-
[show abstract]
[hide abstract]
ABSTRACT: Sepsis is often associated with haemostatic changes ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by widespread microvascular thrombosis and subsequent consumption of platelets and coagulation proteins, eventually causing bleeding manifestations. The key event underlying this life-threatening complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. The latter, along with the micro-organism and its derivatives are now believed to drive the major changes responsible for massive thrombin formation and fibrin deposition, namely 1) the aberrant expression of the TF by different cells (especially monocytes-macrophages), 2) the impairment of physiological anticoagulant pathways, orchestrated mainly by dysfunctional endothelial cells (ECs) and 3) the suppression of fibrinolysis due to overproduction of plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). The ensuing microvascular thrombosis and ischemia are thought to contribute to tissue injury and multiple organ dysfunction syndrome (MODS). Recent evidence indicates that extracellular nuclear materials released from activated and especially apoptotic or necrotic cells, e.g. High Mobility Group Box-1 (HMGB-1) and histones, are endowed with cell toxicity, proinflammatory and clot-promoting properties and thus, during sepsis, they may represent late mediators that propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenesis of DIC and MODS may have implications for the development of new therapeutic agents potentially useful for the management of severe sepsis.
Thrombosis Research 11/2011; 129(3):290-5. · 2.44 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.
Blood 06/2011; 118(7):1952-61. · 9.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To date, no study has tested the effect of different heparin dosages on the hemostatic changes during off-pump coronary artery bypass graft (OPCABG) surgery, and a wide variety of empirical anticoagulation protocols are being applied. We tested the effect of two different heparin dosages on the activation of the hemostatic system in patients undergoing OPCABG procedures.
Forty-two patients eligible for OPCABG procedures were assigned in a randomized fashion to low-dose heparin (150 IU/kg) or high-dose heparin (300 IU/kg). Prothrombin fragment 1+2, plasmin/alpha(2)-plasmin inhibitor complex, D-dimer, soluble tissue factor, tissue factor pathway inhibitor, total thrombin activatable fibrinolysis inhibitor (TAFI), and activated TAFIa were assayed by specific enzyme-linked immunosorbent assays at six different timepoints, before, during, and after surgery. Platelet function was evaluated by means of an in vitro bleeding time test, platelet function analyzer-100.
The OPCABG surgery was accompanied by significant changes of all plasma biomarkers, indicative of systemic activation of coagulation and fibrinolysis. A significant increase in circulating TAFIa was detected perioperatively and postoperatively, and multiple regression analysis indicated that prothrombin F1+2 but not plasmin/alpha(2)-antiplasmin complex was independently associated with TAFIa level. Platelet function analyzer-100 values did not change significantly after OPCABG. All hemostatic changes were similar in the two heparin groups, even perioperatively, when the difference in anticoagulation was maximal.
Both early and late hemostatic changes, including TAFI activation, are similarly affected in the low-dose and high-dose heparin groups, suggesting that the increase in heparin dosage is not accompanied by a better control of clotting activation during OPCABG surgery.
The Annals of thoracic surgery 02/2010; 89(2):421-7. · 3.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Sepsis is almost invariably associated with haemostatic abnormalities ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by massive thrombin formation and widespread microvascular thrombosis, partly responsible of the multiple organ dysfunction syndrome (MODS), and subsequent consumption of platelets and coagulation proteins causing, in most severe cases, bleeding manifestations. There is general agreement that the key event underlying this life-threatening sepsis complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. Mechanistically, the latter, together with the micro-organism and its derivatives, causes DIC by 1) up-regulation of procoagulant molecules, primarily tissue factor (TF), which is produced mainly by stimulated monocytes-macrophages and by specific cells in target tissues; 2) impairment of physiological anticoagulant pathways (antithrombin, protein C pathway, tissue factor pathway inhibitor), which is orchestrated mainly by dysfunctional endothelial cells (ECs); and 3) suppression of fibrinolysis due to increased plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). Notably, clotting enzymes non only lead to microvascular thrombosis but can also elicit cellular responses that amplify the inflammatory reactions. Inflammatory mediators can also cause, directly or indirectly, cell apoptosis or necrosis and recent evidence indicates that products released from dead cells, such as nuclear proteins (particularly extracellular histones), are able to propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenetic mechanisms of DIC and MODS may have important implications for the development of new therapeutic agents that could be potentially useful particularly for the management of severe sepsis.
Mediterranean Journal of Hematology and Infectious Diseases 01/2010; 2(3):e2010024.
-
[show abstract]
[hide abstract]
ABSTRACT: Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.
