Platelet activation induced by human antibodies to interleukin-8

Abstract

Some cases of heparin-induced thrombocytopenia (HIT) have been reported to be associated with antibodies against interleukin-8 (IL-8), a chemokine related to platelet factor 4. We found that sera from 5 HIT patients containing immunoglobulin G (IgG) or IgM antibodies to IL-8, as evidenced using surface plasmon resonance spectroscopy, were able to trigger IL-8-dependent activation of washed platelets, leading to procoagulant activity. This activation occurred at IL-8 concentrations achievable in vivo and was facilitated by heparin (0.1 U/mL). Activation was also induced by affinity-purified anti-IL-8 IgG and involved Fc␥RIIa. In the 2 patients who could be followed up, antibodies were no longer detectable 4 months after heparin withdrawal. One additional patient with paraneoplastic recurrent thrombosis without thrombocytopenia was found to have platelet-activating anti-IL-8 IgM, but in this case heparin was inhibitory. This is another example of potentially pathogenic platelet activation by antibodies. IntroductionIn most patients with heparin-induced thrombocytopenia (HIT), antibodies bind to complexes of heparin and platelet factor 4 (PF4). If these antibodies are immunoglobulin G (IgG), they interact with Fc␥RIIa receptors and trigger platelet activation. 1,2 However, previous investigators using enzyme-linked immunosorbent assay (ELISA) have reported that a few HIT patients have antibodies specific to another, PF4-related ␣-chemokine-interleukin-8 (IL-8), alone 3,4 or in combination with antibodies to heparin-PF4 complexes. 5 These antibodies were infrequently detected in patients with heparin exposure but no thrombocytopenia and in patients with thrombocytopenia from another etiology. 3 The main known property of IL-8 is to induce neutrophil activation, and anti-IL-8 autoantibodies have been shown to inhibit IL-8 interaction with its specific receptors on neutrophils. 6 IL-8 also binds to heparin, 7 and there may be IL-8 receptors on platelets. [8][9][10] This study was undertaken to investigate whether antibodies to IL-8 are able to induce platelet activation in vitro and whether they are dependent on heparin. Study designSera were obtained from 5 selected patients (P1-P5) who fulfilled the diagnostic criteria for HIT 1,11 (suggestive temporal pattern of platelet counts in relation to heparin therapy and evidence for heparin-dependent platelet-activating antibodies) but without IgG to heparin-PF4 complexes. 3 Patient 6 had typical HIT with IgG to heparin-PF4 only. One additional patient (P7), who had experienced fatal recurrent paraneoplastic thromboses while on heparin therapy without thrombocytopenia, 12 was also studied. Small volumes of blood were collected from samples required for routine patient management and were withdrawn after heparin therapy was stopped; heparin was no longer detectable with anti-Xa assay. ELISA (Asserachrom HPIA; Diagnostica Stago, Asnières, France) was used to detect antibodies to heparin-PF4. 13 IgG was purified by affinity chromatography on protein G-Sepharose (Pharmacia Bi...

Platelet factor 4 binding to lipid A of Gram-negative bacteria exposes PF4/heparin-like epitopes

“…We identified positively charged amino acids in the heparin binding site of PF4 (or the related chemokine IL-8, which also binds heparin 43 and has been reported to induce the production of antibodies in HIT patients 44,45 ), to be highly conserved in all vertebrates, including fish species. The interaction of PF4 and lipid A is inhibited by heparin (supplemental Figures 1 and 2, available on the Blood Web site; see the Supplemental Materials link at the top of the online article), suggesting that the amino acids known to contribute to heparin binding are also involved in binding to lipid A.…”

Heparin-Induced Thrombocytopenia: Frequency and Pathogenesis

“…These patients had no increased risk for new thrombosis as compared to HIT antibody negative patients. [76] Although the first report that anti-IL8 and anti-NAP-2 antibodies can induce HIT [77] was later confirmed [78], little additional evidence has been reported which allows to estimate the prevalence of these antibodies in HIT patients.…”

Experts’ opinion or the serotonin release assay as a gold standard for the diagnosis of heparin‐induced thrombocytopenia (HIT)?

“…We read with interest the paper by Roeters van Lennep et al [1] and the response from Patel et al [2]. The Dutch group has assessed a Ôlow doseÕ low-molecular-weight heparin (LMWH) regimen, finding it to be insufficient to prevent recurrent venous thromboembolic events in women at high risk of recurrence.…”

“…Immunoassays (IAs) detect the presence of antibodies directed towards PF4-heparin complexes, whereas functional assays aim at detecting heparin-dependent platelet activation induced by these antibodies. While IAs are sensitive for detecting anti-PF4-heparin antibodies, although they do not detect antibodies directed against other chemokines than PF4 [2], their major drawback is a poor specificity as many patients exposed to heparin develop anti-PF4-heparin antibodies detectable by IA in the absence of HIT [3]. Platelet activation assays such as the platelet serotonin release assay (SRA) detect heparin-dependent antibodies that can bind to and activate platelets via their Fc gamma receptors.…”