Human tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA ) plays an important role in the removal of intravascular fibrin deposits and has several physiological roles and pathological activities in the brain. Its production by many other cell types suggests that t-PA has additional functions outside the vascular and central nervous system. Activity of t-PA is regulated at the level of its gene transcription, its mRNA stability and translation, its storage and regulated release, its interaction with cofactors that enhance its activity, its inhibition by inhibitors such as plasminogen activator inhibitor type 1 or neuroserpin, and its removal by clearance receptors. Gene transcription of t-PA is modulated by a large number of hormones, growth factors, cytokines or drugs and t-PA gene responses may be tissue-specific. The aim of this review is to summarise current knowledge on t-PA function and regulation of its pericellular activity, with an emphasis on regulation of its gene expression.

NF-κB is activated from endosomal compartments in antiphospholipid antibodies-treated human monocytes.

BACKGROUND: The antiphospholipid antibody syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated that Toll-like receptor 2 (TLR2) and CD14 contribute to monocyte activation of aPLA. OBJECTIVE: To study the mechanisms of cell activation by aPLA, leading to pro-coagulant and pro-inflammatory responses. METHODS AND RESULTS: For this study, we used purified antibodies from the plasmas of 10 different patients with APS and healthy donors. We demonstrate that aPLA, but not control IgG, co-localizes with TLR2 and TLR1 or TLR6 on human monocytes. Blocking antibodies to TLR2, TLR1 or TLR6, but not to TLR4, decreased TNF and tissue factor (TF) responses to aPLA. Pharmacological and siRNA approaches revealed the importance of the clathrin/dynamin-dependent endocytic pathway in cell activation by aPLA. In addition, soluble aPLA induced NF-κB activation, while bead-immobilized aPLA beads, which cannot be internalized, were unable to activate NF-κB. Internalization of aPLA in monocytes and NF-κB activation were dependent on the presence of CD14. CONCLUSION: We show that TLR2 and its co-receptors, TLR1 and TLR6, contribute to the pathogenicity of aPLA, that aPLA are internalized via clathrin- and CD14-dependent endocytosis and that endocytosis is required for NF-κB activation. Our results contribute to a better understanding of the APS and provide a possible therapeutic approach.

Retinoids and activation of PKC induce tissue-type plasminogen activator expression and storage in human astrocytes.

BACKGROUND: Emerging data demonstrate important roles for tissue-type plasminogen activator (t-PA) in the central nervous system (CNS). In contrast to endothelial cells, little is known about the regulation of t-PA gene expression and secretion in astrocytes. OBJECTIVES: The aims of the present study were to investigate whether t-PA gene expression is regulated by retinoids and the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) in human astrocytes, and to study whether t-PA is stored and subject to regulated release from these cells, as with endothelial cells. METHODS: Native human astrocytes were treated with RA and/or PMA. mRNA was quantified by real-time RT-PCR and protein secretion determined by ELISA. Intracellular t-PA immunoreactivity in astrocytes was examined by immunocyto- and histochemistry. RESULTS: RA and/or PMA induced a time-dependent increase in t-PA mRNA and protein levels in astrocytes, reaching 10-fold after combined treatment. This was associated with increased amounts of t-PA storage in intracellular granular structures. Both forskolin and histamine induced regulated release of t-PA. The presence of t-PA in reactive astrocytes was confirmed in human brain tissue. CONCLUSIONS: These data show that RA and PKC activation induce a strong up-regulation of t-PA expression in astrocytes, and increased intracellular storage pools. Moreover, a regulated release of t-PA can be induced from these cells. This raises the possibility that astrocytes contribute to the regulation of extracellular t-PA levels in the CNS.

Characterization of endothelial-like cells derived from human mesenchymal stem cells.