Nature medicine 11/2009; 15(11):1318-21. · 27.14 Impact Factor
-
Thrombosis and Haemostasis 05/2009; 101(4):782-5. · 5.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Thrombin is the main activator of the fibrinolysis inhibitor TAFI (thrombin activatable fibrinolysis inhibitor) and heightened clotting activation is believed to impair fibrinolysis through the increase of thrombin activatable fibrinolysis inhibitor activation. However, the enhancement of thrombin generation by soluble tissue factor was reported to have no effect on plasma fibrinolysis and it is not known whether the same is true for cell-associated tissue factor. The aim of this study was to evaluate the effect of tissue factor-expressing monocytes on plasma fibrinolysis in vitro.
Tissue factor expression by human blood mononuclear cells (MNC) and monocytes was induced by LPS stimulation. Fibrinolysis was spectrophotometrically evaluated by measuring the lysis time of plasma clots containing LPS-stimulated or control cells and a low concentration of exogenous tissue plasminogen activator.
LPS-stimulated MNC (LPS-MNC) prolonged fibrinolysis time as compared to unstimulated MNC (C-MNC) in contact-inhibited but not in normal citrated plasma. A significantly prolonged lysis time was observed using as few as 30 activated cells/microL. Fibrinolysis was also impaired when clots were generated on adherent LPS-stimulated monocytes. The antifibrinolytic effect of LPS-MNC or LPS-monocytes was abolished by an anti-tissue factor antibody, by an antibody preventing thrombin-mediated thrombin activatable fibrinolysis inhibitor activation, and by a TAFIa inhibitor (PTCI). Assays of thrombin and TAFIa in contact-inhibited plasma confirmed the greater generation of these enzymes in the presence of LPS-MNC. Finally, the profibrinolytic effect of unfractionated heparin and enoxaparin was markedly lower (approximately 50%) in the presence of LPS-MNC than in the presence of a thromboplastin preparation displaying an identical tissue factor activity.
Our data indicate that LPS-stimulated monocytes inhibit fibrinolysis through a tissue factor-mediated enhancement of thrombin activatable fibrinolysis inhibitor activation and make clots resistant to the profibrinolytic activity of heparins, thus providing an additional mechanism whereby tissue factor-expressing monocytes/macrophages may favor fibrin accumulation and diminish the antithrombotic efficacy of heparins.
Haematologica 05/2009; 94(6):819-26. · 6.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Anticoagulants have been shown to stimulate fibrinolysis principally via inhibition of thrombin-mediated activation of TAFI (thrombin activatable fibrinolysis inhibitor). Their profibrinolytic effect, however, may vary according to their mechanism of action and to the clot composition. We compared the fibrinolytic activity of the direct thrombin inhibitor melagatran with that of unfractionated heparin in platelet-poor (PPP) and platelet-rich (PRP) models consisting of tissue-factor-induced clots exposed to exogenous t-PA (25 ng/ml). In the PPP clot model, both heparin (0.1-0.6 U/ml) and melagatran (20-320 ng/ml) caused a concentration-dependent shortening of lysis time. However, when drug profibrinolytic activity (lysis ratio) was expressed in function of the aPTT prolongation (aPTT ratio), melagatran was more efficient than heparin. In the PRP clot model, melagatran displayed a fibrinolytic activity fairly comparable to that observed in PPP whilst heparin caused a modest reduction of lysis time only at the highest concentrations. Assay of thrombin and TAFIa generation in defibrinated plasma showed that the presence of platelets markedly reduced the ability of heparin, but not that of melagatran, to inhibit the formation of these enzymes. Altogether these data indicate that melagatran is more efficient than heparin in promoting fibrinolysis, particularly in plateletrich clots, and may thus grant a greater antithrombotic activity by enhancing thrombus dissolution.
Thrombosis and Haemostasis 01/2008; 98(6):1208-14. · 5.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The prothrombin gene mutation G20210A is a common risk factor for thrombosis and has been reported to cause APC resistance. However, the inhibition of thrombin formation by APC not only limits fibrin formation but also stimulates fibrinolysis by reducing TAFI activation. We evaluated the influence of prothrombin G20210A mutation on the anticoagulant and fibrinolytic activities of APC (1 microg/ml). Thirty-two heterozygous carriers and 32 non carriers were studied. APC anticoagulant activity was assessed by aPTT prolongation whereas APC fibrinolytic activity was determined by a microplate clot lysis assay. APC-induced aPTT prolongation was markedly less pronounced in carriers than in non carriers. On the contrary, fibrinolysis time was shortened by APC to a comparable extent in both groups. Accordingly, prothrombin levels were strongly correlated with APC-induced aPTT prolongation but not with APC-induced shortening of lysis time. The addition of purified prothrombin to normal plasma (final concentration 150%) caused APC resistance in the clotting assay over the whole range of tested APC concentrations (0.125-1.5 microg/ml). In the fibrinolytic assay, instead, prothrombin supplementation made the sample resistant to low but not to high concentrations of APC (>0.5 microg/ml). Thrombin and TAFIa determination in the presence of 1 microg/ml APC revealed that hyperprothrombinemia, although capable of enhancing thrombin generation, was unable to induce detectable TAFIa formation. It is suggested that APC resistance caused by hyperprothrombinaemia does not translate in impaired fibrinolysis, at least in the presence of high APC levels, because the increase in thrombin formation is insufficient to activate the amount of TAFI required to inhibit plasminogen conversion. These data might help to better understand the relationship between thrombin formation and fibrinolysis down-regulation.
