Leukocyte- and platelet-derived microparticles correlate with thrombus weight and tissue factor activity in an experimental mouse model of venous thrombosis

SummaryMicroparticles (MP) are lipid vesicles from platelets, leukocytes and endothelial cells that are involved in early thrombogenesis. We evaluated a detailed time-course analysis of MPs on thrombogenesis and the associated tissue factor (TF) activity in wild-type, in gene-deleted for E- and P-selectins and with high levels of P-selectin expression after the initiation of venous thrombosis in mice.Inferior vena cava (IVC) ligation was performed on C57BL/6 mice (n =191, 59 = wild-type [WT], 55 = gene-deleted for E- and P – selectins [knock-outs, EPKO] and 77 = elevated levels of soluble P-selectin, named Delta Cytoplasmic Tail (ΔCT). Animals were euthanised at various time points to assess MP production, origin and thrombus weight. MPs were re-injected into separate mice at concentrations of 80,000 and 160,000 units, as well as from different ages. In addition, MPs from thrombosed animals were pooled and TF activity quantitated using a chromogenic assay. Thrombus weight correlated negatively with MPs derived from leukocytes, and positively with MPs derived from platelets for WT animals (p<0.05), while MPs from platelets presented a positive correlation to thrombus weight in the WT and EPKO groups (p<0.01). Total MPs correlated negatively with thrombus weight in the ΔCT group (p<0.05). MP re-injections led to greater thrombus weight, while older MP reinjections tended to form larger thrombus than younger. Finally, TF bearing MPs showed a significant correlation to MP concentrations (R=0.99). In conclusion, MPs appear to be an important element in venous thrombogenesis.

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Severe factor VII deficiency caused by mutations abolishing the cleavage site for activation and altering binding to tissue factor

Blood ◽

10.1182/blood.v83.12.3524.3524 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3524-3535 ◽

Cited By ~ 37

Author(s):

S Chaing ◽

B Clarke ◽

S Sridhara ◽

K Chu ◽

P Friedman ◽

...

Keyword(s):

Amino Acid ◽

Tissue Factor ◽

Time Course ◽

Binding Assay ◽

Factor Vii ◽

Wild Type ◽

Vitro Binding ◽

Epidermal Growth ◽

A Site

Abstract Factor VII (F.VII) is a vitamin-K-dependent serine protease required in the early stages of blood coagulation. We describe here a patient with severe F.VII deficiency, with a normal plasma F.VII antigen level (452 ng/mL) and F.VII activity less than 1%, who is homozygous for two defects: a G-->A transition at nucleotide 6055 in exon 4, which results in an Arg-->Gln change at amino acid 79 (R79Q); and a G-->A transition at nucleotide 8961 in exon 6, which results in an Arg-->Gln substitution at amino acid 152 (R152Q). The R79Q mutation occurs in the first epidermal growth factor (EGF)-like domain, which has previously been implicated in binding to tissue factor. The R152Q mutation occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated F.VII (F.VIIa). Analysis of purified F.VII from patient plasma shows that the material cannot be activated by F.Xa and cofactors. In addition, in an in vitro binding assay using relipidated recombinant tissue factor, patient plasma showed markedly reduced binding to tissue factor at all concentrations tested. In an effort to separate the contributions of the two mutations, three recombinant variants, wild-type, R79Q, and R152Q, were prepared and analyzed. The R152Q variant had markedly reduced activity in a clotting assay, whereas R79Q showed a milder, concentration-dependent reduction. The R152Q variant exhibited nearly normal binding in the tissue factor binding assay, whereas the R79Q variant had markedly reduced binding. The time course of activation of the R79Q variant was slowed compared with wild-type. Our results suggest that the first EGF-like domain is required for binding to tissue factor and that the F.VII zymogen lacks activity and requires activation for expression of biologic activity.

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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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Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

Blood ◽

10.1182/blood-2012-01-402156 ◽

2012 ◽

Vol 119 (23) ◽

pp. 5543-5552 ◽

Cited By ~ 114

Author(s):

Jian-Guo Wang ◽

Julia E. Geddings ◽

Maria M. Aleman ◽

Jessica C. Cardenas ◽

Pichika Chantrathammachart ◽

...

