Viec Antibodies In

There are limited experimental data to indicate that EC-reactive antibodies or immune complexes contribute to the development of thrombosis in patients with HIT. Over 10 yr ago, one group reported that sera from essentially all patients with HIT deposit increased amounts of IgG, IgM, or IgA on HUVEC (Cines et al., 1987). Binding was reduced when the cells were pretreated with enzymes that degrade heparin or heparan sulfate, whereas addition of chondroitinase was without effect. HIT sera induced ECs to express the procoagulant tissue factor, and the expression of procoagulant activity was enhanced further in the presence of platelets. These observations were confirmed and extended by Visentin and colleagues (1994), who demonstrated that the binding of HIT antibodies of HUVEC was dependent on PF4, but not on exogenous heparin, in contrast to the requirement for both to be added for antibody binding to platelets. This is consistent with the concept that PF4, released from activated platelets, can form a competent antigenic complex on the pericellular matrix of the endothelium.

The role of EC-reactive antibodies was also explored in an animal model that simulates certain aspects of HIT (Blank et al., 1997). Mice injected with IgG fractions obtained from HIT patients developed anti-idiotypic antibodies that recognized complexes between hPF4 and heparin. Furthermore, the anti-idiotypic antibodies competed with the immunizing antibodies for binding to the antigenic complex. These effects were not noted when antibodies obtained from mice immunized with control IgG were studied. Additionally, mice immunized with HIT-IgG developed thrombocytopenia when exposed to heparin. Affinity-purified anti-PF4-heparin antibodies bound to murine endothelioma cells in the presence of PF4, but not b2GPI. Of interest, immunized mice did not develop overt thrombi on exposure to heparin, possibly because of insufficient circulating PF4, intrinsic differences in the balance between the procoagulant and fibrinolytic systems compared with humans, or differences in signal transduction through murine and human platelet Fcg receptors. However, it is also possible that the difference lies in a reduced capacity of otherwise healthy mouse ECs to respond to the procoagulant stimulus induced by these antibodies, compared with the responsiveness of patients with HIT, who, in the main, comprise a more elderly population with underlying vascular disorders.

Recent studies suggest that requirements for EC activation by HIT antibodies may differ based on the vascular origin. Blank et al. (2002) reported that anti-PF4-heparin IgG could activate certain microvascular ECs directly. However, antibody-mediated platelet activation (Herbert et al., 1998) or stimulation with cytokines such as tumor necrosis factor-alpha (TNF-a) (Blank et al., 2002) may be necessary to activate macrovascular ECs. In the presence of platelets or TNF-a, sera or IgG from patients with HIT stimulated expression of E-selectin, VCAM, intercellular adhesion molecule-1 (ICAM-1), and tissue factor, release of IL-01, IL-6, TNF-a, and PAI-1, and adhesion of platelets and monocytes to the activated ECs. EC activation was inhibited by SR121566a (a platelet glycoprotein IIb/IIIa antagonist) and to some extent by apyrase and ATPgS, implicating expression of endogenous platelet fibrin(ogen) and release of adenosine diphosphate (ADP). The mechanism(s) by which platelet or cytokine activation facilitate binding of HIT antibodies to cell-associated PF4-heparin requires further investigation, but may be related to released stores of platelet PF4.

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