Thrombosis and Haemostasis 04/2006; 95(4):606-11. · 5.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hypoxia in rodents and humans is associated with a reduction of body fat on the one hand, and with enhanced expression of plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of the fibrinolytic system, on the other hand. It was the objective of this study to investigate whether impairment of adipose tissue development by hypoxia may be mediated by PAI-1. Five week old male wild-type (WT) C57Bl/6 mice were fed a standard (SFD) or high fat (HFD) diet and kept under normoxic or hypoxic (10% O(2)) conditions. In addition, PAI-1 deficient mice and WT littermates were kept on HFD under normoxia or hypoxia. In vitro, the effect of hypoxia (2% O(2)) was investigated on differentiation of 3T3-L1 cells into adipocytes. Hypoxia induced a significant reduction of weight gain in WT mice on either SFD or HFD, accompanied by lower weights of subcutaneous (SC) and gonadal (GON) fat. Under hypoxic conditions, adipocytes in the adipose tissues were significantly smaller, whereas blood vessel size and density were larger. Serum PAI-1 levels were enhanced in hypoxic mice on SFD but not on HFD, and overall did not correlate with the observed changes in adipose tissue composition. Furthermore, the effects of hypoxia on adipose tissue in mice on HFD were not affected by deficiency of PAI-1. The inhibiting effect of hypoxia on in vitro preadipocyte differentiation was not mediated by PAI-1 activity. In conclusion, impairment of in vivo adipose tissue development and in vitro differentiation of preadipocytes by hypoxia is not mediated by PAI-1.
Thrombosis and Haemostasis 02/2006; 95(1):174-81. · 5.04 Impact Factor
-
Angelo Vacca,
Roberto Ria, Fabrizio Semeraro,
Francesca Merchionne,
Mauro Coluccia,
Angela Boccarelli,
Claudio Scavelli,
Beatrice Nico,
Angela Gernone,
Feliciana Battelli,
Antonio Tabilio,
Diego Guidolin,
Maria Teresa Petrucci,
Domenico Ribatti,
Franco Dammacco
[show abstract]
[hide abstract]
ABSTRACT: Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.
Blood 12/2003; 102(9):3340-8. · 9.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In tumors, vascular endothelial growth factor-A (VEGF-A) stimulates angiogenesis and vascular permeability by activating the tyrosine kinase receptor-2 (VEGFR-2 or KDR/Flk-1) and-1 (VEGFR-1 or Flt-1).
The distribution and function of VEGF homologs and their receptors on bone marrow plasma cells, endothelial cells, and other stromal cells (residual stromal cells) were examined in patients with multiple myeloma (MM).
Plasma cells secrete VEGF-A (and VEGF-B, VEGF-C and VEGF-D, albeit marginally) into their conditioned medium (CM). CM VEGF-A stimulates proliferation and chemotaxis in endothelial cells (both being mandatory for angiogenesis) via VEGF receptor-2 (VEGFR-2), and in residual stromal cells via the VEGFR-1. Residual stromal cells secrete VEGF-C and VEGF-D, but little of the other homologs. Their CM VEGF-C and VEGF-D increase in response to plasma cell CM and trigger plasma cell proliferation via VEGFR-3. Proliferation in all cell types parallels VEGFR and extracellular signal-regulated protein kinase-2 (ERK-2) phosphorylation. The homologs and receptors are weakly or inconstantly expressed in patients with monoclonal gammopathies of undetermined significance or vitamin B12/iron deficiency anemias.
This study shows that the VEGF pathway is directly involved in tumor angiogenesis and growth in MM. A paracrine VEGF loop for MM progression is suggested. This, in turn, provides a further indication that the VEGF pathway and its signaling proteins may be appropriate targets in the management of MM.
Haematologica 03/2003; 88(2):176-85. · 6.42 Impact Factor