BACKGROUND: Blood-derived endothelial progenitor cells (EPC) have been used to treat ischemic disease. However, the number of EPC that can be obtained from adult blood is limited. OBJECTIVE: To characterize endothelial-like cells obtained from human bone marrow and determine their ability to stimulate new blood vessel formation in vivo. METHODS: Mononuclear cells (MNC) were isolated from human bone marrow or umbilical cord blood and cultured in endothelial growth medium (EGM-2). Mesenchymal stem cells (MSC) were isolated from bone marrow and induced to differentiate into endothelial-like cells (MSCE), or adipocytes or osteocytes by growth in EGM-2, adipogenic or osteogenic medium. RESULTS: Cells obtained by culturing bone marrow MNC in EGM-2 formed cord- or tube-like structures when grown on Matrigel(TM) and expressed several endothelial marker proteins. However, cell morphology and the profile of endothelial marker protein expression were different from those of cord blood-derived EPC (cbEPC). Cells with a similar phenotype were obtained by differentiation of MSC into MSCE, which was accompanied by an increase of endothelial marker proteins and a diminished capacity to differentiate into adipocytes. Subcutaneous implantation of MSCE in collagen plugs in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice resulted in formation of functional blood vessels that had incorporated the MSCE. CONCLUSIONS: Our results show that MSCE and cbEPC are different cell types. The formation of functional blood vessels by MSCE, combined with high yields and a reduced capacity to differentiate into other cell types compared with MSC, makes these cells potentially useful for autologous therapy of ischemic disease.

Aspirin inhibits endothelial cell activation induced by antiphospholipid antibodies.

BACKGROUND: Antiphospholipid antibodies (APLA) have been shown to activate endothelial cells (EC) in vitro, as documented by an increased expression of tissue factor as well as leukocyte adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin. Currently, treatment of patients with the antiphospholipid syndrome includes aspirin, particularly for women with recurrent fetal loss. OBJECTIVE: The present study was undertaken to investigate whether aspirin interferes with EC activation induced by APLA in vitro. METHODS: IgG from 14 patients with APLA, and suffering from thrombotic complications and/or pregnancy morbidity, and control IgG were tested for their ability to modify the expression of VCAM-1 in human umbilical vein endothelial cells. VCAM-1 antigen was measured by flow cytometry and its mRNA by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Incubation of EC with IgG from most of the patients led to a higher VCAM-1 expression compared with incubation with control IgG. The effect of aspirin was studied for the eight IgG samples that induced a more than 50% increase in VCAM-1. Aspirin (10 mm) treatment of the cells significantly reduced the VCAM-1 response to these APLA. CONCLUSIONS: Our results indicate that besides its antiplatelet properties, aspirin exerts a protective effect towards APLA at the EC level by decreasing leukocyte adhesion molecule expression at the cell surface.

Effects of statins on adhesion molecule expression in endothelial cells.

BACKGROUND: Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis. OBJECTIVES: We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms. METHODS: Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4-6 h activation with tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277). RESULTS: Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-alpha and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-alpha-mediated overexpression of E-selectin. CONCLUSIONS: Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-alpha or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.

Synergistic induction of t-PA by vascular endothelial growth factor and basic fibroblast growth factor and localization of t-PA to Weibel-Palade bodies in bovine microvascular endothelial cells.

Endothelial cell migration is stimulated by members of the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) families, and is dependent on extracellular proteolytic activity provided by enzymes of the plasminogen activator (PA) system. Here we report that in bovine microvascular endothelial cells (BME cells), bFGF principally increased urokinase-type PA (u-PA) while tissue-type PA (t-PA) was increased mainly by VEGF. In bovine aortic endothelial cells (BAE cells), bFGF increased u-PA, whereas VEGF had no effect. Co-added bFGF and VEGF increased t-PA mRNA levels and enzyme activity in both cell types in a synergistic manner. Tissue-type plasminogen activator (t-PA) immunoreactivity colocalized with von Willebrand factor, a marker for Weibel-Palade bodies. Co-added bFGF and VEGF increased the number of t-PA-positive cells as well as the number of t-PA-positive granules per cell. Localization of t-PA in regulated storage granules endows endothelial cells with the potential to rapidly increase proteolytic activity in the pericellular environment.

Localization of beta2-glycoprotein 1 in late endosomes of human endothelial cells.

Antiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards beta2-glycoprotein 1 (beta2GP1)phospholipid complexes, we investigated the localisation of beta2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti-beta2GP1 antibodies, we detected beta2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti-beta2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti-beta2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti-beta2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

The tetraspanin CD63/lamp3 cycles between endocytic and secretory compartments in human endothelial cells.

In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular-multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel-Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel-Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel-Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel-Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel-Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel-Palade bodies.

Cellular degradation of free and inhibitor-bound tissue-type plasminogen activator--requirement for a co-receptor?

