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

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 Xabased 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. IntroductionHeparin-induced thrombocytopenia (HIT) is a common complication of unfractionated heparin (UFH) therapy, often associated with arterial or venous thrombosis. 1 Such thrombocytopenia results from platelet activation caused by antibodies specific for complexes composed of heparin and platelet factor 4 (FP4). [2][3][4][5] The platelet activation involves the binding of HIT IgG to specific Fc receptors (Fc␥RIIA), 6 partly explaining why HIT differs from most other types of drug-induced immune thrombocytopenia, which are mainly complicated by hemorrhage.The most frequent manifestation in HIT is thrombosis, sometimes associated with disseminated intravascular coagulation, and one of the mechanisms proposed to explain the hypercoagulability in HIT is the production of phospholipid platelet microparticles after platelet activation. 7 However, HIT antibodies might also bind to endothelial cells and trigger the synthesis of tissue factor (TF), 4,8,9 a transmembrane glycoprotein that initiates the coagulation pathway. 10,11 TF can be produced by monocytes when activated by lipopolysaccharides, cytokines, or immune complexes, 12 and it can be triggered in the presence of activated platelets. 13 We therefore investigated whether antibodies to PF4 developed in patients with HIT to induce the synthesis of TF by human monocytes. Study design Plasma and purification of immunoglobulin GPlasma from 5 patients with definite HIT complicated by venous (n ϭ 5) and arterial (n ϭ 1) thromboses was studied. Every sample contained significant levels of antibodies to H-PF4 complexes (mean A 492 , 1.7; range, 1.0-2.1) (Asserachrom HPIA, Stago; Asnières, France) 14 able to induce heparin-dependent platelet activation in serotonin release assay (SRA). 15 Normal human plasma without antibodies to H-PF4 complexes (mean A 492 Ͻ 0.2) was collected from 5 healthy volunteers not receiving heparin. All samples were heated at 56°C for 1 hour, centrifuged ...

Thrombocytopenic conditions—autoimmunity and hypercoagulability: Commonalities and differences in ITP, TTP, HIT, and APS

“…Blank et al [57] have demonstrated that in vitro, anti-PF4-heparin antibodies can directly activate endothelial cells via binding of the Fab portion of the IgG. This activation was manifested by an increased release of IL-6, vWf, and thrombomodulin [57] and increased expression of adhesion molecules (p-selectin, e-selectin, and VCAM-1), inflammatory cytokines (IL-1B, IL-6, TNFa, and PAI-1), increased monocyte adhesion, and increased expression of tissue factor [56][57][58][59]. Interestingly, in a recent clinical study on patients with acute coronary syndrome treated with heparin, the development of HIT antibodies was associated with a significant increase in recurrent myocardial infarction within 30 days [60].…”

Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia

“…17 Leukocyte-platelet aggregates and leukocyte activation has been identified in the circulation of affected patients, [18][19][20] and HIT antibodies have been shown to induce elaboration of tissue factor (TF) in several cell types, 21 including monocytes. 22,23 Yet, the involvement of monocytes in the pathogenesis of thrombosis has not been demonstrated directly.…”

“…17 Leukocyte-platelet aggregates and leukocyte activation has been identified in the circulation of affected patients, 18-20 and HIT antibodies have been shown to induce elaboration of tissue factor (TF) in several cell types, 21 including monocytes. 22,23 Yet, the involvement of monocytes in the pathogenesis of thrombosis has not been demonstrated directly.Participation of monocytes in the pathogenesis of HIT may be mediated through their proclivity to bind PF4 released from activated platelets. Monocytes, unlike platelets, also express GAG side chains composed of dermatan sulfate (DS) and heparan sulfate (HS) as well, 24,25 both of which bind PF4 with higher affinity than CS, 1 making the bound PF4 more resistant to elution by heparin.…”

“…In a prior study, monocyte activation and TF expression by HIT antibodies occurred with isolated monocytes when PF4 was added alone without the addition of heparin. 23 When whole blood was studied, both PF4 and heparin had to be added to see activation by the HIT antibody. These studies are consistent with the data in Figure 1B, if there were released endogenous PF4 in addition to the exogenous PF4, in the whole blood samples.…”

Risk of thrombosis in patients with malignancy and heparin‐induced thrombocytopenia

“…For example, platelet activation, suppression of fibrinolytic activity and coagulation cascade activation, all of which have been described in cancer related thrombosis, may interact with circulating procoagulant platelet microparticles previously described in HIT [11,12]. Also, induction of monocyte tissue factor expression has been described in in vitro experiments with HIT antibodies [10].…”