FcRn augments induction of tissue factor activity by IgG-containing immune complexes

Abstract Thromboembolism complicates disorders caused by immunoglobulin G (IgG)–containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), β-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

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Complement mediates binding and procoagulant effects of ultra-large HIT immune complexes

Blood ◽

10.1182/blood.2020009487 ◽

2021 ◽

Author(s):

Sanjay Khandelwal ◽

Ayiesha Barnes ◽

Lubica Rauova ◽

Amrita Sarkar ◽

Ann H Rux ◽

...

Keyword(s):

Tissue Factor ◽

Complement Activation ◽

Immune Complexes ◽

Classical Pathway ◽

Tissue Factor Expression ◽

Platelet Factor ◽

Effector Functions ◽

Igg Binding ◽

Factor Expression ◽

Monocyte Cell

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing IgG antibodies to a multivalent antigen composed of platelet factor 4 (PF4) and heparin. The limitations of current anti-thrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcgRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote co-deposition of IgG and activated C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates Fcg receptor(R)-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcgRs and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway, abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex-mediated thrombotic disorders.

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Induction of monocyte tissue factor expression by antibodies to heparin–platelet factor 4 complexes developed in heparin-induced thrombocytopenia

Blood ◽

10.1182/blood.v97.10.3300 ◽

2001 ◽

Vol 97 (10) ◽

pp. 3300-3302 ◽

Cited By ~ 136

Author(s):

Claire Pouplard ◽

Sophie Iochmann ◽

Benoit Renard ◽

Olivier Herault ◽

Philippe Colombat ◽

...

Keyword(s):

Tissue Factor ◽

Mononuclear Cells ◽

Factor Xa ◽

Platelet Factor 4 ◽

Peripheral Blood Mononuclear ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Chromogenic Assay ◽

High Concentrations

Abstract The pathogenesis of thrombosis in heparin-induced thrombocytopenia (HIT) was studied by investigating whether antibodies to heparin-platelet factor 4 (H-PF4) induced tissue factor (TF) synthesis by monocytes. Plasma from 5 patients with HIT containing IgG to H-PF4 was incubated with peripheral blood mononuclear cells without or with purified PF4 and heparin. Significant TF-dependent procoagulant activity (PCA) expressed by monocytes, measured with a factor Xa-based chromogenic assay, was induced after incubation of each HIT plasma sample. This monocyte PCA required the presence of PF4 and was inhibited by high concentrations of heparin. Furthermore, purified HIT IgG added to whole blood with PF4 and heparin also provoked significant synthesis of TF mRNA by monocytes, demonstrated by RT-PCR, and this effect was not observed with normal IgG. These findings strongly support the hypothesis that antibodies to PF4 developed in HIT trigger the production of tissue factor by monocytes, and this effect could account in vivo for hypercoagulability and thrombotic complications in affected patients.

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Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657643 ◽

1997 ◽

Vol 78 (02) ◽

pp. 864-870 ◽

Cited By ~ 9

Author(s):

Hideki Nagase ◽

Kei-ichi Enjyoji ◽

Yu-ichi Kamikubo ◽

Keiko T Kitazato ◽

Kenji Kitazato ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Free Form ◽

Initial Reaction ◽

Extrinsic Pathway ◽

Factor Xa Inhibition ◽

Tissue Factor Pathway

SummaryDepolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

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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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Recombinant full-length tissue factor pathway inhibitor fails to bind to the cell surface: implications for catabolism in vitro and in vivo

Blood ◽

10.1182/blood.v95.6.1973 ◽

2000 ◽

Vol 95 (6) ◽

pp. 1973-1978 ◽

Cited By ~ 7

Author(s):

Guyu Ho ◽

Masaaki Narita ◽

George J. Broze ◽

Alan L. Schwartz

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Feedback Inhibition ◽

Factor Xa ◽

Full Length ◽

Pharmacokinetic Studies ◽

Dependent Manner ◽

Carboxy Terminus ◽

Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) plays a key role in the regulation of tissue factor-initiated blood coagulation secondary to loss of the integrity of the blood vessel wall. TFPI is a naturally occurring Kunitz-type protease inhibitor that inhibits coagulation factor Xa and, in a factor Xa-dependent manner, mediates feedback inhibition of the factor VIIa/tissuefactor catalytic complex. In vivo full-length TFPI is thought to be primarily bound to the vascular endothelium and the high affinity binding requires an intact carboxy terminus. Here we describe a full-length TFPI molecule, expressed in mouse C127 cells (TFPIC127), which exhibits virtually no cellular binding yet contains the intact carboxy terminus. This TFPI (TFPIC127) is neither internalized nor degraded via the TFPI endocytic receptor, LDL-receptor–related protein. Pharmacokinetic studies of TFPIC127 in vivo demonstrate a 10-fold prolongation in the plasma half-life, compared with that of bacterial recombinant TFPI.

