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Prostacyclin prevents pulmonary vascular injury and shock by inhibiting increases in lung tissue levels of TNF in rats administered endotoxin. We previously reported that NO derived from eNOS increases endothelial production of prostacyclin. Because neutrophil elastase has been shown to decrease endothelial production of prostacyclin by inhibiting NOS activity, we examined whether neutrophil elastase inhibitors reduce pulmonary vascular injury and hypotension by inhibiting the decrease in pulmonary endothelial production of prostacyclin in rats administered endotoxin. Animals were pretreated with sivelestat or L-658,758, neutrophil elastase inhibitors, before endotoxin administration. Lung tissue levels of 6-keto-prostaglandin F1were markedly increased after endotoxin administration, followed by a rapid decrease to baseline levels. Sivelestat and L-658,758 inhibited these decreases as well as inhibiting increases in lung tissue levels of TNF and lung wet-to-dry weight ratios in animals administered endotoxin. These inhibitors also reduced hypotension and inhibited increases in lung tissue levels of mRNA of the inducible form of NOS in animals administered endotoxin. The effects of neutrophil elastase inhibitors were completely reversed by pretreatment with nitro-l-arginine methyl ester, an inhibitor of NOS, or indomethacin, a nonspecific cyclooxygenase inhibitor. These observations suggested that neutrophil elastase might decrease the pulmonary endothelial production of prostacyclin by inhibiting endothelial NO production, thereby contributing to the development of pulmonary vascular injury and shock through increases in lung tissue levels of TNF in rats administered endotoxin.

Antithrombin (AT) supplementation in patients with severe sepsis has been shown to improve organ failures in which activated leukocytes are critically involved. However, the precise mechanism(s) for the therapeutic effects of AT is not well understood. We examined in rats whether AT reduces ischemia/reperfusion (I/R)–induced renal injury by inhibiting leukocyte activation. AT markedly reduced the I/R-induced renal dysfunction and histologic changes, whereas neither dansyl glutamylglycylarginyl chloromethyl ketone–treated factor Xa (DEGR-F.Xa), a selective inhibitor of thrombin generation, nor Trp49-modified AT, which lacks affinity for heparin, had any effect. Renal tissue levels of 6-keto-PGF1α, a stable metabolite of prostacyclin (PGI2), increased after renal I/R. AT enhanced the I/R-induced increases in renal tissue levels of 6-keto-PGF1α, whereas neither DEGR-F.Xa nor Trp49-modified AT had any effect. AT significantly inhibited I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability. Ischemia/reperfusion-induced increases in renal tissue levels of tumor necrosis factor-α, cytokine-induced neutrophil chemoattractant, and myeloperoxidase were significantly inhibited in animals given AT. Pretreatment of animals with indomethacin reversed the effects induced by AT. Iloprost, an analog of PGI2, produced effects similar to those induced by AT. These observations strongly suggest that AT reduces the I/R-induced renal injury by inhibiting leukocyte activation. The therapeutic effects of AT might be mainly mediated by PGI2released from endothelial cells through interaction of AT with cell surface glycosaminoglycans.

Antithrombin (AT) supplementation in patients with severe sepsis has been shown to improve organ failures in which activated leukocytes are critically involved. However, the precise mechanism(s) for the therapeutic effects of AT is not well understood. We examined in rats whether AT reduces ischemia/reperfusion (I/R)–induced renal injury by inhibiting leukocyte activation. AT markedly reduced the I/R-induced renal dysfunction and histologic changes, whereas neither dansyl glutamylglycylarginyl chloromethyl ketone–treated factor Xa (DEGR-F.Xa), a selective inhibitor of thrombin generation, nor Trp49-modified AT, which lacks affinity for heparin, had any effect. Renal tissue levels of 6-keto-PGF1α, a stable metabolite of prostacyclin (PGI2), increased after renal I/R. AT enhanced the I/R-induced increases in renal tissue levels of 6-keto-PGF1α, whereas neither DEGR-F.Xa nor Trp49-modified AT had any effect. AT significantly inhibited I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability. Ischemia/reperfusion-induced increases in renal tissue levels of tumor necrosis factor-α, cytokine-induced neutrophil chemoattractant, and myeloperoxidase were significantly inhibited in animals given AT. Pretreatment of animals with indomethacin reversed the effects induced by AT. Iloprost, an analog of PGI2, produced effects similar to those induced by AT. These observations strongly suggest that AT reduces the I/R-induced renal injury by inhibiting leukocyte activation. The therapeutic effects of AT might be mainly mediated by PGI2released from endothelial cells through interaction of AT with cell surface glycosaminoglycans.

