Abelacimab Reduces Thrombus Propagation in a Baboon Model of Vascular Graft Thrombosis

Abstract Background: Factor XI (FXI) inhibition demonstrated strong efficacy in preventing thrombus formation in preclinical and clinical models of arterial and venous thrombosis. However, the effect of FXI inhibition in halting the progression of a formed clot remains largely unknown. Aims: This study aims to test whether abelacimab, a dual-acting FXI and activated FXI (FXIa) monoclonal antibody, is effective in halting clot formation and downstream growth when administered before or during active clot formation in an established baboon femoral arterio-venous (AV) shunt model. Methods: Three baboons had a chronic femoral AV shunt put in place; platelet and fibrin deposition inside and distal to collagen- or collagen + tissue factor (TF)-coated vascular grafts were measured at baseline (control), in a therapeutic setting, where abelacimab (1 mg/kg, intravenously) was administered 30 minutes after thrombus initiation, and in a preventative setting within the first 48 h and 1 week (144 - 216 h) post-administration. Pharmacodynamic effect was measured by activated partial thromboplastin time (aPTT). Results: Consistent with its half-life of 20 to 30 days, single iv administration of abelacimab at a dose of 1 mg/kg resulted in long-lasting (> 4-week) aPTT prolongation (> 2-fold). Administration of abelacimab 30 minutes after initiation of thrombosis using grafts coated either with collagen or with collagen + TF quickly halted downstream propagation of platelet and fibrin deposition compared to control. Further, downstream propagation of platelet and fibrin deposition was markedly reduced when clotting was induced by collagen, or collagen + tissue factor after abelacimab administration. Conclusions: These data suggest that abelacimab, a dual-acting anti-FXI/FXIa monoclonal antibody with a single long-lasting iv injection has the potential to slow down the growth and reduce the size of thrombi when admistered before or after clot induction. Data indicate a potential for therapeutic benefit of targeting FXI both in therapeutic and preventive settings. Sponsored by: Anthos Therapeutics Inc., 55 Cambridge Parkway, Suite 103, Cambridge, MA 02142 Figure 1 Figure 1. Disclosures Wallisch: Aronora Inc,: Current Employment. Khder: Anthos Therapeutics: Consultancy; Novartis: Current equity holder in publicly-traded company, Other: Retiree. Bloomfield: Anthos Therapeutics: Current Employment. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

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Factor XI Deficient Mice Have Reduced Platelet Accumulation and Fibrin Deposition after Laser Injury.

Blood ◽

10.1182/blood.v104.11.218.218 ◽

2004 ◽

Vol 104 (11) ◽

pp. 218-218

Author(s):