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Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽

10.1182/blood-2009-02-203604 ◽

2009 ◽

Vol 114 (2) ◽

pp. 452-458 ◽

Cited By ~ 96

Author(s):

Dmitri V. Kravtsov ◽

Anton Matafonov ◽

Erik I. Tucker ◽

Mao-fu Sun ◽

Peter N. Walsh ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Factor Xa ◽

Factor Ix ◽

Factor Xii ◽

Factor Xi ◽

Factor Xii Deficiency ◽

Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

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Thrombin generation is not increased in the blood of hemophilia B patients after the infusion of a purified factor IX concentrate

Blood ◽

10.1182/blood.v76.12.2540.2540 ◽

1990 ◽

Vol 76 (12) ◽

pp. 2540-2545 ◽

Cited By ~ 41

Author(s):

PM Mannucci ◽

KA Bauer ◽

A Gringeri ◽

S Barzegar ◽

B Bottasso ◽

...

Keyword(s):

Significant Risk ◽

Factor Xa ◽

Factor Ix ◽

Hemophilia B ◽

Coagulation Cascade ◽

Platelet Factor ◽

Sensitive Index ◽

Before And After ◽

Fibrin Monomers

Abstract Prothrombin complex concentrates (PCC), licensed for the treatment of hemophilia B, are known to carry a significant risk of thromboembolic complications. Although the reasons for thrombogenicity are not completely understood, several manufacturers have developed purified factor IX concentrates that contain negligible amounts of the other vitamin K-dependent factors. To evaluate whether or not the infusion of such a factor IX concentrate is followed by lesser activation of the hemostatic system than by the infusion of a PCC, we performed a series of coagulation assays on 11 hemophilia B patients before and after the administration of these two types of concentrate using a randomized cross-over design. The levels of prothrombin fragment F1 + 2, a sensitive measure of the in vivo cleavage of prothrombin by factor Xa, was significantly increased in plasma after PCC, but not after factor IX concentrate. Plasma fibrinopeptide A, a sensitive index of the enzymatic activity of thrombin on fibrinogen, also increased significantly after PCC but not after factor IX concentrate. The fragment B beta 15–42, a sensitive index of the enzymatic action of plasmin on fibrin II, did not change after either concentrate. There were also no differences in less sensitive coagulation measurements, such as plasma fibrinogen, antithrombin III, and fibrin monomers, nor in indices of platelet activation, such as beta-thromboglobulin and platelet factor 4. These findings show that the infusion of a purified factor IX concentrate can result in substantially less activation of the coagulation cascade than may be seen with PCC.

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The Effect of Grp78 Inhibition on Tissue Factor Gene Expression, Procoagulant Activity, and Factor Xa Generation.

Blood ◽

10.1182/blood.v104.11.1924.1924 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1924-1924

Author(s):

Gourab Bhattacharjee ◽

Jasimuddin Ahamed ◽

Brian Pedersen ◽

Amr El-Sheikh ◽

Cheng Liu ◽

...

Keyword(s):

Gene Expression ◽

Cell Surface ◽

Tissue Factor ◽

Factor Xa ◽

Cell Types ◽

Procoagulant Activity ◽

Coagulation Cascade ◽

Clotting Factor ◽

Tf Gene

Abstract In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.

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The Role of Fcγ Receptor Signaling, Tissue Factor Expression and Monocyte-Platelet Cross-Talk in the Initiation of Thrombosis by Immune Complexes.

