Increased tissue factor expression on circulating monocytes in chronic HIV infection: relationship to in vivo coagulation and immune activation

Abstract HIV infection is associated with an increased risk of thrombosis; and as antiretroviral therapy has increased the lifespan of HIV-infected patients, their risk for cardiovascular events is expected to increase. A large clinical study found recently that all-cause mortality for HIV+ patients was related to plasma levels of interleukin-6 and to D-dimer products of fibrinolysis. We provide evidence that this elevated risk for coagulation may be related to increased proportions of monocytes expressing cell surface tissue factor (TF, thromboplastin) in persons with HIV infection. Monocyte TF expression could be induced in vitro by lipopolysaccharide and flagellin, but not by interleukin-6. Monocyte expression of TF was correlated with HIV levels in plasma, with indices of immune activation, and with plasma levels of soluble CD14, a marker of in vivo lipopolysaccharide exposure. TF levels also correlated with plasma levels of D-dimers, reflective of in vivo clot formation and fibrinolysis. Thus, drivers of immune activation in HIV disease, such as HIV replication, and potentially, microbial translocation, may activate clotting cascades and contribute to thrombus formation and cardiovascular morbidities in HIV infection.

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Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles

Blood Advances ◽

10.1182/bloodadvances.2020002998 ◽

2021 ◽

Vol 5 (6) ◽

pp. 1682-1694

Author(s):

Nadim Tawil ◽

Rayhaan Bassawon ◽

Brian Meehan ◽

Ali Nehme ◽

Laura Montermini ◽

...

Keyword(s):

Cancer Cells ◽

Tissue Factor ◽

Extracellular Vesicles ◽

Coagulation System ◽

Platelet Factor ◽

Increased Risk ◽

Cell Subpopulations ◽

Cancer Associated Thrombosis

Abstract Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type–based diagnosis and antithrombotic intervention.

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Abstract 14366: T-lymphocytes May Contribute to Thrombus Formation in Patients With Acute Coronary Syndrome via Expression of Tissue Factor

Circulation ◽

10.1161/circ.132.suppl_3.14366 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Giovanni Cimmino ◽

Giovanni Ciccarelli ◽

Stefano Conte ◽

Grazia Pellegrino ◽

Luigi Insabato ◽

...

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Tissue Factor ◽

Thrombus Formation ◽

Specific Antigen ◽

Activated T Cells ◽

Coronary Syndrome ◽

Stable Cad

Background: Activation of T-cells plays an important role in the pathophysiology of acute coronary syndromes (ACS). We have previously shown that plaques from ACS patients are characterized by a selective oligoclonal expansion of T-cells, indicating a specific, antigen-mediated recruitment of T-cells within the unstable lesions. We have also previously reported that activated T-cells in vitro express functional Tissue Factor (TF) on their surface. At the moment, however it is not known whether expression of TF by T-cells may contribute to thrombus formation in vivo. Methods: Blood was collected from the aorta and the coronary sinus of 13 NSTEMI and 10 stable CAD patients. CD3+ cells were selectively isolated and TF gene expression (real time PCR)and protein levels (western blot) were evaluated. In additional 7 STEMI patients, thrombotic formation material was obtained from the occluded coronary artery by catheter aspiration during primary PCI. TF expression in CD3+ cells was evaluated by immunohistochemistry and confocal microscopy. Results: Transcardiac TF expression in CD3+ cells was significantly higher in NSTEMI patients as compared to CD3+ cells obtained from stable CAD patients. Interestingly, thrombi aspirated from STEMI patients resulted enriched in CD3+cells, which expressed TF on their surface as shown in the figure. Conclusions: Our data demonstrate that in patients with ACS, T-lymphocytes may express surface TF, thus contributing to the process of thrombus formation. This finding may shed new light into the pathophysiologyof ACS.

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Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall

Blood ◽

10.1182/blood-2004-06-2225 ◽

2005 ◽

Vol 105 (1) ◽

pp. 192-198 ◽

Cited By ~ 212

Author(s):

Sharlene M. Day ◽

Jennifer L. Reeve ◽

Brian Pedersen ◽

Diana M Farris ◽

Daniel D. Myers ◽

...

