Mechanisms of Platelet Activation by Antithymocyte Globulin (ATG).

We have previously shown that polyclonal rabbit ATG from Fresenius (ATG-F) can induce non-overt DIC in patients undergoing hematopoietic stem cell transplantation (HSCT) [Weber et al. 2003]. The compensated state of the coagulopathy suggested that the severe drop in platelet counts (>60%) observed in patients with normal pretreatment levels was not solely due to thrombin generation and platelet consumption. We thus further investigated the mechanisms of ATG-F-induced thrombocytopenia, hypothesizing that ATG-F had direct platelet-stimulating activity. Using an indirect flow cytometric binding assay we found that, compared to control IgG, ATG-F dose-dependently (1–100 μg/ml) bound to the surface of resting and TRAP-activated platelets, showing an up to 50fold increase in MFI at 50–100 μg/ml. In a washed platelet system, in which baseline positivity for CD62P was generally <15%, ATG-F alone had no effect on platelet aggregation. However, when platelets were primed with low concentrations of ADP (1–2 μM) or epinephrine (0.5 μM), ATG-F induced strong and stable aggregation of up to 80–100% in a dose-dependent manner (25–100 μg/ml), whereas control IgG did not. The priming effect of ADP was dependent on both P₂Y₁ and P₂Y₁₂ as evidenced by respective ADP receptor antagonists. Preincubation of platelets with inhibitory CD32 mAb completely abolished ATG-F-induced platelet aggregation, suggesting that clustering of, and signalling through, FcγRIIA was crucial for this platelet response. Similarly, ATG-F-derived F(ab′)₂ had no effect on ADP-triggered platelet aggregation. Furthermore, preliminary studies indicated that responsiveness of platelet donors to ATG-F was associated with the FcγRIIA-R/R131 polymorphism, which has previously been shown to confer increased in vitro platelet responsiveness to heparin/PF4 antibodies from patients with HIT. In a ¹⁴C-serotonin release assay, in which prolonged incubation at 37⁰C under shaking conditions increases baseline platelet activation and ensures constant platelet-platelet contacts, ATG-F (100 μg/ml), but not control IgG, induced strong FcγRIIA-dependent dense-granule release of up to 80%. In contrast, using diluted platelet-rich plasma under static conditions, ATG-F had only minor effects on platelet activation, increasing CD62P+ platelets from 2±1 to 15±5% (n=5). In a cohort of 16 consecutive HSCT patients receiving ATG-F, two patients were identified with normal pretreatment platelet counts and soluble CD62P (sCD62P) plasma levels of >51 ng/ml (mean+2SD of 23 controls). In these patients, an ATG-F-induced drop in platelet count to 38 and 19% of baseline was associated with a 1.7 and 2.2fold increase in sCD62P, respectively, indicating further platelet activation. In summary, strong binding of ATG-F to resting platelets may accelerate their clearance by the reticulo-endothelial system, thus contributing to the pathophysiology of thrombocytopenia. While ATG-F alone had negligible effects on resting platelets, it significantly enhanced activation of prestimulated platelets. Patients with normal-to-high platelet counts and evidence of in vivo platelet activation may especially be prone to this potentially hazardous side effect.