Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8

Thrombosis is a life-threatening complication that occurs in a subset of patients with heparin-induced thrombocytopenia (HITT). The pathogenic mechanisms underlying the variable occurrence of thrombosis in HITT is poorly understood. It was hypothesized that monocyte activation leading to tissue factor expression may play a role in promoting a thrombogenic state in HITT. This study demonstrates that a human platelet factor 4 (PF4)/heparin-specific murine monoclonal antibody (KKO) binds to peripheral blood-derived human monocytes in a PF4-dependent manner. KKO and antibodies from patients with HITT induce monocytes to synthesize and secrete interleukin-8 and induce cell-surface procoagulant activity, which is abrogated following treatment with antihuman tissue factor antibody. The findings suggest a novel mechanism by which PF4/heparin antibodies may promote a hypercoagulable state in patients with HITT.

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PF4/heparin complexes are T cell–dependent antigens

Blood ◽

10.1182/blood-2004-12-4955 ◽

2005 ◽

Vol 106 (3) ◽

pp. 929-931 ◽

Cited By ~ 38

Author(s):

Shayela Suvarna ◽

Lubica Rauova ◽

Emily K. E. McCracken ◽

Christina M. Goss ◽

Bruce S. Sachais ◽

...

Keyword(s):

T Cell ◽

Dependent Manner ◽

Athymic Mice ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Thymic Function ◽

Preferential Binding ◽

Heparin Complexes ◽

Life Threatening ◽

Autoantibody Formation

Abstract Heparin-induced thrombocytopenia (HIT) is a life-threatening, thrombotic disorder associated with development of anti–platelet factor 4 (anti-PF4)/heparin autoantibodies. Little is known about the antigenic and cellular requirements that initiate the immune response to these complexes. To begin to delineate mechanisms of autoantibody formation in HIT, we studied the immunizing effects of murine PF4 (mPF4)/heparin in mice with and without thymic function. Euthymic mice were injected with mPF4/heparin complexes, mPF4, heparin, or buffer. Mice injected with mPF4/heparin, but not mPF4 or heparin alone, developed heparin-dependent autoantibodies that shared serologic and functional characteristics of human HIT antibodies, including preferential binding to mPF4/heparin complexes and causing heparin- and FcRγIIA-dependent platelet activation. In contrast, athymic mice did not develop HIT-like antibodies. Taken together, these studies establish that PF4/heparin complexes are highly immunogenic and elicit self-reacting anti-PF4/heparin antibodies in a T cell–dependent manner.

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PF4/heparin-antibody complex induces monocyte tissue factor expression and release of tissue factor positive microparticles by activation of FcγRI

Blood ◽

10.1182/blood-2011-06-359430 ◽

2012 ◽

Vol 119 (22) ◽

pp. 5285-5293 ◽

Cited By ~ 56

Author(s):

Raj S. Kasthuri ◽

Sam L. Glover ◽

William Jonas ◽

Troy McEachron ◽

Rafal Pawlinski ◽

...

Keyword(s):

Tissue Factor ◽

Mononuclear Cells ◽

Ex Vivo ◽

Tissue Factor Expression ◽

Drug Induced ◽

Peripheral Blood Mononuclear ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Factor Expression ◽

Antibody Complex

Heparin-induced thrombocytopenia (HIT) is a potentially devastating form of drug-induced thrombocytopenia that occurs in patients receiving heparin for prevention or treatment of thrombosis. Patients with HIT develop autoantibodies to the platelet factor 4 (PF4)/heparin complex, which is termed the HIT Ab complex. Despite a decrease in the platelet count, the most feared complication of HIT is thrombosis. The mechanism of thrombosis in HIT remains poorly understood. We investigated the effects of the HIT Ab complex on tissue factor (TF) expression and release of TF-positive microparticles in peripheral blood mononuclear cells and monocytes. To model these effects ex vivo, we used a murine mAb specific for the PF4/heparin complex (KKO), as well as plasma from patients with HIT. We found that the HIT Ab complex induced TF expression in monocytes and the release of TF-positive microparticles. Further, we found that induction of TF is mediated via engagement of the FcγRI receptor and activation of the MEK1-ERK1/2 signaling pathway. Our data suggest that monocyte TF may contribute to the development of thrombosis in patients with HIT.