The low density lipoprotein receptor-related protein (LRP) is a multiligand clearance receptor that removes free tissue-type plasminogen activator (t-PA) or complexes of t-PA with plasminogen activator inhibitor type 1 (PAI-1) from the blood circulation or the pericellular space. Co-receptors are essential for LRP-mediated clearance of several ligands (e.g. glycosaminoglycans for thrombin/protease nexin and lipoprotein lipase, and the urokinase receptor for urokinase/PAI-1 complexes). The present study was undertaken to investigate whether LRP-mediated t-PA clearance requires a co-receptor as well. In five cell lines from different organs and species degradation of t-PA and t-PA/PAI-1 was mediated by LRP (or LRP-like receptors). No degradation of t-PA and t-PA/PAI-1 occurred in THP-1 or U-937 human monocyte-like cells, despite the presence of functional LRP. As glycosaminoglycans can bind t-PA and PAI-1 we investigated whether they are involved in t-PA/PAI-1 degradation. Pre-treatment of COS cells or HT1080 cells with chlorate, an inhibitor of glycosaminoglycan sulfation, did not decrease t-PA/PAI-1 degradation. Furthermore, CHO cells genetically deficient in glycosaminoglycans efficiently degraded t-PA/PAI-1. Thus it is unlikely that glycosaminoglycans are co-receptors for degradation of t-PA or t-PA/PAI-1. This study indicates that THP-1 and U-937 cells lack a critical component (co-receptor?) for the LRP-mediated degradation of t-PA.

Interaction of anti-phospholipid antibodies with late endosomes of human endothelial cells.

Anti-phospholipid antibodies (APLAs) are associated with thrombosis and/or recurrent pregnancy loss. APLAs bind to anionic phospholipids directly or indirectly via a cofactor such as beta(2)-glycoprotein 1 (beta(2)GPI). The lipid target of APLA is not yet established. Recently, we observed that APLAs in vitro can bind lysobisphosphatidic acid (LBPA). The internal membranes of late endosomes are enriched in this phospholipid. The current study was undertaken to determine to what extent binding of APLA to LBPA is correlated with binding to cardiolipin and to beta(2)GPI and to determine whether patient antibodies interact with late endosomes of human umbilical vein endothelial cells (HUVECs) and thus modify the intracellular trafficking of proteins. Binding of patient immunoglobulin G (n=37) to LBPA was correlated significantly with binding to cardiolipin. Although LBPA binding was correlated to a lesser extent with beta(2)GPI binding, we observed that beta(2)GPI binds with high affinity to LBPA. Immunofluorescence studies showed that late endosomes of HUVECs contain LBPA. Patient but not control antibodies recognized late endosomes, but not cardiolipin-rich mitochondria, even when we used antibodies that were immunopurified on cardiolipin. Incubation of HUVECs with patient plasma samples immunoreactive toward LBPA resulted in an accumulation of the antibodies in late endosomes and led to a redistribution of the insulinlike growth factor 2/mannose-6-phosphate receptor from the Golgi apparatus to late endosomes. Our results suggest that LBPA is an important lipid target of APLA in HUVECs. These antibodies are internalized by the cells and accumulate in late endosomes. By modifying the intracellular trafficking of proteins, APLA could contribute to several of the proposed pathogenic mechanisms leading to the antiphospholipid syndrome.

Regulated von Willebrand factor (vWf) secretion is restored by pro-vWf expression in a transfectable endothelial cell line.

Von Willebrand factor (vWf) is a glycoprotein involved in primary hemostasis and synthesized in endothelial cells (EC). vWf is stored in secretory granules specific for EC called Weibel-Palade bodies (WPb). Studies on the molecular mechanisms of vWf storage and acute release are hampered by the limitations of the available endothelial cell culture models. We created a suitable model by stable transfection of the vWf-negative ECV304 endothelial cell line with pro-vWf cDNA. Pro-vWf was normally cleaved to mature vWf and stored in WPb. Acute vWf release occurred in response to the calcium ionophore A23187. Thus, vWf expression is sufficient to restore functional secretory granules in ECV304 cells. We used this model to study the role of WPb in the storage of tissue-type plasminogen activator (t-PA), a key fibrinolytic enzyme that is acutely released by EC, but whose intracellular storage compartment is still a matter of debate. We observed that restoration of WPb in ECV304 cells results in the targeting of t-PA to these storage granules.