Keyword(s):

Venous Thrombosis ◽

Tissue Factor ◽

Cell Lines ◽

Nude Mice ◽

Culture Medium ◽

Vena Cava ◽

Human Pancreatic Cancer ◽

Tumor Bearing ◽

Mouse Xenograft Model ◽

Increased Risk

Abstract Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

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Tissue factor activity in hela cells measured with a continuous chromogenic assay and elisa reader

Thrombosis Research ◽

10.1016/0049-3848(86)90228-8 ◽

1986 ◽

Vol 41 (2) ◽

pp. 185-195 ◽

Cited By ~ 28

Author(s):

Steven D. Carson ◽

Philip G. Archer

Keyword(s):

Tissue Factor ◽

Hela Cells ◽

Factor Activity ◽

Tissue Factor Activity ◽

Chromogenic Assay

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RECOMBINANT HIRUDIN INHIBITS EXPERIMENTAL VENOUS THROMBOSIS INDUCED BY INJECTION OF TISSUE FACTOR AND STASIS

10.1055/s-0038-1643917 ◽

1987 ◽

Author(s):

M Freund ◽

J-P Cazenave ◽

M-L Wiesel ◽

C Roitsch ◽

N Riehl-Bellon ◽

...

Keyword(s):

Venous Thrombosis ◽

Tissue Factor ◽

Large Scale ◽

Recombinant Dna ◽

Specific Activity ◽

Thrombus Formation ◽

Linear Regression Analysis ◽

Vena Cava ◽

Cloning And Expression ◽

Clotting Factors

Hirudin (HIR), a polypeptide of 65 aminoacids, is the most potent natural inhibitor of coagulation by forming rapidly a very stable and specific non covalent 1:1 complex with α-thrombin, independent of antithrombin III. Although natural HIR has in vivo anticoagulant and antithrombotic properties, its limited availability for large scale purification has prevented further clinical testing and potential use; this can now be solved by recombinant DNA technology. We have previously reported the cloning and expression of a cDNA encoding one variant (called HV-2) of Hirudo medicinalis HIR (Proc. Natl. Acad. Sci. USA. 1986, 83, 1084-1088). The main factors responsible for venous thrombosis are stasis and thrombin generation secondary to tissue factor liberation from vascular cells and monocytes by injury, endotoxin, interleukin-1 or cachectin and the subsequent activation and circulation of activated clotting factors. We have studied the antithrombotic properties of recombinant HIR, HV-2, in a rat experiemental model of venous thrombosis. HV-2 was expressed in yeast, extracted from culture supernatant and purified by HPLC. Pure HV-2 had an isoleucine NH2-terminus and a specific activity of 13000 ATU/mg.30 male Wistar rats (225-300g) were anesthetized with pentobarbital. At time t (0 min) an i.v. (penis) injection of 0.4 ml of saline or HV-2 (2000 to 8000 ATU/kg) was given, followed at t (5min) by 25 mg/kg tissue factor (Thromboplastin C, Dade) i.v. ; 10 s later stasis of the exposed vena cava between 2 sutures 0.7 cm apart and at t (15 min) removal, blotting, fixation and weighing of the thrombus. Linear regression analysis showed a correlation (r=0.99) between the dose of HV-2 and thrombus weight and a calculated IC50 = 3000 ATU/kg. Total inhibition of thrombus formation was seen after injection of 6000 ATU/kg HV-2 and lasted up to 15 min of circulation, HV-2 being completely eliminated from blood in 60 min and accumulated in the kidneys as shown by gamma imaging with 131I-HV-2. In conclusion, the recombinant HIR HV-2 is a potent immediate antithrombin which inhibits venous thrombosis induced by tissue factor and stasis.

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Interleukin-18 mediates interleukin-1-induced cardiac dysfunction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00795.2013 ◽

2014 ◽

Vol 306 (7) ◽

pp. H1025-H1031 ◽

Cited By ~ 70

Author(s):

Stefano Toldo ◽

Eleonora Mezzaroma ◽

Laura O'Brien ◽

Carlo Marchetti ◽

Ignacio M. Seropian ◽

...

Keyword(s):