Keyword(s):

Blood Vessel ◽

Tissue Factor ◽

Vessel Wall ◽

Thrombus Formation ◽

Vena Cava ◽

Vascular Wall ◽

Blood Vessel Wall ◽

Wild Type

Abstract Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF–/–, hTF-Tg+, or “low-TF”) demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

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Inhibition of restenosis by tissue factor pathway inhibitor: in vivo and in vitro evidence for suppressed monocyte chemoattraction and reduced gelatinolytic activity

Blood ◽

10.1182/blood-2003-04-1148 ◽

2004 ◽

Vol 103 (5) ◽

pp. 1653-1661 ◽

Cited By ~ 37

Author(s):

Christoph W. Kopp ◽

Thomas Hölzenbein ◽

Sabine Steiner ◽

Rodrig Marculescu ◽

Helga Bergmeister ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Early Stage ◽

Thrombus Formation ◽

Matrix Metalloproteinase 2 ◽

Monocyte Chemoattractant Protein 1 ◽

Markers Of Inflammation ◽

Tissue Factor Pathway

AbstractActivation of inflammatory and procoagulant mechanisms is thought to contribute significantly to the initiation of restenosis, a common complication after balloon angioplasty of obstructed arteries. During this process, expression of tissue factor (TF) represents one of the major physiologic triggers of coagulation that results in thrombus formation and the generation of additional signals leading to vascular smooth muscle cell (VSMC) proliferation and migration. In this study, we have investigated the mechanisms by which inhibition of coagulation at an early stage through overexpression of tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of TF, might reduce restenosis. In a rabbit femoral artery model, percutaneous delivery of TFPI using a recombinant adenoviral vector resulted in a significant reduction of the intimamedia ratio 21 days after injury. Investigating several markers of inflammation and coagulation, we found reduced neointimal expression of monocyte chemoattractant protein-1 (MCP-1), lesional monocyte infiltration, and expression of vascular TF, matrix metalloproteinase-2 (MMP-2), and MMP-9. Moreover, overexpression of TFPI suppressed the autocrine release of platelet-derived growth factor BB (PDGF-BB), MCP-1, and MMP-2 in response to factors VIIa and Xa from VSMCs in vitro and inhibited monocyte TF activity. These results suggest that TFPI exerts its action in vivo through not only thrombotic, but also nonthrombotic mechanisms.

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Vascular Gas6 contributes to thrombogenesis and promotes tissue factor up-regulation after vessel injury in mice

Blood ◽

10.1182/blood-2012-05-433730 ◽

2013 ◽

Vol 121 (4) ◽

pp. 692-699 ◽

Cited By ~ 35

Author(s):

Richard S. Robins ◽

Catherine A. Lemarié ◽

Sandrine Laurance ◽

Meghedi N. Aghourian ◽

Jianqiu Wu ◽

...

Keyword(s):

Tissue Factor ◽

Thrombus Formation ◽

Vascular Wall ◽

Specific Gene ◽

Tissue Factor Expression ◽

Vessel Injury ◽

Factor Expression ◽

Platelet Signaling

Abstract Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (−/−) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5μM ADP and 10μM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6−/− mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6−/− endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism.

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Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Blood ◽

10.1182/blood-2012-06-437772 ◽

2012 ◽

Vol 120 (10) ◽

pp. 2133-2143 ◽

Cited By ~ 143

Author(s):

Roxane Darbousset ◽

Grace M. Thomas ◽

Soraya Mezouar ◽

Corinne Frère ◽

Rénaté Bonier ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Blood Coagulation ◽

Thrombus Formation ◽

Coagulation Cascade ◽

Factor Xii ◽

Long Time ◽

Platelet Accumulation

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

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Abstract 456: Antiphospholipid Antibodies Promote Platelet Activation and Thrombosis by Inhibition of Platelet eNOS

Circulation ◽

10.1161/circ.116.suppl_16.ii_76-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Craig Morrell ◽

AnneMarie Swaim ◽

Tanika Martin ◽

Guillermina Girardi ◽

Jane E Salmon ◽

...

Keyword(s):

Platelet Activation ◽

Antiphospholipid Antibodies ◽

Thrombus Formation ◽

Ldl Receptor ◽

Wild Type ◽

Human Igg ◽

Increased Risk ◽

Receptor Family

The antiphospholipid syndrome (APS) is an autoimmune systemic disorder characterized by the persistent presence of antiphospholipid antibodies (aPL Ab) and increased risk of thrombosis, coronary artery disease and myocardial infarction. Although platelets are known direct targets of aPL Ab action, the molecular basis of aPL Ab actions on platelets remains unclear. Platelet endothelial NO synthase (eNOS) is a key regulator of platelet function, with NO causing blunted activation. We therefore determined whether aPL Ab modulate platelet eNOS. Normal human IgG (NH IgG) and human IgG containing polyclonal aPL Ab were obtained from healthy individuals and APS patients, respectively, and purified using protein G-Sepharose chromatography. Using both human and mouse platelets, we found that aPL Ab increased agonist-induced platelet activation whereas NH IgG did not. In contrast to the enhanced activation by aPL Ab in platelets from wild-type mice, aPL Ab had no effect on platelets isolated from eNOS null mice. Pre-treatment of platelets with aPL Ab also inhibited insulin-mediated eNOS stimulation as evidenced by diminished cGMP production and DAF2 fluorescence. Receptor associated protein (RAP), an antagonist of ligand binding to members of the LDL receptor family, blocked aPL Ab-induced increases in platelet activation. RAP also prevented aPL Ab-mediated antagonism of platelet eNOS, indicating that aPL Ab signal through the platelet ApoER2â ϵ™ (LRP8) to attenuate eNOS activity. Furthermore, using intravital microscopy of the mouse mesenteric circulation, we demonstrated that platelets from wild-type mice treated with aPL Ab have increased rolling on a stimulated endothelium and a decreased time to thrombus formation in vivo versus platelets treated with NH IgG. In contrast, aPL Ab did not alter the in vivo function of platelets from eNOS null mice. These cumulative in vitro and in vivo findings demonstrate that aPL Ab antagonism of platelet eNOS through LDL receptor family member binding underlies aPL Ab-mediated thrombosis.