Antithrombin (AT) prevents Escherichia coli–induced hypotension in animal models of sepsis, and it further reduces the mortality of patients with septic shock. In the present study, we examined whether AT may prevent the endotoxin (ET)-induced hypotension by promoting the endothelial release of prostacyclin (PGI2) in rats. Intravenous administration of AT (250 U/kg) prevented both hypotension and the increases in plasma levels of NO2−/NO3− in rats given ET. Lung expression of messenger RNA (mRNA) for tumor necrosis factor-α (TNF-α) was transiently increased after ET administration, followed by the increases in lung tissue levels of TNF-α. Both the lung activity of the inducible form of nitric oxide synthase (iNOS) and the lung expression of iNOS mRNA in animals administered ET were gradually increased after the TNF-α mRNA expression had peaked. Administration of AT significantly inhibited these increases. Neither DEGR-F.Xa, a selective inhibitor of thrombin generation, nor Trp49-modified AT, which is not capable of promoting the endothelial release of PGI2, showed any effects on these changes induced by ET. Administration of antirat TNF-α antibody produced effects similar to those induced by AT. Indomethacin pretreatment abrogated the effects induced by AT. Iloprost, a stable derivative of PGI2, produced effects similar to those of AT. These findings suggested that AT prevents the ET-induced hypotension by inhibiting the induction of iNOS through inhibiting TNF-α production. These effects of AT could be mediated by the promotion of endothelial release of PGI2 and might at least partly explain the therapeutic effects for septic shock.

Antithrombin (AT) prevents Escherichia coli–induced hypotension in animal models of sepsis, and it further reduces the mortality of patients with septic shock. In the present study, we examined whether AT may prevent the endotoxin (ET)-induced hypotension by promoting the endothelial release of prostacyclin (PGI2) in rats. Intravenous administration of AT (250 U/kg) prevented both hypotension and the increases in plasma levels of NO2−/NO3−in rats given ET. Lung expression of messenger RNA (mRNA) for tumor necrosis factor-α (TNF-α) was transiently increased after ET administration, followed by the increases in lung tissue levels of TNF-α. Both the lung activity of the inducible form of nitric oxide synthase (iNOS) and the lung expression of iNOS mRNA in animals administered ET were gradually increased after the TNF-α mRNA expression had peaked. Administration of AT significantly inhibited these increases. Neither DEGR-F.Xa, a selective inhibitor of thrombin generation, nor Trp49-modified AT, which is not capable of promoting the endothelial release of PGI2, showed any effects on these changes induced by ET. Administration of antirat TNF-α antibody produced effects similar to those induced by AT. Indomethacin pretreatment abrogated the effects induced by AT. Iloprost, a stable derivative of PGI2, produced effects similar to those of AT. These findings suggested that AT prevents the ET-induced hypotension by inhibiting the induction of iNOS through inhibiting TNF-α production. These effects of AT could be mediated by the promotion of endothelial release of PGI2and might at least partly explain the therapeutic effects for septic shock.

We investigated whether antithrombin (AT) can reduce ischemia/reperfusion (I/R)-induced injury of rat liver by promoting prostacyclin release from endothelial cells. Although intravenous administration of AT (250 U/kg) markedly reduced hepatic injury, neither dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, nor Trp49-modified AT, which lacks affinity for heparin, had any effect. Hepatic levels of 6-keto-PGF1?, a stable prostacyclin (PGI2) metabolite, were increased significantly after I/R of the rat liver. AT significantly increased the hepatic level of 6-keto-PGF1?, whereas neither DEGR-Xa nor Trp49-modified AT increased it. Hepatic tissue blood flow was markedly reduced after I/R. Although AT significantly increased the hepatic tissue blood flow after I/R, neither DEGR-Xa nor Trp49-modified AT increased the blood flow. Hepatic levels of cytokine-induced neutrophil chemoattractant (CINC) and myeloperoxidase (MPO) were significantly increased after hepatic I/R. The levels of these two indicators were reduced by AT but were unaffected by either DEGR-Xa or Trp49-modified AT. Pretreatment of animals with indomethacin (IM) completely inhibited the protective effects of AT on the I/R-induced hepatic damage and the leukocyte activation as well as the AT-induced increase in hepatic 6-keto-PGF1? levels after I/R. Iloprost, a stable analog of PGI2, exhibited effects similar to those of AT and also significantly inhibited the exacerbation of liver injury, the decrease in hepatic tissue blood flow, and the increases in hepatic CINC and MPO levels seen in rats subjected to I/R but pretreated with IM. These findings suggest that AT may prevent I/R-induced hepatic injury by increasing the hepatic levels of PGI2 through the interaction of AT with cell-surface glycosaminoglycans, thus increasing hepatic tissue blood flow and inhibiting leukocyte activation in animals subjected to I/R.