Acute release of tissue factor pathway inhibitor after in vivo thrombin generation in baboons.

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.

Plasminogen activation in human acute leukaemias.

Plasminogen activation is implicated in solid tumour growth, invasion and metatastic spread. However, little is known about its role in leukaemia. We investigated the production by leukaemic cells of plasminogen activators [urokinase (uPA) and tissue-type PA (tPA)], cell surface receptor for uPA (uPAR) and PA inhibitors (PAI-1 and PAI-2). Leukaemic cells from 37 patients [26 with acute myeloid leukaemia (AML) and 11 with acute lymphoid leukaemia (ALL)] were analysed for mRNA content and enzymatic activities. High levels of uPA mRNA were found in M1, M2, M3 and M4-M5 AMLs, whereas tPA mRNA was not detected in any of the analysed cases. uPAR mRNA was confined to subtypes M4-M5. PAI-1 mRNA was detected in M3 and M4-M5. PAI-2 mRNA was found predominantly in M2 and M4-M5. SDS-PAGE/zymography analyses of cell extracts and supernatants after 24 and 48 h of culture confirmed the production of active uPA by AML cells (mainly M4-M5). but not by ALL. The finding of uPA, uPAR, PAI-1 and PAI-2 synthesized by leukaemic cells suggests that plasminogen activation may contribute to the invasive behaviour of these cells, the fibrinolytic imbalance observed in leukaemic patients and the differentiation and proliferation of M4-M5 by interaction of uPA with uPAR.

Storage of tissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells.

Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone-containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.

Studies on the effect of fucosylated and non-fucosylated finger/growth-factor constructs on the clearance of tissue-type plasminogen activator mediated by the low-density-lipoprotein-receptor-related protein.

Previously we observed that the finger/growth factor (FG) region of tissue-type plasminogen activator (t-PA) blocked the low-density-lipoprotein-receptor-related protein (LRP)-mediated clearance of t-PA by rat hepatocytes. However, the concentrations needed were much higher than those for intact t-PA. The FG region was expressed in yeast and lacked the fucose on Thr61, which was reported to be important for efficient clearance of t-PA by human hepatoma cells. At position 83 it had a serine, whereas human t-PA has a free cysteine and rodent t-PA an arginine at this position. To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG2-Fc). Two variants were studied, one having Ser83, the other Arg83. The two fucosylated FG2-Fc chimeras were compared with each other, with non-fucosylated FG and with intact t-PA with regard to their effect on the clearance of t-PA and t-PA x plasminogen-activator inhibitor type 1 (PAI-1). For this comparison, LRP-specific clearance models were used. In rat hepatoma cells and in mouse embryonic fibroblasts (MEF-1) the clearance of t-PA and of t-PA x PAI-1 was inhibited more than 95% by receptor-associated protein, an inhibitor of LRP-mediated clearance, whereas no t-PA or t-PA x PAI-1 clearance was observed in LRP-deficient PEA-13 mouse embryonic fibroblasts. The Ser83 and Arg83 FG2-Fc chimeras were equally efficient inhibitors in these models. Their efficacies in inhibiting t-PA and t-PA x PAI-1 degradation (IC50 750 nM and 890 nM, respectively) were similar to those of non-fucosylated FG (IC50 1950 nM and 1560 nM) and 75-fold lower than that of intact t-PA (IC50 9.9 nM and 21.1 nM). The results indicate that the presence of a serine or an arginine at position 83 and the presence of a fucose on Thr61 are not of major importance for the LRP-mediated clearance of t-PA.

Plasminogen: an important hemostatic parameter in septic patients.