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Tissue Factor Reduction and Tissue Factor Pathway Inhibitor Release after Heparin Administration

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614530 ◽

1999 ◽

Vol 81 (04) ◽

pp. 589-593 ◽

Cited By ~ 38

Author(s):

A. M. Gori ◽

G. Pepe ◽

M. Attanasio ◽

M. Falciani ◽

R. Abbate ◽

...

Keyword(s):

Tissue Factor ◽

Plasma Levels ◽

Tissue Factor Pathway Inhibitor ◽

Day Treatment ◽

Procoagulant Activity ◽

Cytokine Gene Expression ◽

Heparin Administration ◽

Tissue Factor Pathway

SummaryElevated plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) and large amounts of monocyte procoagulant activity (PCA) have been documented in unstable angina (UA) patients. In in vitro experiments heparin is able to blunt monocyte TF production by inhibiting TF and cytokine gene expression by stimulated cells and after in vivo administration it reduces adverse ischemic outcomes in UA patients. TF and TFPI plasma levels and monocyte PCA have been investigated in 28 refractory UA patients before and during anticoagulant subcutaneous heparin administration (thrice daily weight- and PTT-adjusted for 3 days) followed by 5000 IU × 3 for 5 days. After 2-day treatment, immediately prior to the heparin injection, TF and TFPI plasma levels [(median and range): 239 pg/ml, 130-385 pg/ ml and 120 ng/ml, 80-287 ng/ml] were lower in comparison to baseline samples (254.5 pg/ml, 134.6-380 pg/ml and 135.5 ng/ml, 74-306 ng/ml). Four h after the heparin injection TF furtherly decreased (176.5 pg/ml, 87.5-321 pg/ml; -32.5%, p<0.001) and TFPI increased (240.5 ng/ml, 140-450 ng/ml; +67%, p<0.0001).After 7-day treatment, before the injection of heparin, TF and TFPI plasma levels (200 pg/ml, 128-325 pg/ml and 115 ng/ml, 70-252 ng/ml) significantly decreased (p<0.05) in comparison to the pre-treatment values. On the morning of the 8th day, 4 h after the injection of heparin TF plasma levels and monocytes PCA significantly decreased (156.5 pg/ml, 74-259 pg/ml and from 180 U/105 monocytes, 109-582 U/105 monocytes to 86.1 U/105 monocytes, 28-320 U/105 monocytes; - 38% and -55% respectively) and TFPI increased (235.6 ng/ml, 152-423 ng/ ml; +70%, p<0.001). In conclusion, heparin treatment is associated with a decrease of high TF plasma levels and monocyte procoagulant activity in UA patients. These actions of heparin may play a role in determining the antithrombotic and antiinflammatory properties of this drug.

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C-Cbl Regulates c-Mpl Receptor Trafficking and Its Internalization

Blood ◽

10.1182/blood.v126.23.2388.2388 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2388-2388

Author(s):

Sebastian Jonas Saur ◽

Melanie Märklin ◽

Manuela Ganser ◽

Kyle Hoehn ◽

James E David ◽

...

Keyword(s):