T. Regan Baird ◽

David Gailani ◽

Bruce Furie ◽

Barbara C. Furie

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Thrombus Formation ◽

Factor Xa ◽

Fibrin Deposition ◽

Wild Type ◽

Factor Xi ◽

Factor Xia ◽

Tissue Factor Pathway ◽

Platelet Accumulation

Abstract Tissue factor exposure at sites of vascular injury results in the generation of factor Xa and thrombin. A current model of blood coagulation suggests that the amount of thrombin generated through this pathway is limited by the inhibition of the factor VIIa-tissue factor complex by tissue factor pathway inhibitor in the presence of factor Xa. The initial thrombin activates a number of hemostatic proteins including factor XI. Factor XIa then activates factor IX leading to generation of the tenase complex to maintain the thrombin flux. While in vitro studies support this hypothesis the importance of factor XI for thrombus formation in vivo remains unclear. We have examined thrombus formation upon laser injury to the arterioles (30–50 μm diameter) of the cremaster muscle in living mice lacking factor XI using digital multi-channel fluorescence intravital microscopy. Platelets were labeled with Alexa 488 conjugated murine CD41 Fab fragments by systemic infusion of the antibody. Maximum platelet accumulation in factor XI null mice (median of 35 thrombi in 4 mice) is only 25% of that of wild type mice (median of 40 thrombi in 4 mice) after injury (p<0.03). The time course of platelet accumulation is similar between both genotypes. Maximum platelet accumulation occurs in approximately 90 seconds (p<0.2). Fibrin deposition was observed simultaneously using an Alexa 660 conjugated anti-fibrin antibody that does not recognize fibrinogen. Maximum fibrin deposition in factor XI null mice is 50% that of wild type mice (p<0.001) and the rate of fibrin generation is slower in factor XI null mice. However, the time to achieve half maximal fibrin deposition is approximately the same in factor XI null mice (77 sec) compared to wild type mice (63.5 sec, p<0.09). These data suggest that the primary difference in response to laser induced injury between the factor XI null mice and wild type mice is the level of thrombin generated and supports the hypothesis that factor XI participates in maintaining thrombin flux after inhibition of the factor VII-tissue factor. The model above postulates a single source of tissue factor, the vessel wall, and further, that the tissue factor-factor VIIa complex formed from the exposed tissue factor is rapidly inactivated by tissue factor pathway inhibitor after the appearance of the initial factor Xa formed. In addition it has been suggested that a rapidly growing thrombus blocks access to vascular wall tissue factor. However we have recently observed that there is a P-selectin and P-selectin glycoprotein ligand 1 dependent pathway of blood coagulation that recruits blood borne tissue factor into a growing thrombus at sites of laser-induced vessel injury. Both vessel wall and blood borne tissue factor are required for normal thrombus formation. Our data suggest that although tissue factor is continuously recruited to the growing thrombus, factor XIa plays a significant role in thrombin generation.

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Temporal Roles of Platelet and Coagulation Pathways in Collagen- and Tissue Factor-Induced Thrombus Formation

International Journal of Molecular Sciences ◽

10.3390/ijms23010358 ◽

2021 ◽

Vol 23 (1) ◽

pp. 358

Author(s):

Stefano Navarro ◽

David Stegner ◽

Bernhard Nieswandt ◽

Johan W. M. Heemskerk ◽

Marijke J. E. Kuijpers

Keyword(s):

Tissue Factor ◽

Platelet Activation ◽

Factor Viia ◽

Thrombus Formation ◽

Flow Chamber ◽

Clot Formation ◽

Pharmacological Intervention ◽

Molecular Pathways ◽

Glycoprotein Vi ◽

Platelet Signaling

In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.

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Retinoic acid reduces induction of monocyte tissue factor and tissue factor/factor VIIa-dependent arterial thrombus formation

Blood ◽

10.1182/blood.v86.1.212.bloodjournal861212 ◽

1995 ◽

Vol 86 (1) ◽

pp. 212-218 ◽

Cited By ~ 35

Author(s):

RM Barstad ◽

MJ Hamers ◽

RW Stephens ◽

KS Sakariassen

Keyword(s):

Retinoic Acid ◽

Tissue Factor ◽

Human Peripheral Blood ◽

Thrombus Formation ◽

Antigen Expression ◽

Fibrin Deposition ◽

Thrombotic Risk ◽

Plaque Disruption ◽

Platelet Thrombus ◽

Arterial Grafting

Agents that downregulate the induction of monocyte/macrophage tissue factor (TF) activity may attenuate the thrombotic risk associated with mechanical restoration of vessel patency or artificial arterial grafting. In such events, procoagulant macrophages in the atherosclerotic plaque and procoagulant monocytes adherent to artificial materials may be exposed to the blood stream. Ishii et al (Blood 80:2556, 1992) reported that induction of endothelial TF is downregulated by all-trans retinoic acid (ATRA), and Conese et al (Thromb Haemost 66:662, 1991) reported that retinoids downregulate monocyte procoagulant activity (PCA). These findings led us to investigate the effect of ATRA on monocyte TF expression, and to study the effect of ATRA on monocyte-induced thrombus formation in a model system of human arterial thrombogenesis. Induction of PCA in human peripheral blood monocytes by 0.5 microgram/mL lipopolysaccharide (LPS) was dose dependently reduced by ATRA, reaching a reduction of 56% at 10(-5) mol/L ATRA (P < .0001). A 38% reduction (P < .0007) in LPS- induced TF antigen expression was observed at an ATRA concentration of 10(-6) mol/L. Adherence of monocytes to plastic cover slips (Thermanox, Miles Laboratories, Naperville, IL) also triggered induction of cellular PCA, which was inhibited by more than 80% by an anti-TF monoclonal antibody (MoAb) (P < .002). Inclusion of ATRA (10(-6) mol/L) reduced this PCA by 40% (P < .03), and the TF antigen expression by 30% (P < .0001). Exposure of Thermanox adherent monocytes to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device at an arterial wall shear rate of 650 s-1 elicited significant fibrin deposition and platelet thrombus formation. Partial interruption of this thrombus formation was achieved by 10(-6) mol/L ATRA, which reduced the fibrin deposition by 80% (P < .02) and platelet thrombus formation by 50% (P < .05). In comparison, incubation of adherent monocytes with the anti-TF MoAb before the blood exposure, reduced the fibrin deposition by 83% (P < .02) and platelet thrombus volume by 75% (P < .0008). Thus, ATRA is an effective down-regulator of monocyte TF- PCA, and may reduce thrombotic complications at sites of plaque rupture, at plaque disruption after percutaneous transluminal angioplasty procedures, or on surfaces introduced by artificial arterial grafting.

