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

Abstract

Heparin-induced thrombocytopenia (HIT) is IntroductionHeparin-induced thrombocytopenia (HIT) is a well-recognized complication of heparin therapy and is one of the most common and potentially devastating causes of drug-induced thrombocytopenia. 1 Millions of Americans are exposed to heparin each year, resulting in thousands of cases of HIT annually. 2 Platelet factor 4 (PF4), a strongly cationic chemokine expressed by megakaryocytes and packaged into the ␣ granules of platelets, is released on platelet activation. 3 PF4 binds with high affinity to highly anionic molecules, such as heparin and endogenous heparin-like glycosaminoglycans (GAGs). 4 The precise physiologic role of PF4 in the regulation of coagulation is not known. 5 A subset of patients exposed to heparin develop autoantibodies (so called HIT Abs) against PF4/heparin complexes. 6,7 These Abs bind to specific epitopes on PF4, leading to the formation of trimeric complexes of PF4/heparin-IgG, which are referred to as HIT Ab complexes. 8,9 These complexes bind to platelets, resulting in platelet activation and the development of thrombocytopenia. 10 Immune complexes exert their effects on cells via engagement of specific Fc receptors. IgG Abs bind to Fc␥ receptors. In humans, 3 different classes of Fc␥Rs have been identified: Fc␥Rs I, II, and III. Fc␥RI is a high-affinity receptor and as such can also bind monomeric IgG as opposed to the low-affinity Fc␥Rs, Fc␥RII and Fc␥RIII, that only bind aggregated immunoglobulins or Ag-Ab complexes. 11 Fc␥RI is expressed on monocytes/macrophages, neutrophils, eosinophils, and dendritic cells. Fc␥RII is further subclassified into Fc␥RIIa and Fc␥RIIb. Fc␥RIIa is an activating receptor and is expressed on monocytes/macrophages, neutrophils, eosinophils, platelets, and Langerhans cells, whereas Fc␥RIIb is expressed on B lymphocytes and mast cells. In terms of function, Fc␥RIIb is the only inhibitory Fc␥R, and all the other Fc␥Rs result in activation of cells. 12 Fc␥RIII is subdivided into Fc␥RIIIa, which is expressed on natural killer cells and macrophages, and Fc␥RIIIb, which is expressed on eosinophils, neutrophils, macrophages, mast cells, and follicular dendritic cells. Binding of immune complexes to the activating Fc␥Rs leads to phosphorylation of the cytoplasmic immunoreceptor tyrosine-based activation motif by src-kinases, leading to activation of downstream signaling. 12 It has been found that platelet activation by HIT Ab complexes is mediated by engagement of the Fc␥RIIa receptor. 10 Most thrombocytopenias are associated with an increased risk of bleeding. However, the most-feared complication in HIT is the development of arterial and venous thrombosis. 13,14 The precise mechanism leading to thrombosis in HIT is unknown. It has been proposed that platelet activation and the release of platelet-derived microparticles (PMPs) may be responsible for thrombosis. 15,16 Microparticles (MPs) are sub-micron-sized membrane vesicles released on cell activation or apoptosis. 17 PMPs generally express phosphatidylserine a...

Comparison of natural and recombinant tissue factor proteins: new insights

“…More recently, it has been suggested that the toll-like receptors TLR4 and TLR6 contribute to the expression of TF on monocytes and monocytic cells Zhou et al , 2011 ;Owens et al , 2012 ) induced by oxidized low-density lipoproteins and anti-β 2 -glycoprotein I/ β 2 -glycoprotein I. It has been also shown that plateletderived growth factor CC (Gebhard et al , 2010 ) and the platelet factor 4/heparin-antibody complex (Kasthuri et al , 2012 ) can induce monocyte TF expression. It has been suggested that the majority of the cell surface TF is latent or ' encrypted ' (Bach , 2006 ), has little (if any) activity, and that cell lysis (Le et al , 1992 ) or treatment (Wolberg et al , 1999 ) can lead to the ' decryption ' .…”

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia

“…On monocytes, however, FcgRI has been implicated in the KKO-mediated generation of TF and monocyte microparticle formation. 31 Both FcgRI and FcgRIIA activation involves ITAM, which is rapidly phosphorylated upon receptor engagement followed by binding of Syk. 32 We previously reported that inhibition of the Syk pathway in platelets using the specific inhibitor PRT318 blocked the development of thrombocytopenia and thrombosis in the murine model of HIT.…”

Excess of heme induces tissue factor-dependent activation of coagulation in mice

“…Isolation of human PBMCs and mouse white blood cells, the 1-stage clotting assay and TF staining are standard procedures in our laboratory, 14,26 and are described in detail in the Online Supplementary Methods.…”