Bone Marrow ◽

Plasma Levels ◽

Thrombus Formation ◽

Surface Expression ◽

Mrna Levels ◽

Wild Type ◽

Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors and cytokines in order to maintain physiological levels of circulating platelets. Thrombopoietin (TPO) signalling via its receptor c-Mpl is a key regulator of megakaryopoiesis driving megakaryocyte differentiation, promoting endomitosis and proplatelet formation. Therefore TPO/c-Mpl signalling needs to be tightly regulated to maintain physiological megakaryopoiesis. One of the most effective mechanisms to permanently disable activated signalling proteins is by targeted degradation via lysosomes or proteasomes. Previous studies have identified c-Cbl as an E3 ligase responsible for the ubiquitination of c-Mpl in cell lines. In this study, we investigated the mechanisms of TPO-mediated c-Mpl degradation in primary mouse cells. In order to determine the potential role of c-Cbl in murine megakaryopoiesis we used a conditional PF4-Cre c-Cbl knockout (ko) mouse model to specifically delete c-Cbl in the megakaryocytic lineage. Megakaryocytes were generated in vitro by culturing bone marrow from WT and PF4-Cre/c-Cbl-floxed (c-Cbl ko) lines for 72 hrs in the presence of rmTPO. C-Cbl ko mice showed significant bone marrow megakaryocyte hyperplasia, however megakaryocyte numbers in the spleen remained unchanged. Platelets counts were significantly elevated as compared to control mice (1.2 x106 vs. 1.7x106 p=0.0001) and in addition, the platelets from the c-Cbl ko mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). Using a method of in vivo double labelling of platelets, we were able to simultaneously follow the survival of both the entire population of platelets and new platelets which were generated during the last 24 hours. There were more new platelets produced within a 24 h period in the c-Cbl ko mice although the half-life of platelets was similar in the both cohorts. Although c-Cbl ko mice exhibited thrombocytosis, they showed a severe defect in thrombus formation using an in vivo thrombus formation model with Fe3Cl. TPO plasma levels, known to be inversely regulated by circulating platelet numbers, were surprisingly increased (250 vs. 420 pg/ml, p=0.005) in the c-Cbl ko mice. There was no difference in liver mRNA levels in the two cohorts. We therefore looked at c-Mpl protein and mRNA expression in megakaryocytes and found c-Cbl ko mice to express more c-Mpl compared with wild type controls. Surprisingly, we found c-Mpl surface expression to be reduced and internalization of the receptor significantly impaired following TPO stimulation in c-Cbl ko mice. Incubating platelets in vitro with TPO for 2 hours to evaluate the TPO uptake capacity of platelets, we found c-Cbl ko platelets to show a severe uptake defect compared with wild type control platelets. Taken together, we have successfully ablated c-Cbl specifically from the megakaryocyte lineage and demonstrated that this has profound effects on platelet counts and size. In addition, we showed that c-Cbl ablation leads to reduced c-Mpl surface expression and impaired internalization, which culminates in increased TPO plasma levels causing increased megakaryopoiesis in the c-Cbl ko mice. In summary, our data enhance our understanding of the regulation of TPO signalling and the physiological role of c-Cbl in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

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Sustained hypothermia accelerates microvascular thrombus formation in mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00425.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. H2680-H2687 ◽

Cited By ~ 35

Author(s):

Nicole Lindenblatt ◽

Michael D. Menger ◽

Ernst Klar ◽

Brigitte Vollmar

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Specific Protein ◽

Microvascular Thrombosis ◽

Fibrinogen Binding ◽

Increased Risk ◽

Systemic Hypothermia ◽

Risk For Thrombosis

Cold is supposed to be associated with alterations in blood coagulation and a pronounced risk for thrombosis. We studied the effect of clinically encountered systemic hypothermia on microvascular thrombosis in vivo and in vitro. Ferric chloride-induced microvascular thrombus formation was analyzed in cremaster muscle preparations from hypothermic mice. Additionally, flow cytometry and Western blot analysis was used to evaluate the effect of hypothermia on platelet activation. To test whether preceding hypothermia predisposes for enhanced thrombosis, experiments were repeated after hypothermia and rewarming to 37°C. Control animals revealed complete occlusion of arterioles and venules after 742 ± 150 and 824 ± 172 s, respectively. Systemic hypothermia of 34°C accelerated thrombus formation in arterioles and venules (279 ± 120 and 376 ± 121 s; P < 0.05 vs. 37°C). This was further pronounced after cooling to 31°C (163 ± 57 and 281 ± 71 s; P < 0.05 vs. 37°C). Magnitude of thrombin receptor activating peptide (TRAP)-induced platelet activation increased with decreasing temperatures, as shown by 1.8- and 3.0-fold increases in mean fluorescence after PAC-1 binding to glycoprotein (GP)IIb-IIIa and 1.6- and 2.9-fold increases of fibrinogen binding on incubation at 34°C and 31°C. Additionally, tyrosine-specific protein phosphorylation in platelets was increased at hypothermic temperatures. In rewarmed animals, kinetics of thrombus formation were comparable to those in normothermic controls. Concomitantly, spontaneous and TRAP-enhanced GPIIb-IIIa activation did not differ between rewarmed platelets and those maintained continuously at 37°C. Moderate systemic hypothermia accelerates microvascular thrombosis, which might be mediated by increased GPIIb-IIIa activation on platelets but does not cause predisposition with increased risk for microvascular thrombus formation after rewarming.

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