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Effects of Ionic and Nonionic Contrast Media on Endothelium and on Arterial Thrombus Formation

Acta Radiologica ◽

10.1177/02841851960373p2102 ◽

1996 ◽

Vol 37 (3P2) ◽

pp. 954-961 ◽

Cited By ~ 16

Author(s):

R. M. Barstad ◽

M. S. Buchmann ◽

M. J. A. G. Hamers ◽

L. Örning ◽

U. Ørvim ◽

...

Keyword(s):

Extracellular Matrix ◽

Contrast Media ◽

Tissue Factor ◽

Plasminogen Activator ◽

Thrombus Formation ◽

In Vitro Assays ◽

Fibrin Deposition ◽

Perfusion Chamber ◽

Arterial Thrombus ◽

Nonionic Contrast Media

Background: The aims of the present study were to investigate whether ionic and nonionic contrast media (CM) affect: 1) the procoagulant and fibrinolytic activities of cultured human vessel endothelium; and 2) early events of tissue-factor-induced arterial thrombus formation under conditions which may follow a percutaneous transluminal coronary angioplasty (PTCA) procedure. The following 3 CM were studied: iohexol (nonionic monomer, Omnipaque); iodixanol (nonionic dimer, Visipaque); and ioxaglate (ionic dimer, Hexabrix). Saline (0.9%) and glucose (40 vol%) were used as control. Methods and Results: Exposing endothelium to 40 vol% CM for 10 min did not affect the selected parameters of cellular procoagulant (tissue factor), anticoagulant (thrombomodulin), fibrinolytic (tissue plasminogen activator) or antifibrinolytic (plasminogen activator inhibitor-1) activity or antigen. However, ioxaglate had a profound impact on the cell morphology, which was noted already after one minute of exposure. The cells contracted and rounded, exposing large areas of extracellular matrix. Iohexol showed this phenomenon to a considerably lesser extent, whereas iodixanol induced a slight swelling of the cells without detectable exposure of extracellular matrix. The effect of the respective CM on tissue-factor-driven thrombus formation at an arterial shear rate of 2600 s−1 was studied in an ex vivo parallel-plate perfusion chamber device. In this model, human native blood was passed over a tissue factor/phospholipid-rich surface following 30 s exposure to 100% CM. The CM was washed out by nonanticoagulated blood drawn directly from an antecubital vein by a pump positioned distal to the perfusion chamber. Such a pre-exposure of the procoagulant surface to iodixanol reduced the fibrin deposition around the platelet thrombi by 50% (p<0.01). However, iohexol and ioxaglate did not affect fibrin deposition. None of the 3 CM affected the recruitment of platelets in the thrombi, since similar values were obtained with pre-exposure to 40 vol% of saline. Conclusion: Iodixanol appears to be most biocompatible with endothelium, and has a moderate inhibitory effect on fibrin deposition in flowing blood. This differs from iohexol, and in particular from ioxaglate, which induce endothelial changes in morphology with no effect on fibrin deposition. Since none of the CM affected the platelet aggregate formation, and since ioxaglate has been reported to have stronger anticoagulant and antithrombotic properties than iodixanol or iohexol in in vitro assays, it is apparent that these properties were not reflected in thrombus formation under the experimental conditions of high arterial shear.

