Investigations of the immunoglobulin subtype transformation of anti-heparin-platelet factor 4 antibodies during treatment with a low-molecular-weight heparin (clivarin) in orthopedic patients

Abstract

Context: It is now widely accepted that the pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated by the generation of a wide array of functional and molecularly heterogeneous anti-heparin-platelet factor 4 (AHPF4) antibodies that may mediate platelet and/or endothelial cell activation/destruction.

Objective: We investigated the differential prevalence and functionality of AHPF4 immunoglobulin subtypes (IgA, IgG, and IgM) in plasmas obtained from orthopedic patients immobilized with Plaster-Cast and treated with clivarin (a low-molecular-weight heparin) in comparison to a placebo for the prophylaxis of deep-vein thrombosis.

Design and methods: Clivarin was administered subcutaneously at a fixed daily dosage of 1750 U without any adjustment or loading dosage. Citrated plasmas were obtained at baseline, at 10 to 14 days, and at postbrace procedure (5-12 weeks). An enzyme-linked immunosorbent assay (ELISA) was used to quantitate the AHPF4 antibody titers. The functionality of the ELISA-positive samples was determined by a 14C-serotonin release assay (SRA).

Results: In the ELISA test, 16 of 1073 samples (1.5%; 6 in clivarin and 10 in placebo groups) were positive for AHPF4 antibodies (mean optical density [OD] = 0.46 +/- 0.02). None of the ELISA-positive samples for AHPF4 antibodies could mediate platelet activation responses as determined by the SRA (0%-3% serotonin release, P >.10, n = 16). Through differential immunoglobulin subtype analysis of the samples positive for (cumulative) AHPF4 antibodies, we determined that their relative prevalence in plasma were as follows: IgM (mean OD = 0.71 +/- 0.13) > IgG (0.31 +/- 0.08) > IgA (0.14 +/- 0.02). Although there was no significant difference in the total antibody titers between clivarin and placebo groups, the antibody subtyping data showed conversion trends (ie, IgA [clivarin to placebo], IgG [placebo to clivarin], and IgM [clivarin to placebo]).

Conclusion: These observations indicate that even at reduced dosages, clivarin can shift the immunogenic up-regulation toward the IgG subpopulation; however, the IgG subtype is of a nonfunctional type of AHPF4 antibody and thus may not cause any HIT-related pathogenic responses.