BACKGROUND: Previously we observed in some but not all septic patients a low plasma concentration of plasminogen. OBJECTIVES: To investigate prospectively whether plasma levels of plasminogen or the ratio of plasminogen to alpha-2-antiplasmin have a prognostic value for survival from sepsis and to study the variation of other hemostatic parameters during septicemia. PATIENTS: The study population consisted of 45 consecutive patients with septicemia, 15 non-septic patients from the same intensive care unit and 30 healthy volunteers. MEASUREMENTS AND MAIN RESULTS: Plasminogen concentrations were significantly lower (p < 0.001) in plasma of septic patients (median 0,62 IU/ml range: 0.15-1,06) than in plasma of healthy controls (median 1.00 IU/ml, range: 0.75-1.10) or of non-septic intensive care patients (median 1.00 IU/ml, range: 0.82-1.08). Among the other parameters tested, plasminogen activator inhibitor (PAI-1) antigen concentration and PAI activity were similar in septic and non-septic intensive care patients, but higher than in healthy controls. Concentrations of elastase-alpha-1-protease inhibitor or of thrombin-antithrombin complexes were higher in septic patients than in non-septic intensive care patients or healthy controls. A degraded form of plasminogen of 38 kDa was detected by Western blot analysis in the plasma of septic patients, but not in plasma of non-septic intensive care patients or controls. Plasminogen alone or the ratio of plasminogen to antiplasmin were good markers for survival from septicemia. E.g. for plasminogen at a cut off of 0.65 IU/ml, sensitivity was 90.5% and specificity 66.7%, whereas for the ratio of plasminogen over antiplasmin at a cut off ratio of 0,65 IU/ml, sensitivity was 95.2% and specificity 70.8%. CONCLUSION: Plasminogen or the ratio of plasminogen to antiplasmin are sensitive markers for survival in patients with septicemia.

The coagulation and fibrinolytic responses of baboons after in vivo thrombin generation--effect of interleukin 6.

Disseminated intravascular coagulation (DIC) may lead to severe thrombotic or hemorrhagic complications. The present work was undertaken to study the effect of interleukin 6 (IL-6) on variations of key coagulation and fibrinolytic parameters in plasma in a baboon model of experimental DIC induced by injection of factor Xa and phospholipids at dosages leading to partial (48%) or complete fibrinogen depletion. Transient increases of D-dimer, fibrinopeptide A, thrombin-antithrombin and the activated partial thromboplastin time were observed. Each parameter had a particular (time and Xa/phospholipid dose dependent) pattern of changes. The principal effect of IL-6 was a more rapid restoration of fibrinogen concentrations and of overall coagulation tests. Injection of factor Xa/phospholipids led also to a rapid increase of tissue-type plasminogen activator (t-PA) the extent of which was dependent on Xa/phospholipid dose. Pretreatment with IL-6 induced a threefold increase of basal t-PA and a corresponding increase of the t-PA response. Plasminogen activator inhibitor type 1 (PAI-1) concentrations did not change after low dose Xa/phospholipids, but increased eightfold after high dose Xa/phospholipids, IL-6 pretreatment induced within 8 h a twentyfold increase of PAI-1 but no further increase was observed after injection of factor Xa/phospholipids. Thus, in vivo thrombin generation leads to dynamic modifications of the coagulation and fibrinolytic systems. The principal effect of IL-6 is a more rapid normalization of overall coagulation tests, due to normalization of fibrinogen, and an increased t-PA release response which is partially counteracted by increased PAI-1 concentrations.

Effects of intermittent pneumatic compression on venous haemodynamics and fibrinolytic activity.

Pneumatic intermittent compression is an effective method to prevent postoperative venous thromboembolism. Its efficacy has been ascribed to both a haemodynamic action (increase of blood flow velocity) and a stimulation of endogenous fibrinolytic activity [via the production of tissue-type plasminogen activator (t-PA) by the vascular endothelium]. The relative contribution of these two effects is still debated. In a randomized, cross-over study in ten healthy volunteers, we compared the haemodynamic and fibrinolytic effects of two different pneumatic intermittent compression devices: a classical, low-pressure, whole-leg boots system, and a novel, high-pressure, plantar compression system. The study was performed at rest, to compare haemodynamics and fibrinolytic activity modifications, and under induced venous leg stasis, in order to compare the two compression systems in experimental conditions mimicking laparoscopic surgery. Our data show that (1) a pneumatic compression device that exerts its compression on the plantar venous plexus only induced an increase of venous blood peak velocity and flow in the common femoral vein that is very similar to that induced by the classical whole-leg boots compression system; (2) the venous stasis induced by an external pressure mimicking the conditions of laparoscopic surgery further increased the absolute velocity and flow increase, with the two intermittent compression systems tested; (3) no changes of t-PA or plasminogen activator-inhibitor 1 antigens were observed with either pneumatic compression device. In conclusion, the present study indicates that the antithrombotic effect of mechanical prophylaxis is probably mainly due to its ability to increase venous peak velocity and flow, especially under venous stasis conditions.