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A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries.

Circulation Research ◽

10.1161/01.res.74.1.56 ◽

1994 ◽

Vol 74 (1) ◽

pp. 56-63 ◽

Cited By ~ 80

Author(s):

A B Pawashe ◽

P Golino ◽

G Ambrosio ◽

F Migliaccio ◽

M Ragni ◽

...

Keyword(s):

Monoclonal Antibody ◽

Tissue Factor ◽

Carotid Arteries ◽

Thrombus Formation ◽

Rabbit Tissue

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Relationship between Endothelial Tissue Factor and Thrombogenesis under Blood Flow Conditions

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649812 ◽

1995 ◽

Vol 74 (02) ◽

pp. 778-783 ◽

Cited By ~ 24

Author(s):

Armelle Diquélou ◽

Dominique Dupouy ◽

Dominique Gaspin ◽

Jacques Constans ◽

Pierre Sié ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Flow ◽

Tissue Factor ◽

Thrombus Formation ◽

Luminal Surface ◽

Fibrin Deposition ◽

Flow Conditions ◽

The Impact ◽

The Relationship ◽

Endothelial Tissue

SummaryWe have evaluated the relationship between the level of tissue factor (TF) expression by stimulated endothelial cells and thrombus formation under blood flow conditions. Cultures of human umbilical venous endothelial cells (HUVECs) were treated in order to express different levels of TF activity. They were stimulated for 4 h with either I) lipopolysaccharides (LPS, 10 µg/ml), II) recombinant interleukin Iß (IL1ß, 50 Ul/ml) or III) simultaneously with LPS and IL1ß (LPS + IL1ß). TF activity was low on confluent HUVECs or on the corresponding extracellular-matrix (ECM prepared by exposure of HUVECs to 0.1 N NH4OH). In contrast, it was high when HUVECs were stimulated with LPS or IL1ß, and significantly higher (p <0.05) with LPS+IL1ß. The TF activity associated with the stimulated ECM was 2-fold higher (p <0.05) than that expressed on the luminal surface of the stimulated HUVECs, irrespective of the agonist or combination of agonists used.These surfaces were exposed to non-anticoagulated human blood at a venous (50 s-1) and an arterial (650 s-1) wall shear rate in parallel-plate perfusion chambers for 5 min. Thrombus formation was morphologically quantified by measuring the deposition of platelets and fibrin. Fibrin deposition was also immunologically quantified. Fibrin deposition was related to the level of TF expression. Non-stimulated HUVECs and corresponding ECMs were not thrombogenic. The luminal surface of HUVECs stimulated with LPS or IL1ß alone expressed low levels of TF activity and was a poor inducer of platelet deposition and fibrin deposition (<15%) at 50 s-1. In contrast, fibrin deposition increased to 80% when the cells were stimulated with LPS and IL1ß simultaneously. This fibrin deposition was comparable to that found on the corresponding ECM, despite a two-fold lower TF activity. However, at 650 s-1, platelet and fibrin deposition on HUVECs stimulated with LPS + IL1ß were significantly lower than that observed on the corresponding ECM. In all circumstances, the thrombogenicity was TF-dependent, since fibrin deposition was totally blocked by anti-TF antibodies. Thus, it appears that the level of TF activity expressed on endothelial cells governs thrombus formation. However, the impact of TF expression on thrombus formation is also affected by the blood flow.

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Novel Plasma Based Microfluidic Assay for Measuring Fibrin Formation,Thrombin Generation and Fibrinolysis Under Flow: Monitoring Patients with Hemophilia on Inhibitors

Blood ◽

10.1182/blood.v128.22.2579.2579 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2579-2579

Author(s):