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Diagnosis and treatment of heparin-induced thrombocytopenia

Perfusion ◽

10.1191/0267659103pf637oa ◽

2003 ◽

Vol 18 (1) ◽

pp. 47-53 ◽

Cited By ~ 6

Author(s):

William J DeBois ◽

Junli Liu ◽

Leonard Y Lee ◽

Leonard N Girardi ◽

Charles Mack ◽

...

Keyword(s):

New York ◽

Platelet Inhibition ◽

Heparin Therapy ◽

Serotonin Release ◽

Antibody Detection ◽

Thrombin Inhibitor ◽

Heparin Infusion ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Life Threatening

Heparin-induced thrombocytopenia (HIT) is a major side effect secondary to the administration of heparin. This syndrome is serious and potentially life threatening. This response is the result of antibodies formed against the platelet factor 4 (PF4)/heparin complex. The incidence of this immune-mediated syndrome has been estimated to be 1-3% of all patients receiving heparin therapy. The occurrence of HIT in patients requiring full anticoagulation for cardiopulmonary bypass (CPB), therefore, presents a serious challenge to the cardiac surgery team. The diagnosis of HIT should be based on both clinical and laboratory evidence. While functional assays, platelet aggregation tests, and the serotonin release assay can be used to support the diagnosis, the negative predictive value of these tests is generally less than 50%. In contrast, although non-functional antibody detection assays are more sensitive, they have a low specificity. HIT can be treated in several ways, including cessation of all heparin and giving an alternative thrombin inhibitor, platelet inhibition followed by heparin infusion, and the use of low molecular weight heparins. In this presentation, the pathology and current diagnostic tests, as well as the successful management of patients with HIT undergoing CPB at New York Presbyterian Hospital, are reviewed.

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Adenosine Inhibits Tissue Factor Expression by LPS-stimulated Human Monocytes: Involvement of the A3 Adenosine Receptor

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613164 ◽

2002 ◽

Vol 88 (07) ◽

pp. 123-130 ◽

Cited By ~ 32

Author(s):

Matthieu Broussas ◽

Pascale Cornillet-Lefèbvre ◽

Gérard Potron ◽

Philippe Nguyên

Keyword(s):

Tissue Factor ◽

Rank Order ◽

Mrna Level ◽

Dependent Manner ◽

Human Monocytes ◽

Extrinsic Pathway ◽

A3 Adenosine Receptor ◽

Factor Expression ◽

Cardioprotective Effects ◽

Dose Dependent

SummaryTissue Factor (TF), an integral membrane glycoprotein that initiates the extrinsic pathway of blood coagulation, is thought to play a major part in coronary acute events. Adenosine, an endogenous nucleoside produced by the degradation of intracellular adenosine triphosphate, has been shown to exert many cardioprotective effects via an inhibition of platelets and neutrophils. This study was conducted to determine whether adenosine (ADO) could modulate the expression of TF by human monocytes. We found that ADO inhibited TF antigen and activity on endotoxin-stimulated monocytes in a dose-dependent manner. The mechanism was at least pre-translational since ADO caused a change in the TF mRNA level. Using ADO receptor-specific analogs, we showed that highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5’-N’-methyluronamide (IB-MECA) inhibited LPSinduced TF activity expression more potently than A1 agonist R-phenylisopropyladenosine (R-PIA) and A2 agonist CGS 2180. Furthermore, A1/A3 antagonist, xanthine amine congener (XAC) blocked the effect of ADO whereas A2a, A2b and A1 antagonists were ineffective. In addition, we observed that ADO agonists inhibited monocyte TF expression in LPS-stimulated whole blood. The rank order of agonist potency suggested that A2 and A3 receptors might be involved (2-Cado > CGS = IB-MECA > R-PIA). This was supported by the fact that A2 and A3 antagonists reversed the action of 2-Cado. We conclude that TF inhibition by ADO on human purified monocytes involved A3 receptors.

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Serologic Results in >1000 Patients With Suspected Heparin-Induced Thrombocytopenia

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029607304721 ◽

2007 ◽

Vol 14 (4) ◽

pp. 410-414 ◽

Cited By ~ 8

Author(s):

Suresh G. Shelat ◽

Anne Tomaski ◽

Eleanor S. Pollak

Keyword(s):

Laboratory Diagnosis ◽

Serotonin Release ◽

Enzyme Linked Immunosorbent Assay ◽

Functional Assay ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Life Threatening ◽

Release Assay ◽

Pathogenic Antibodies

Heparin-induced thrombocytopenia (HIT) can lead to life-threatening and limb-threatening thrombosis. HIT is thought to be initiated by the interaction of pathogenic antibodies toward a complex platelet factor 4 (PF4) and heparin (PF4:H), which can activate platelets and predispose to thrombosis. As such, the laboratory diagnosis of HIT includes antigenic and functional assays to detect antibodies directed at PF4:H complexes. We performed a retrospective analysis of 1017 consecutive samples tested by serotonin-release assay and by enzyme-linked immunosorbent assay (ELISA). Most samples showed no serologic evidence of HIT, whereas 4% to 5% of samples demonstrated both antigenic and functional serological evidence for HIT. Approximately 12% to 18% of samples showed immunologic evidence of anti-PF4:H antibodies but without functional evidence of serotonin release in vitro. Interestingly, a small minority of samples (0.7%) caused serotonin release but were negative in the ELISA. The results are presented using cutoff values established at our hospital and for the ELISA manufacturer. This study provides a pretest probability of the serologic results from an antigenic assay (ELISA) and a functional assay (serotonin-release assay) in patients clinically suspected of having HIT.

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Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells

Biochemical Journal ◽

10.1042/bj3141027 ◽

1996 ◽

Vol 314 (3) ◽

pp. 1027-1033 ◽

Cited By ~ 30

Author(s):

Karine AUPEIX ◽

Florence TOTI ◽

Nathalie SATTA ◽

Pierre BISCHOFF ◽

Jean-Marie FREYSSINET

Keyword(s):

Tissue Factor ◽

Annexin V ◽

Antigen Expression ◽

Resting Cells ◽

Dependent Manner ◽

Anionic Phospholipid ◽

Monocytic Cells ◽

Anionic Phospholipids ◽

Phosphatidylserine Exposure ◽

And Control

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7β-hydroxycholesterol (7β-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7β-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may result from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.

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Dexamethasone enhances LPS induction of tissue factor expression in human monocytic cells by increasing tissue factor mRNA stability

Journal of Leukocyte Biology ◽

10.1189/jlb.0204068 ◽

2004 ◽

Vol 76 (1) ◽

pp. 145-151 ◽

Cited By ~ 28

Author(s):

K. Veera Reddy ◽

Gourab Bhattacharjee ◽

Gernot Schabbauer ◽

Angela Hollis ◽

Kevin Kempf ◽

...

Keyword(s):

Tissue Factor ◽

Mrna Stability ◽

Tissue Factor Expression ◽

Monocytic Cells ◽

Factor Expression

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Management of Heparin-Induced Thrombocytopenia and Heparin-Induced Thrombocytopenia and Thrombosis Syndrome

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029697003001s08 ◽

1997 ◽

Vol 3 (1_suppl) ◽

pp. S53-S63 ◽

Cited By ~ 6

Author(s):

Jeanine M. Walenga ◽

Bruce E. Lewis ◽

Debra A. Hoppensteadt ◽

Jawed Fareed ◽

Mamdouh Bakbos

Keyword(s):

Antibody Titer ◽

Platelet Factor 4 ◽

Positive Patient ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Comprehensive Account ◽

Immune Mediated ◽

Life Threatening ◽

Pros And Cons ◽

Sensitivity Specificity

Summary: Heparin-induced thrombocytopenia (HIT) is an immune mediated response to heparin in which antibody driven thrombosis can have a dramatic life-threatening expression. There is much interest on this subject including studies on the pathophysiologic mechanism, the clinical managements of the initial stages of HIT versus the HIT-positive patient requiring continued anticoagulation versus the HIT patient with thrombosis, the pros and cons of available alterr~aci~~ anticoagulants, and the laboratory assays to aid in the diagnosis of HIT with particular reference to the sensitivity/specificity of the new heparin-platelet factor 4 antibody titer assay. A comprehensive account of these timely issues is given in this article.

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