Systolic Dysfunction ◽

Interleukin 1 ◽

Left Ventricular ◽

Wild Type ◽

Blocking Antibody ◽

Experimental Mouse Model ◽

Patients With Heart Failure ◽

Experimental Mouse ◽

Genetic Deletion ◽

Fractional Shortening

Patients with heart failure (HF) have enhanced systemic IL-1 activity, and, in the experimental mouse model, IL-1 induces left ventricular (LV) systolic dysfunction. Whether the effects of IL-1 are direct or mediated by an inducible cytokine, such as IL-18, is unknown. Recombinant human IL-18-binding protein (IL-18BP) or an IL-18-blocking antibody (IL-18AB) was used to neutralize endogenous IL-18 after challenge with the plasma of patients with HF or with recombinant murine IL-1β in adult male mice. Plasma levels of IL-18 and IL-6 (a key mediator of IL-1-induced systemic effects) and LV fractional shortening were measured in mice sedated with pentobarbital sodium (30–50 mg/kg). Mice with genetic deletion of IL-18 or IL-18 receptors were compared with matching wild-type mice. A group of mice received murine IL-18 to evaluate the effects on LV fractional shortening. Plasma from HF patients and IL-1β induced LV systolic dysfunction that was prevented by pretreatment with IL-18AB or IL-18BP. IL-1β failed to induce LV systolic dysfunction in mice with genetic deletion of IL-18 signaling. IL-1β induced a significant increase in plasma IL-18 and IL-6 levels. Genetic or pharmacological inhibition of IL-18 signaling failed to block the induction of IL-6 by IL-1β. In conclusion, IL-1 induces a release of active IL-18 in the mouse that mediates the LV systolic dysfunction but not the induction of IL-6. IL-18 blockade may therefore represent a novel and more targeted therapeutic approach to treat HF.

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Polymorphisms of the drug transporter gene ABCB1 predict side effects of treatment with cabergoline in patients with PRL adenomas

Acta Endocrinologica ◽

10.1530/eje-12-0198 ◽

2012 ◽

Vol 167 (3) ◽

pp. 327-335 ◽

Cited By ~ 20

Author(s):

A P Athanasoulia ◽

C Sievers ◽

M Ising ◽

A C Brockhaus ◽

A Yassouridis ◽

...

Keyword(s):

Side Effects ◽

Mouse Model ◽

Blood Brain Barrier ◽

Human Study ◽

Brain Barrier ◽

Wild Type ◽

Max Planck ◽

Blood Brain ◽

Experimental Mouse Model ◽

Experimental Mouse

IntroductionTreatment with dopamine agonists in patients with prolactin (PRL) adenomas and Parkinson's disease is associated with central side effects. Central side effects may depend on a substance's ability to pass the blood–brain barrier, which can be actively controlled by transporter molecules such as the P-glycoprotein (P-gp) encoded by theABCB1gene.Materials and methodsWe aimed to determine whether cabergoline is transported by the P-gp and whether polymorphisms of its encodingABCB1gene predict central side effects of cabergoline therapy in patients with PRL adenomas. i) In an experimental mouse model lacking the homologues of the humanABCB1gene (Abcb1abdouble knockout mouse model), we examined whether cabergoline is a substrate of the P-gp using eight mutant and eight wild-type mice. ii) In a human case–control study including 79 patients with PRL adenomas treated with cabergoline at the Max Planck Institute of Psychiatry in Munich, we investigated the association of four selectedABCB1gene single nucleotide polymorphisms (SNPs) (rs1045642, rs2032582, rs2032583 and rs2235015), with the occurrence of central side effects under cabergoline therapy.Resultsi) In the experimental mouse model, we observed that brain concentrations of cabergoline were tenfold higher in the mutant mice compared with their wild-type littermates, implying that cabergoline is indeed a substrate of the transporter P-gp at the blood–brain barrier level. ii) In the human study, we observed significant negative associations under cabergoline for the C-carriers and heterozygous CT individuals of SNP rs1045642 with two central side effects (frequency of fatigue and sleep disorders) and for the G-carriers of SNP rs2032582 with the enhancement of dizziness. For the SNPs rs2235015 and rs2032583, no associations with central side effects under cabergoline were found.DiscussionThis is the first study demonstrating that individualABCB1gene polymorphisms, reflecting a different expression and function of the P-gp, could predict the occurrence of central side effects under cabergoline. Our findings can be viewed as a step into personalised therapy in PRL adenoma patients.

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The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis

Thrombosis and Haemostasis ◽

10.1160/th16-05-0351 ◽

2017 ◽

Vol 117 (01) ◽

pp. 83-89 ◽

Cited By ~ 3

Author(s):

Simon F. Stämpfli ◽

Martin F. Reiner ◽

Yi Shi ◽

Stephan Keller ◽

Alexander Akhmedov ◽

...

Keyword(s):

Map Kinase ◽

Tissue Factor ◽

Cigarette Smoke ◽

Arterial Thrombosis ◽

Molecular Mechanisms ◽

Public Awareness ◽

Smoking Ban ◽

Factor Activity ◽

Wild Type ◽

Tissue Factor Activity

SummaryDespite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2−/− mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2−/− mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2−/− and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2−/− mice (n=7; p<0.05 for wild-type mice and n=5–6; p=NS for JNK2−/− mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.

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