Abimbola Jarvis ◽

Katherine Ruegg ◽

Linda Jacobson ◽

Brian Branchford ◽

Elizabeth Villalobos-Menuey

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Substrate Surface ◽

Lag Time ◽

Clot Formation ◽

Plasma Samples ◽

Flow Monitoring ◽

Fibrin Formation

Abstract INTRODUCTION The unpredictable clinical response of patients to bypassing therapy and the lack of a proper laboratory tools to measure clot formation and stability renders prophylaxis and surgery on these patients a huge challenge. These patients are at a risk for bleeding or thromboembolic complications. AIMS In this study we introduce a novel plasma based microfluidic assay that can qualitatively and quantitatively measure fibrin deposition, thrombin and plasmin generation, and fibrinolysis under flow. We then examined the dynamics of thrombus formation in patients with hemophilia and their response to replacement and bypass therapies under flow conditions. METHODS Coagulation in the plasma based assay was initiated by spherical 1µm lipidated- Tissue Factor biomimetic silica beads which were patterned into 200µm circles on a substrate surface. Plasma samples were obtained from Hemophilia patients and inhibitor patients, before and after replacement or bypassing treatment and perfused over the tissue factor rich surfaces at a sheer rate of 100 s-1 Fibrinolysis was initiated with the addition of tPAto the plasma samples before perfusion. RESULTS The microfluidic assay was sensitive enough to measure the activation of coagulation triggered by the bypassing agents. Fibrin generation and thrombin generation were measurable both qualitatively and quantitatively using three metrics; lag time, rate of production and maximum quantity produced. Individuals on replacement therapy showed normalized fibrin formation with a 69% increase in fibrin formation, a decrease in lag time and an overall increase in maximum fibrin and thrombin production (See attached Figure). The microfluidic assay was also able to show an increase in overall fibrin generation in certain Individuals that were given more bypassing treatment than needed. Compared to healthy controls the rate of fibrin generation and maximum fibrin was greater, thereby identifying a risk for prothrombotic state. (See attached Figure). Finally, using the microfluidic assay we were able to observe both clot formation and lysis and asses the the stability of the fibrin clot produced when these inhibitor patients were on and off treatments, which reflects a more complete picture of the coagulation process. CONCLUSION We are able to show that individuals, treated by replacement therapies showed normalized clot formation. Individuals with hemophilia treated by bypassing therapies also showed normalized clot formation. Sometimes however, the fibrin production is more than a normal control, which could lead to a risk of prothrombosis. By using microfluidic assay, the treatment can be given in doses and fibrin production observed, to decrease the overall fibrin formation from a hypocoagulable to a hemostatic state, avoiding hypercoagulability. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Dietary α-Linolenic Acid Inhibits Arterial Thrombus Formation, Tissue Factor Expression, and Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.111.226118 ◽

2011 ◽

Vol 31 (8) ◽

pp. 1772-1780 ◽

Cited By ~ 45

Author(s):

Erik W. Holy ◽

Marc Forestier ◽

Eva K. Richter ◽

Alexander Akhmedov ◽

Florian Leiber ◽

...

Keyword(s):

Tissue Factor ◽

Linolenic Acid ◽

Platelet Activation ◽

Thrombus Formation ◽

Tissue Factor Expression ◽

Arterial Thrombus ◽

Factor Expression

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

Blood ◽

10.1182/blood-2009-03-210179 ◽

2010 ◽

Vol 115 (2) ◽

pp. 161-167 ◽

Cited By ~ 166

Author(s):

Nicholas T. Funderburg ◽

Elizabeth Mayne ◽

Scott F. Sieg ◽

Robert Asaad ◽

Wei Jiang ◽

...

Keyword(s):

Hiv Infection ◽

Tissue Factor ◽

Immune Activation ◽

Interleukin 6 ◽

Plasma Levels ◽

Thrombus Formation ◽

Hiv Replication ◽

Increased Risk

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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Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model

Blood ◽

10.1182/blood.v68.3.783.bloodjournal683783 ◽

1986 ◽

Vol 68 (3) ◽

pp. 783-786 ◽

Cited By ~ 1

Author(s):

BS Coller ◽

JD Folts ◽

LE Scudder ◽

SR Smith

Keyword(s):

Monoclonal Antibody ◽

Animal Model ◽

Platelet Aggregation ◽

Ex Vivo ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Antithrombotic Effect ◽

Antithrombotic Agent ◽

Antithrombotic Effects ◽

Platelet Thrombus

A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.

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