In normal individuals microparticles (MPs) derived from leukocytes, erythrocytes, platelets and endothelial cells are found in the circulation. When the thrombogenicity of microparticles was measured in vitro by their capacity to generate thrombin (thrombin generating activity, TGA) in normal control plasma we found the highest in vitro activity to be generated by red blood cell derived MPs and the lowest by monocyte derived MPs. While all MPs exhibited Annexin V binding, indicating the presence of negatively charged phospholipids on the surface, only monocyte and smooth muscle cell (SMC) derived MPs expressed tissue factor and their TGA was dependent on the “extrinsic” factor VII dependent pathway. All other MPs utilized the “intrinsic” factor XI, factor IX and VIII dependent pathway. In vivo, however, only MPs activating the “extrinsic” pathway were active as shown by TAT formation upon injection of MPs in wt C57/bl6 mice (3 minutes after injection). When, however, not directly thrombogenic MPs derived from red blood cells were co-injected with tissue factor bearing MPs, TAT formation was significantly enhanced, indicating that non-directly thrombogenic MPs need tissue factor “priming” to become thrombogenic. To prove this hypothesis, we injected red blood cell (RBC) derived MPs (106–108) into wild type C57BL/6 mice and into ApoE−/− or LDLR−/ − deficient mice, fed a high lipid diet. In wild type C57BL6 mice red blood cell derived MPs did not significantly increase TAT complexes as compared to PBS (RBC derived MPs 108 6.2±0.8 vs. PBS 4.1±1.3 mg/L). When, however, such RBC derived MPs were injected into LDLR−/− mice fed a high lipid diet, TAT complexes significantly increased (PBS 4.4±0.5 vs RBC derived MPs 108 15.8±1.9mg/L). Similar results were obtained also in ApoE−/− mice. This indicates that tissue factor “priming” also occurs in atherosclerotic mice, likely by tissue factor expressed on the plaque or by plaque derived tissue factor bearing MPs. To determine whether local tissue factor expression causes also local fibrin formation upon injection of red blood cell derived MPs, the aortic arch of such mice were analyzed for local fibrin deposition after injection of red blood cell derived MPs. In fact red blood cell derived MPs did not only induce higher TAT complexes in LDLR−/− mice but also increased the number of fibrin positive plaques as compared to PBS. This indicates that red blood cell derived MPs are thrombogenic in animal models of atherosclerosis. When MPs isolated form the arterial blood of atherosclerotic patients undergoing carotid endarterectomy were analyzed, it was found that MPs from symptomatic patients were more thrombogenic and that this increased thrombogenicity was due to increased number of red blood cell derived MPs. This indicates that red blood cell derived MPs might also cause thrombus formation in atherosclerotic patients.

