An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14--the LPS receptor on the macrophage surface--or to high-density lipoprotein (HDL) particles. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from the circulation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.

A clinically important adverse drug reaction, heparin-induced thrombocytopenia (HIT), is induced by antibodies specific for complexes of the chemokine platelet factor 4 (PF4) and the polyanion heparin. Even heparin-naive patients can generate anti-PF4/heparin IgG as early as day 4 of heparin treatment, suggesting preimmunization by antigens mimicking PF4/heparin complexes. These antibodies probably result from bacterial infections, as (1) PF4 bound charge-dependently to various bacteria, (2) human heparin-induced anti-PF4/heparin antibodies cross-reacted with PF4-coated Staphylococcus aureus and Escherichia coli, and (3) mice developed anti-PF4/heparin antibodies during polymicrobial sepsis without heparin application. Thus, after binding to bacteria, the endogenous protein PF4 induces antibodies with specificity for PF4/polyanion complexes. These can target a large variety of PF4-coated bacteria and enhance bacterial phagocytosis in vitro. The same antigenic epitopes are expressed when pharmacologic heparin binds to platelets augmenting formation of PF4 complexes. Boosting of preformed B cells by PF4/ heparin complexes could explain the early occurrence of IgG antibodies in HIT. We also found a continuous, rather than dichotomous, distribution of anti-PF4/heparin IgM and IgG serum concentrations in a cross-sectional population study (n ‫؍‬ 4029), indicating frequent preimmunization to modified PF4. PF4 may have a role in bacterial defense, and HIT is probably a misdirected antibacterial host defense mechanism. (Blood. 2011;117(4): 1370-1378) IntroductionThe chemokine platelet factor 4 (PF4, CXCL4) is stored within platelet ␣-granules 1 and released during platelet activation. Although its biologic role is poorly understood, PF4 commands attention in clinical medicine because it binds charge-dependently to the anticoagulant heparin, one of the most frequently used anticoagulants in clinical medicine, thereby neutralizing heparin's anticoagulant effect, 2,3 but also forming highly antigenic multimolecular complexes. [4][5][6] The resulting antibody response 7 induces the most frequent immune-mediated adverse drug reaction involving human blood cells, heparin-induced thrombocytopenia (HIT). 8 The pathogenic antibodies bind to PF4/heparin complexes at the platelet surface, and the resulting immune complexes induce Fcreceptor-mediated platelet activation and enhanced thrombin generation. 8 In a subset of patients, this causes thrombocytopenia and triggers paradoxical thrombosis, which is aggravated by continuation of heparin treatment.The immune response of HIT has several atypical features. Even in patients who receive heparin for the first time, there is rapid induction (as early as 4 days) of anti-PF4/heparin antibodies of IgG isotype. Moreover, IgG antibodies are the predominant class of immunoglobulins formed. 9,10 We and others discovered that up to 50% of patients after cardiopulmonary bypass surgery and 20% to 30% of orthopedic surgery patients (many of whom have not been previously exposed to heparin) devel...

Transfusion-related acute lung injury (TRALI) is a frequent cause of transfusion-associated morbidity and mortality. Severe TRALI is often due to antibodies in blood components directed against the human neutrophil alloantigen-3a (HNA-3a). We show here that the HNA-3a antigen arises from a nucleotide polymorphism in the choline transporter-like protein-2 gene (SLC44A2), with the resulting variation at amino acid position 154 determining the reactivity of the protein with HNA-3a-specific antibodies; the variant with an arginine at this position, rather than a glutamine, constitutes the HNA-3a antigen. The molecular identification of this antigen should facilitate the development of assays for blood donor screening to lower the risk of TRALI.

Key Points• PF4 binds to nucleic acids and thereby exposes the epitope to which anti-PF4/ heparin antibodies bind.• PF4/aptamer complexes can induce an immune response resembling heparin-induced thrombocytopenia.The tight electrostatic binding of the chemokine platelet factor 4 (PF4) to polyanions induces heparin-induced thrombocytopenia, a prothrombotic adverse drug reaction caused by immunoglobulin G directed against PF4/polyanion complexes. This study demonstrates that nucleic acids, including aptamers, also bind to PF4 and enhance PF4 binding to platelets. Systematic assessment of RNA and DNA constructs, as well as 4 aptamers of different lengths and secondary structures, revealed that increasing length and double-stranded segments of nucleic acids augment complex formation with PF4, while single nucleotides or single-stranded polyA or polyC constructs do not. Aptamers were shown by circular dichroism spectroscopy to induce structural changes in PF4 that resemble those induced by heparin. Moreover, heparin-induced anti-human-PF4/ heparin antibodies cross-reacted with human PF4/nucleic acid and PF4/aptamer complexes, as shown by an enzyme immunoassay and a functional platelet activation assay. Finally, administration of PF4/44mer-DNA protein C aptamer complexes in mice induced anti-PF4/aptamer antibodies, which cross-reacted with murine PF4/heparin complexes. These data indicate that the formation of anti-PF4/heparin antibodies in postoperative patients may be augmented by PF4/nucleic acid complexes. Moreover, administration of therapeutic aptamers has the potential to induce anti-PF4/polyanion antibodies and a prothrombotic diathesis. (Blood. 2013;122(2):272-281) IntroductionThe chemokine platelet factor 4 (PF4) is released from platelet a-granules during platelet activation 1 and binds, due to its high positive charge, to many negatively charged polyanions, including heparin. PF4 forms large multimolecular complexes with heparin that are highly immunogenic. 2,3 The resulting immunoglobulin G (IgG) antibodies are the cause of heparin-induced thrombocytopenia (HIT), a prothrombotic adverse drug effect. 4 In HIT, multimolecular complexes composed of PF4, heparin, and anti-PF4/heparin IgG cross-link platelet FcgIIa receptors, 5 triggering platelet activation, microparticle formation, and thrombin generation, with ;50% of affected patients developing thrombosis. 6 Recently, we showed that PF4 binds to polyanions on the surface of bacteria, thereby forming multimolecular complexes that are recognized by human anti-PF4/heparin antibodies. More specifically, we identified the PF4 binding site on gram-negative bacteria as the phosphate groups of lipid A. 7 Based on this observation, we hypothesized that nucleic acids might also form multimolecular complexes with PF4 because they also expose multiple negatively charged phosphate groups. This idea was fostered by previous findings that salmon sperm DNA could substitute heparin in HIT-IgG-induced platelet activation. 8Plasma levels of extracellular nucleic acids are re...

Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations.We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxa