A Unique Biomarker Is Associated with the Prevalence of Functional Heparin Induced Thrombocytopenia (HIT) Associated Antibodies: Results from Proteomic (ProteinChip Array) Profiling of ELISA Positive HIT Plasma Samples.

Abstract

Heparin induced thrombocytopenia (HIT) represents a complex immunopathologic syndrome resulting in the activation of coagulation, inflammatory and cellular processes, which contribute to the overall pathogenesis. Protease mediated transformation of endogenous proteins resulting in the generation of certain mediators and specific cleavage products may be used to understand the pathogenesis of this syndrome. It was hypothesized that unique biomarkers may be generated in patients who develop HIT antibodies upon exposure to heparins. The identification of unique biomarkers and their prevalence in heparin exposed patients may provide an additional parameter in the prognosis of this syndrome. To profile the proteomic biomarkers in serum, specimens were selected from archived sera that had been referred to Loyola University Medical Center for the quantitation of HIT antibodies and 14C Serotonin Release Assay (SRA). All of these specimens were positive in the GTI PF4 Enhanced ELISA for the presence of anti-heparin platelet factor 4 antibodies with a broad range of antibody titers absorbance range (0.4 – 2.5). Eleven of these specimens were positive in the SRA. All of these samples were proteomic profiled utilizing a ProteinChip Array method using a Gold (AU) Chip in the molecular weight range of 0 – 150 kDa. The normal sera samples (n = 40) were also analyzed simultaneously in a crossover fashion along with the patients' specimens. Surface enhanced laser desorption/ionization (SELDI) technique utilizing Ciphergen PBS II (Fremont, CA) was used for mass profiling. Proteomic profiling is widely used to identify unique biomarkers in various hemotologic disorders. Although electrophoretic methods identify biomarkers at molecular weights ≥ 25 kDa, the SELDI technique provides a complete spectrum in the lower molecular weight range. The ProteinChip Software for mass profiling was used to analyze the data. Proteins such as albumin were used as control proteins throughout the study. Of the 54 HIT samples profiled, 36 showed a unique biomarker peak at 11.9 kDa (67%) whereas none of the normal sera (n = 40) showed this peak. All of the SRA positive samples also showed the 11.9 kDa biomarker peak. Several biomarker peaks in the range of 5 – 10 kDa were present in the HIT positive sera suggesting increased proteolysis. While no other unique biomarkers were obvious in the HIT samples, the relative proportion of some of the biomarker peaks in the range of 15 – 20 kDa were differentiable in the HIT group. These studies demonstrate that HIT patients' sera exhibit a unique biomarker profile, which is distinguishable from normal serum samples. Combined usage of the ProteinChip Array analysis of the HIT patients' sera with other diagnostic methods may further enhance the diagnostic process for this syndrome. Additional molecular characterization of these biomarkers will also help in the understanding of the pathogenesis of HIT with particular reference to the protease activation processes.