In normal individuals microparticles (MPs) derived from leukocytes, erythrocytes, platelets and endothelial cells are found in the circulation. When the thrombogenicity of microparticles was measured in vitroby their capacity to generate thrombin (thrombin generating activity, TGA) in normal control plasma we found the highest in vitroactivity to be generated by red blood cell derived MPs and the lowest by monocyte derived MPs. While all MPs exhibited Annexin V binding, indicating the presence of negatively charged phospholipids on the surface, only monocyte and smooth muscle cell (SMC) derived MPs expressed tissue factor and their TGA was dependent on the “extrinsic” factor VII dependent pathway. All other MPs utilized the “intrinsic” factor XI, factor IX and VIII dependent pathway. In vivo, however, only MPs activating the “extrinsic” pathway were active as shown by TAT formation upon injection of MPs in wt C57/bl6 mice (3 minutes after injection). When, however, not directly thrombogenic MPs derived from red blood cells were co-injected with tissue factor bearing MPs, TAT formation was significantly enhanced, indicating that non-directly thrombogenic MPs need tissue factor “priming” to become thrombogenic. To prove this hypothesis, we injected red blood cell (RBC) derived MPs (106–108) into wild type C57BL/6 mice and into ApoE−/−or LDLR−/ −deficient mice, fed a high lipid diet. In wild type C57BL6 mice red blood cell derived MPs did not significantly increase TAT complexes as compared to PBS (RBC derived MPs 1086.2±0.8 vs. PBS 4.1±1.3 mg/L). When, however, such RBC derived MPs were injected into LDLR−/−mice fed a high lipid diet, TAT complexes significantly increased (PBS 4.4±0.5 vs RBC derived MPs 10815.8±1.9mg/L). Similar results were obtained also in ApoE−/−mice. This indicates that tissue factor “priming” also occurs in atherosclerotic mice, likely by tissue factor expressed on the plaque or by plaque derived tissue factor bearing MPs. To determine whether local tissue factor expression causes also local fibrin formation upon injection of red blood cell derived MPs, the aortic arch of such mice were analyzed for local fibrin deposition after injection of red blood cell derived MPs. In fact red blood cell derived MPs did not only induce higher TAT complexes in LDLR−/−mice but also increased the number of fibrin positive plaques as compared to PBS. This indicates that red blood cell derived MPs are thrombogenic in animal models of atherosclerosis. When MPs isolated form the arterial blood of atherosclerotic patients undergoing carotid endarterectomy were analyzed, it was found that MPs from symptomatic patients were more thrombogenic and that this increased thrombogenicity was due to increased number of red blood cell derived MPs. This indicates that red blood cell derived MPs might also cause thrombus formation in atherosclerotic patients.

Microparticles (MPs) are released from cells during processes such as apoptosis or during cell activation. These MPs contain phospholipids, proteins and even nucleic acids derived from their parent cells. They are found circulating in plasma but also in tissues such as atherosclerotic plaques. It is thought that MPs contain and transfer tissue factor and can thereby induce blood clotting. In this study we analyzed clot promoting properties of MPs generated from vascular cells in vitro. MPs were generated from endothelial cells (EC), smooth muscles cells (SMC), monocytes (U937), erythrocytes (RBC) or platelets (Pl) by inducing apoptosis or by calcium ionophore activation; they were subsequently isolated by differential centrifugation. Thrombogenicity of the MPs was evaluated using a thrombin generation assay (Technothrombin® TGA) and MP free plasma as substrate. MPs displayed a different thrombin generating potential depending on the parent cells. MPs derived from RBCs (~400nM peak thrombin/105 MPs/ml plasma), ECs (~300nM), SMCs (~300nM) and Pls (~300nM) were more thrombogenic than MPs derived from U937 (~200 nM). In addition EC, SMC and U937 MPs all expressed tissue factor but EC MPs induced thrombin generation in a tissue factor and FVII independent manner. EC MPs even expressed active tissue factor pathway inhibitor and functionally inhibited tissue factor dependent thrombin generation. Since the higher thrombin generation induced by MPs derived from EC as compared U937 derived MPs could not be explained by a different activity of tissue factor, we were interested whether lipids contained in the microparticles could account for the differences in thrombin generation. We therefore analyzed thrombin generation induced by lipids isolated from MPs and parent cells and could show that lipids from EC MPs and SMC MPs exhibited higher thrombin generation than those from U937 MPs. Upon analysis of lipids by thin layer chromatography and mass spectrometry we found that in general microparticles are enriched in cholesterol, sphingomyeline and phosphatidylserine over the parent cells and that EC and SMC MPs were enriched in negatively charged phospholipids (different species of phosphatidylserine and phosphatiylglycerol) as compared to MPs derived from U937 cells. When thrombogenicity was, however, evaluated in vivo by injecting MPs into mice it was found that the highest capability to induce thrombin-antithrombin (TAT) complexes had MPs derived from SMCs; also U937 MPs induced an increase in TAT levels, while EC MPs – although more thrombogenic than U937 MPs in vitro – did not induce TAT complex formation by themselves but were only synergistic in vivo. From these data we conclude that thrombin formation in vivo depends on the initiation of the tissue factor FVII pathway, while the extent of thrombin formation is dependent on negatively charged phospholipids contained to a higher extent in MPs derived e.g. from ECs.

