548 (1mg/kg), a selective thromboxane receptor antagonist. In addition, transfusion of platelets in SQ29548 treated-mice excluded the likelihood of a redundant role of platelet-TP receptor in this context.Finally, incubation of macrophages and neutrophils with SQ29548 prevented TF activity and/or NETs formation induced by supernatant of activated platelets or by IBOP, a selective thromboxane analogue.In conclusion, ASA, suppressing TXA2, prevents macrophages and neutrophils activation and markedly reduces thrombus size with a mechanism most likely dependent of the inhibition of TF activity and NETs formation. These results provide a new link between platelet-produced thromboxane and the occurrence of venous thrombosis.

Tissue factor activity. A marker of alveolar macrophage maturation in rabbits. Effects of granulomatous pneumonitis.

Experiments were carried out to examine relationships between alveolar macrophage maturity and amounts of tissue factor (Clotting Factor III) in these cells under physiologic conditions and during immunologically induced pneumonitis. Using discontinuous density gradient centrifugation, alveolar macrophages from healthy rabbits were rapidly isolated into five subpopulations at different stages of maturation, as demonstrated by morphologic and morphometric evaluation. Very large amounts of tissue factor activity were found in fully mature cells that were purified in the lowest density subpopulation and assayed without preliminary in vitro stimulation or culture. In the remaining four subpopulations of increasing density, amounts of tissue factor were found to progressively diminish in direct correlation with declines of cell maturity. These differences at mean levels were as great as 35-fold. In addition, blood monocytes had less than 1/219 and less than 1/6 of the activity of the fully mature and the least mature subpopulations, respectively. After 16 h culture of the five isolated subpopulations in the absence of lymphokines or of significant numbers of lymphocytes, tissue factor activity increased in inverse correlation with the preincubation stage of cell maturity (2,387 and 109% in the least mature and most mature subpopulations, respectively). These increases required protein synthesis and were accompanied by morphologic and morphometric changes which indicated cellular maturation during the period of tissue factor activity generation in vitro, thus further demonstrating relationships between macrophage maturity and tissue factor content. In additional experiments, direct correlations between cell maturity and tissue factor activity content were also found in activated alveolar macrophage populations from rabbits with Bacillus Calmette Guering (BCG)-induced granulomatous pneumonitis. However, as compared with controls, the BCG populations had increased total amounts of tissue factor activity due to the presence of large numbers of mature alveolar macrophage forms that had high levels of the procoagulant. Thus, tissue factor activity in alveolar macrophages is a marker of cellular maturation in vivo and in vitro. Increased amounts of this initiator of the extrinsic clotting pathway, as found in alveolar macrophage populations from animals with granulomatous pneumonitis induced by BCG hypersensitivity, suggest that alveolar macrophage tissue factor may contribute to the pathology of immune lung diseases.