Blood ◽

10.1182/blood.v106.11.2167.2167 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2167-2167

Author(s):

Peter Casasanto ◽

Michael P. Reilly ◽

Ming-Lin Liu ◽

Kevin Jon Williams ◽

Steven E. McKenzie

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Immune Complex ◽

Cross Talk ◽

Immune Complexes ◽

Thrombus Formation ◽

Procoagulant Activity ◽

Monocytic Cell ◽

Platelet Factor ◽

Activated Platelets

Abstract Thrombus formation in response to physical disruption of the vascular endothelium is an essential response to vessel injury. In contrast, thrombus formation is pathologic when the endothelium is physically intact but blood and endothelial cells are activated by inflammation. Thrombosis secondary to immune complexes is a major cause of morbidity and mortality in hospitalized patients. We recently generated and characterized the first transgenic mouse model of heparin-induced thrombocytopenia/thrombosis (HIT/T) to recapitulate the salient features of the disease and confirmed that complexes of heparin and platelet factor 4, antibodies to the complex, and FcγRIIa-dependent platelet activation are both necessary and sufficient to model the disease in vivo. It is also likely that immune complex activation of monocytes and endothelial cells occurs in HIT/T. However, the interaction between activated blood and endothelial cells and tissue factor positive microparticles (TF+-MP) that may result in thrombin generation is not clear. Recent studies (del Conde et al., Blood 2005) showed that phosphatidylserine and PSGL-1 on the surface of monocyte-derived TF+-MP enables their fusion with activated platelets. Collagen-activated platelets incubated with TF+-MP were reported to cause increased TF-VIIa procoagulant activity (PCA) compared to non-activated platelets. We hypothesized that platelets activated by HIT/T immune complexes would also result in increased TF-PCA when incubated with monocyte-derived TF+-MP. To test this hypothesis we generated TF+-MP from THP-1 cells, a human monocytic cell line, stimulated with LPS (6 hr) and A23187 (subsequent 15 min). TF+-MPs were co-incubated with untreated or agonist-treated platelets. The HIT/T immune complex was prepared by incubating optimal ratios of heparin and recombinant human PF4 with KKO, a mouse monoclonal anti-heparin-PF4 antibody. Other agonists included anti-CD9 (producing a particulate immune complex) and collagen. Using a chromogenic assay of Xa generation we found that TF+-MPs were necessary to detect TF-PCA. PCA increased by 12–25% when TF+-MPs were incubated with platelets stimulated by collagen or anti-CD9 as compared to untreated platelets. When TF+-MPs were incubated with platelets stimulated by the HIT/T immune complex, there was a 2-fold increase in the PCA. The increase in TF-PCA was observed to be proportional to the concentration of microparticles added. The results suggest an important role in platelet-monocyte cross-talk in initiating and increasing TF procoagulant activity upon immune complex stimulation.

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Dual Effect of Platelet Factor Four on the Activities of Factor Xa

Blood ◽

10.1182/blood.v112.11.1034.1034 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1034-1034

Author(s):

Martine Marie Fiore ◽

Ian J Mackie

Keyword(s):

Factor Xa ◽

Fold Increase ◽

Peptide Hydrolysis ◽

Dual Effect ◽

Platelet Factor ◽

Paradoxical Effects ◽

Heparin Activity ◽

High Concentrations ◽

Prothrombin Activation

Abstract Platelet Factor 4 (PF4) is a cationic molecule that binds to heparin with high affinity and neutralises the activity of the latter. Our recent studies indicate that heparin can promote an interaction of fXa with PF4 since neutralization of heparin activity by PF4 was dependent on the concentration of protease. To examine the contribution of PF4 in protease function, fXa activity was determined in chromogenic assays. Upon preincubation with fXa and heparin, PF4 (at a concentration of 100 nM) decreased the kcat of S2765 peptide hydrolysis 4-fold and that of prothrombin activation about 2-fold. These results suggested an effect of PF4 on the primary specificity of the protease. In fact, PF4 exerted a mild effect (30 % decrease) on the Na+ dependence of fXa, consistent with linkage between Na+ and S1. PF4 preincubation with fXa also prevented the binding of the S1 probe p-aminobenzamidine (pAB) while simultaneous addition of PF4 and pAB diminished the contribution of PF4. In the presence of excess fVa (relative to fXa), kinetic parameters measuring fXa amidolytic activity in the presence of PF4 were restored to control values in the absence of PF4. Interestingly, high concentrations of PF4 (> 1 μM) totally restored fXa activity toward peptidyl substrate and strongly enhanced prothrombin activation, indicating a dual effect of PF4 on fXa activities. The inhibitory contribution of PF4 during prothrombin activation was due to a three-fold decreased affinity of fXa for fVa while enhancement of prothrombin activation was accompanied by a three-fold increase in fVa-dependent cofactor activity. Thus, the effects of PF4 possibly involved a region of the heparin/fVabinding exosite that is linked to the S1 and Na+ sites. These findings suggest that PF4 is a probe of fVa-dependent changes occurring in the active site of fXa and provide an explanation for the in vivo paradoxical effects of PF4 reported in the literature.

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