Activation of neutrophils by antibodies generated in heparin-induced thrombocytopenia

Abstract number: P1164

Jeske^* W., Sinno† S., Thoreson† A., Prechel† M. M., Walenga† J. M.

^*Loyola University Medical Center, USA; †USA

The pathogenesis of thrombosis associated with heparin-induced thrombocytopenia (HIT), a potentially devastating complication of heparin (H) therapy, is unclear as a large proportion of H treated patients develop antibodies against the H-platelet factor 4 complex without developing clinical symptoms consistent with HIT. This study examines the interaction of platelets with leukocytes and the activation of neutrophils induced by sera from individuals with HIT using flow cytometric assays. These sera had previously been shown to activate platelets as measured by the expression of P-selectin, the generation of platelet microparticles and using the serotonin release assay. Platelet-leukocyte complexes were identified as the percentage of leukocytes that colabeled with a platelet specific marker. An increase in platelet binding to monocytes and neutrophils was observed upon incubation of normal whole blood with HIT serum and H compared to when incubations were carried out with HIT serum in the absence of H or with normal human serum (NHS). Platelet-monocyte complexes increased from 22.3 ± 16.8% in the presence of NHS and H to 53.1 ± 12.6% in the presence of HIT serum and H (P < 0.05). Platelet-neutrophil complexes were also increased in the presence of HIT serum and H (16.9 ± 3.4%) compared to NHS and H (6.4 ± 1.1%; P < 0.05). Supratherapeutic concentrations of H eliminated these responses. Neutrophil activation, measured as an increase in CD11b expression [MFI; 403 ± 97 with NHS and 718 ± 131 with HIT serum (P < 0.05)] and a decrease in l-selectin expression [MFI; 657 ± 86 with NHS and 531 ± 67 with HIT serum], was observed. Neutrophils were labeled with dihydrorhodamine (DHR) in order to assess the effect of HIT antibodies on neutrophil oxidative activity. An increase in DHR fluorescence was observed in the presence of HIT sera compared to NHS (MFI; 2621 ± 567 vs. 22.6 ± 1.1; P < 0.05). This effect was significantly, though not completely, inhibited by the anti-P-selectin antibody AK-4 (MFI; 578 ± 206; P < 0.05). Interestingly, platelet activation measured as the generation of platelet microparticles was also inhibited in the presence of AK-4 (23.5 ± 3.0% vs. 11.0 ± 2.1%; P < 0.05). These results suggest an importance for inflammatory activation, in combination with platelet activation, in mediating the pathology associated with HIT. If such effects can be demonstrated in patients with HIT, new avenues for the diagnosis, treatment or prevention of HIT may be opened.