Three out of eight human HLA-specific monoclonal antibodies induced activation of HLA-matched platelets from healthy donors as evidenced by enhanced -granule release, aggregation, and IIbb3 activation. as F(ab)2 fragments of HLA monoclonal antibodies were unable to induce platelet activation. Mixing experiments revealed that Mouse monoclonal to KSHV ORF26 activation of platelets occurred in an intra-platelet dependent manner. Accordingly, a proportion of sera from refractory patients with HLA antibodies induced FcRIIa-dependent platelet activation. Our data show that a subset of HLA antibodies is usually capable of crosslinking HLA and FcRIIa thereby promoting platelet activation and enhancing these cells phagocytosis by macrophages. Based on these findings we suggest that FcRIIa-dependent platelet activation may contribute to the decreased platelet survival in platelet-transfusion-dependent patients with HLA antibodies. Introduction Antibodies against human leukocyte antigen (HLA) can be induced by pregnancy, blood transfusion or transplantation.1C3 During or after a pregnancy, 15C50% (depending on the quantity of pregnancies) of women develop HLA antibodies.4C6 Platelet reactive alloantibodies commonly directed toward HLA of the donor platelets develop in 20C30% of chronic platelet transfusion recipients.2,3,7 Although platelet refractoriness is more commonly caused by non-immune factors2,7,8, 30C50% of platelet-transfusion-dependent recipients with HLA antibodies become refractory to platelet transfusions due to alloimmunization.2,7 HLA antibodies in this setting are primarily composed of immunoglobulin G (IgG) and are directed toward HLA- A and B.3 Binding of antibodies to HLA class I on donor platelets results in the formation of IgG-opsonized platelets which are rapidly cleared from your circulation. Several parameters may contribute to the efficacy of HLA antibody-induced platelet clearance. Firstly, HLA density on platelets may differ between individuals. A recent study showed that platelets from VU0364289 donors with consistently low HLA-B8, B12 or B35 displayed a strongly reduced antibody-mediated internalization by macrophages.9 Furthermore, low levels of HLA antibodies were not associated with platelet refractoriness VU0364289 in the TRAP (Trial to Reduce Alloimmunization to Platelets) study.10 Results from the same study revealed that high levels of anti-HLA antibodies were clearly related to refractoriness to platelet transfusion.10 Transfusions with HLA-compatible platelets have been shown to be effective in patients with preexisting HLA antibodies.11 In an early clinical trial no beneficial effect of treatment with HLA-matched platelet concentrates was observed.12 These findings indicate that platelet refractoriness is of non-immune origin in a significant number of patients and, collectively, suggest that HLA antibodies, dependent on their titer and the HLA density on donor platelets, can induce platelet refractoriness. Whether additional mechanisms contribute to the observed clinical effects of anti-HLA antibodies has not yet been clearly delineated. Apart from VU0364289 the transfusion setting, a pathogenic role for platelet-specific antibodies has been described in several diseases. Patients with immune thrombocytopenia often have autoantibodies against glycoprotein (GP)Ib/IX or GPIIbIIIa, frequently coinciding with refractoriness.13C15 Anti-GPIb has been associated with Fc receptor IIa (FcRIIa)-independent platelet activation, through loss of sialic acid and subsequent clearance via VU0364289 the Ashwell Morell receptor localized on hepatocytes.16 Alternatively, in heparin-induced thrombocytopenia, antibodies directed to platelet factor 4/heparin complex induce platelet clearance and FcRIIa-dependent platelet activation.17,18 Previous studies have explained on FcRIIa-dependent activation of platelets by (non-physiological) crosslinking of the murine pan-HLA class VU0364289 I antibody W6/32.19 Complement-dependent platelet aggregation induced by HLA antibodies has also been reported.20 Based on these findings we hypothesized that a subset of human HLA antibodies may be able to activate platelets. To address this issue we tested a panel of well-characterized human monoclonal HLA antibodies and HLA antibody-containing sera from platelet-transfusion refractory patients for their ability to activate platelets. Methods HLA monoclonal antibodies and patients sera Human HLA-specific monoclonal antibodies,.