单克隆抗体在肝素诱导的血小板减少症发病机制中的作用

Treverton Jared, Arnold Donald M, Ivanov Daniil G, Ivetic Nikola, Zhang Yi, Ali Hossam A, Clare Rumi, Kaltashov Igor A, Kelton John G, Nazy Ishac

Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.

Michael G. DeGroote Centre for Transfusion Research, McMaster University, Hamilton, ON, Canada.

N Engl J Med. 2025 Sep 4;393(9):879-886. doi: 10.1056/NEJMoa2507175.

BACKGROUND

Heparin-induced thrombocytopenia (HIT) is an immune-mediated platelet disorder caused by antibodies that target complexes of platelet factor 4 (PF4) and heparin. HIT has been characterized as a polyclonal immune response; however, studies of other rare anti-PF4 disorders have identified clonally restricted antibodies.

METHODS

In this study, we investigated the clonality of pathogenic HIT antibodies. Antibodies against PF4-heparin were affinity-purified with the use of PF4-heparin beads from serum samples obtained from nine patients with clinically and serologically confirmed HIT. Antibody clonality was assessed by means of immunofixation electrophoresis and mass spectrometry. Antibody binding to PF4 was evaluated by an enzyme immunoassay, and functional platelet activation was evaluated with the use of a P-selectin expression assay. HIT antibody epitopes were mapped in two patients with the use of a PF4 mutant library.

RESULTS

Serum samples from all nine patients with HIT were positive for platelet-activating antibodies against PF4-heparin by enzyme immunoassay, as well as the P-selectin expression assay, and six samples (67%) had a monoclonal antibody detectable by immunofixation electrophoresis. The affinity-purified antibodies against PF4-heparin from all nine samples activated platelets in the P-selectin expression assay, and mass spectrometry showed monoclonality. After affinity purification, antibody-depleted serum samples lost binding activity in the enzyme immunoassay and functional activity in the P-selectin expression assay, which confirmed the removal of the pathogenic antibodies. The epitopes on PF4 targeted by anti-PF4-heparin antibodies from serum samples were the same as those targeted by the affinity-purified monoclonal antibodies.

CONCLUSIONS

The pathogenic antibodies in all nine patients with HIT were found to be monoclonal. This finding provides insight into the pathogenesis of HIT and has implications for improved diagnostics and targeted therapeutics. (Funded by the Canadian Institutes of Health Research and the National Institutes of Health.).

背景

肝素诱导的血小板减少症（HIT）是一种由针对血小板因子4（PF4）与肝素复合物的抗体引起的免疫介导的血小板疾病。HIT被认为是一种多克隆免疫反应；然而，对其他罕见的抗PF4疾病的研究已经鉴定出克隆受限抗体。

方法

在本研究中，我们调查了致病性HIT抗体的克隆性。使用PF4-肝素磁珠从9例临床和血清学确诊为HIT的患者血清样本中亲和纯化抗PF4-肝素抗体。通过免疫固定电泳和质谱评估抗体克隆性。通过酶免疫测定评估抗体与PF4的结合，并使用P-选择素表达测定评估功能性血小板活化。使用PF4突变体文库在2例患者中绘制HIT抗体表位。

结果

通过酶免疫测定以及P-选择素表达测定，所有9例HIT患者的血清样本中针对PF4-肝素的血小板活化抗体均为阳性，6份样本（67%）通过免疫固定电泳可检测到单克隆抗体。在P-选择素表达测定中，从所有9份样本中亲和纯化的抗PF4-肝素抗体均能激活血小板，质谱显示为单克隆性。亲和纯化后，抗体去除的血清样本在酶免疫测定中失去结合活性，在P-选择素表达测定中失去功能活性，这证实了致病性抗体已被去除。血清样本中抗PF4-肝素抗体靶向的PF4上的表位与亲和纯化的单克隆抗体靶向的表位相同。