Functional Microparticles Are up Regulated in Patients with Anti-Heparin/Platelet Factor 4 Antibodies: A Potential Mechanism of Thrombogenesis in the Heparin-Induced Thrombocytopenia Syndrome.

Heparin-induced thrombocytopenia (HIT) is one of the most catastrophic adverse effects of heparin therapy, representing a complex syndrome involving immunopathologic and hemostatic disorders. Vascular and blood cellular damage results in the generation of microparticles (MP). These MP are formed from stress conditions/cellular disruption and apoptosis. Cellular MP mediated pathophysiologic responses include platelet activation, up regulation of adhesion molecules, monocyte activation, up regulation of tissue factor and endothelial dysfunction. Several methods based on flow cytometric and other immunologic probes have been used to measure MP in the HIT syndrome. Recently, a functional method based on the complexation of MP with annexin V promoting the generation of factor Xa and thrombin has become available (Hyphen Biomedical, Neuville-Oise, France). To validate the hypothesis that functional MP are elevated in the HIT syndrome, this method was utilized for the quantitation of MP in sera ELISA positive for anti-heparin/platelet factor 4 (HIT) antibodies. Specimens (n = 53) were selected from archived samples that had been referred to Loyola University Medical Center for the laboratory diagnosis of HIT by quantitating anti-heparin/PF4 antibodies by ELISA and by evaluating HIT antibody induced platelet activation using the ¹⁴C Serotonin Release Assay (SRA). All selected specimens were positive for HIT antibodies in the GTI PF4 Enhanced ELISA with a broad range of antibody titers (absorbance range of 0.4 – 2.5). Eleven of these specimens were positive in the SRA. In addition, serial samples from HIT patients treated with argatroban (from the ARG-911 clinical study) were included (n = 23). The normal samples represented control sera obtained from healthy human volunteers (n = 25) and processed in the same manner as the clinical samples. Test samples were added to microtiter plates coated with streptavidin and biotinylated annexin V. MP present in the test sample bound to annexin V via exposed surface phospholipids. Following incubation and washing steps, a FXa – FVa mixture containing calcium and prothrombin was added. The assay was optimized so that MP associated phospholipid was the limiting factor for the generation of thrombin. In normal non-HIT sera, the MP levels ranged 5.6 – 10.1 nM (6.1 ± 2.8 nM). The pre-treatment, baseline levels of circulating MP in the suspected HIT patients ranged from 4.2 – 26.8 nM (15.8 ± 7.3 nM). Interestingly, SRA positive/ELISA positive samples had relatively higher levels of MP (19.9 ± 7.7 nM; range 11.5 – 29.8 nM) than SRA negative/ELISA positive samples (14.2± 4.6; range 6.8–21.2). In the ARG-911 study, sequential blood samples exhibited MP levels at the baseline ranging from 8.2 – 38.6 nM (21.8 ± 10.8 nM), whereas after 3 days of argatroban treatment were reduced to 5.1 – 19.2 nM (12.6 ± 6.3). The results of these studies suggest that circulating functional MP are increased in patients with ELISA positive HIT antibodies. Anticoagulation with such direct thrombin agents as argatroban effectively decreases the circulating functional MP levels. Since the elevated MP levels may mediate thrombin and FXa generation, the therapeutic effects of these drugs in HIT may be related to the decreased activation of coagulation and related thrombogenic processes.