Microparticles (MPs) are released from cells during processes such as apoptosis or during cell activation. These MPs contain phospholipids, proteins and even nucleic acids derived from their parent cells. They are found circulating in plasma but also in tissues such as atherosclerotic plaques. It is thought that MPs contain and transfer tissue factor and can thereby induce blood clotting.

Apoptotic microparticles (MPs) have been shown to be produced in atherosclerotic plaques and could be involved in the development of atherothrombosis. In the present study, we compared the procoagulant activities of apoptotic MPs derived from cultured endothelial cells (ECs), smooth muscle cells (SMCs), and monocytes, the major constituents of the plaque formation. Apoptotic MPs were generated from ECs, SMCs, monocytes and monocytic cell line, U937 cells, collected and purified by differential centrifugation. MPs were analyzed for the expression of phosphatidylserine and tissue factor (TF). Procoagulant activities were measured using a novel thrombin generation assay (TECHNOTHROMBIN® TGA) with normal and factor deficient plasmas. All MPs derived from ECs, SMCs, and U937 cells bound with annexin V, expressed TF, and induced thrombin generation (TG) dose dependently, indicating the thormbogenic properties. Among these MPs, SMC-MPs and EC-MPs showed the higher levels of TG activities than those of monocytic MPs. When antibodies against TF were used to inhibit TG generated by MPs, maximum level of TG of monocytic MPs was quenched dose dependently, and that of SMC-MPs was partially inhibited, but that of EC-MPs was not inhibited. To further investigate this phenomenon, factor VII, VIII, IX, XI and XII deficient plasmas were used. Although the initiation of TG induced by all MPs was dependent on factor VII, the amplification mechanisms of TG were different among these MPs. EC-MP induced amplification of TG was dependent on factors VIII, IX, XI and XII, and not dependent on factor VII. SMC-MPs and monocytic MPs induced TG dependently on an amplification loop including factor VII, and independently of factors VIII, IX, XI and XII. From these data we conclude that, apoptotic MPs derived from cultured ECs, SMCs and monocytes could induce TG via different coagulant pathways and could critically contribute to the process leading to thrombus formation after plaque rapture.

Apoptotic microparticles (MPs) have been shown to be produced in atherosclerotic plaques and could be involved in the development of atherothrombosis. In the present study, we compared the procoagulant activities of apoptotic MPs derived from cultured endothelial cells (ECs), smooth muscle cells (SMCs), and monocytes, the major constituents of the plaque formation. Apoptotic MPs were generated from ECs, SMCs, monocytes and monocytic cell line, U937 cells, collected and purified by differential centrifugation. MPs were analyzed for the expression of phosphatidylserine and tissue factor (TF). Procoagulant activities were measured using a novel thrombin generation assay (TECHNOTHROMBIN® TGA) with normal and factor deficient plasmas. All MPs derived from ECs, SMCs, and U937 cells bound with annexin V, expressed TF, and induced thrombin generation (TG) dose dependently, indicating the thormbogenic properties. Among these MPs, SMC-MPs and EC-MPs showed the higher levels of TG activities than those of monocytic MPs. When antibodies against TF were used to inhibit TG generated by MPs, maximum level of TG of monocytic MPs was quenched dose dependently, and that of SMC-MPs was partially inhibited, but that of EC-MPs was not inhibited. To further investigate this phenomenon, factor VII, VIII, IX, XI and XII deficient plasmas were used. Although the initiation of TG induced by all MPs was dependent on factor VII, the amplification mechanisms of TG were different among these MPs. EC-MP induced amplification of TG was dependent on factors VIII, IX, XI and XII, and not dependent on factor VII. SMC-MPs and monocytic MPs induced TG dependently on an amplification loop including factor VII, and independently of factors VIII, IX, XI and XII. From these data we conclude that, apoptotic MPs derived from cultured ECs, SMCs and monocytes could induce TG via different coagulant pathways and could critically contribute to the process leading to thrombus formation after plaque rapture.