Heparin-induced thrombocytopenia (HIT)is an antibody-mediated disorder that occurs with variable frequency in patients exposed to heparin. HIT antibodies preferentially recognize large macromolecular complexes formed between PF4 and heparin over a narrow range of molar ratios, but the biophysical properties of complexes that initiate antibody production are unknown. To identify structural determinants underlying PF4/heparin immunogenicity, we characterized the in vitro interactions of murine PF4 (mPF4) and heparin with respect to light absorption, size, and surface charge (zeta potential). We show that PF4/heparin macromolecular assembly occurs through colloidal interactions, wherein heparin facilitates the growth of complexes through charge neutralization. The size of PF4/heparin macromolecules is governed by the molar ratios of the reactants. Maximal complex size occurs at molar ratios of PF4/ heparin at which surface charge is neutral. When mice are immunized with complexes that differ in size and/or zeta potential, antibody formation varies inversely with heparin concentration and is most robust in animals immunized with complexes displaying a net positive zetapotential. These studies suggest that the clinical heterogeneity in the HIT immune response may be due in part to requirements for specific biophysical parameters of the PF4/heparin complexes that occur in settings of intense platelet activation and PF4 release. IntroductionHeparin-induced thrombocytopenia (HIT) is a drug-dependent immune disorder caused by antibodies to complexes between platelet factor 4 (PF4) and heparin. PF4/heparin antibodies are detected in virtually all patients with HIT, but can also occur in the absence of disease in patients treated with unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), or the synthetic pentasaccharide, fondaparinux, and other heparin-like molecules. [1][2][3] The prevalence of PF4/heparin antibodies varies widely among patient populations and appears to be significantly influenced by the clinical context of drug exposure and heparin formulation. Antibodies are detected most commonly (27%-61%) in patients undergoing cardiopulmonary bypass (CPB) surgery, 4-9 but occur far less frequently in uremic, obstetric, and pediatric patients even after prolonged heparin exposure. [10][11][12] General medical and surgical patients are reported to have intermediate rates of seroconversion, ranging from 8% to 21%. 1 Why only a subset of patients with anti-PF4/heparin antibodies develops HIT is not known. One likely determinant is antigen concentration. Patients undergoing CPB have high circulating levels of both PF4 (560-750 ng/mL 13,14 ) and heparin (3-4 Units/ mL 15 ). Another determinant may be the physical composition of the antigen. We recently reported that when PF4 and UFH associate over a narrow range of molar ratios approximating 1:1, they form ultralarge macromolecular complexes (ULCs, Ͼ 670 kDa). 16 Assembly of macromolecular complexes is influenced profoundly by small changes in the stoichiome...

Agonist-induced phosphorylation of ␤-adrenergic receptors (␤ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization؊ /GRK ؊ ␤ 1 AR was independent of ␤-arrestin recruitment. Importantly, clathrin inhibitors abolished agonistdependent internalization for both the WT␤ 1 AR and PKA ؊ ␤ 1 AR, whereas caveolae inhibitors prevented internalization only of the GRK ؊ ␤ 1 AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the ␤ 1 AR and 2) the pathway selected for ␤ 1 AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits. ␤-Adrenergic receptors (␤ARs)1 belong to the large family of G protein-coupled receptors (GPCRs) characterized by a typical structure of seven transmembrane domains (1, 2). Three types of ␤ARs, designated ␤ 1 , ␤ 2 , and ␤ 3 ARs, have been cloned from mammalian tissues (1, 3). Both ␤ 1 and ␤ 2 ARs contain phosphorylation sites located in the third intracellular loop and the C-terminal tail of the receptor, which serve as targets for cAMPdependent protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs) (2). Furthermore, site-specific mutagenesis studies of the human ␤ 2 AR suggest that low concentrations of agonist preferentially induce phosphorylation at PKA sites, whereas higher concentrations of agonist induce phosphorylation at both PKA and GRK sites (4).Continuous exposure of cells to a stimulus causes ␤ARs to undergo rapid phosphorylation in a process that dampens receptor signaling known as desensitization (4 -8). ␤ARs demonstrate two different mechanisms of desensitization. Agonistspecific or homologous desensitization of ␤ARs consists of a two-step process in which phosphorylation at the C terminus of the ␤AR is mediated by GRKs followed by binding to an arrestin protein, which sterically interrupts signaling to the G protein (5,8). Heterologous or non-agonist-specific desensitization is mediated by the second messenger-stimulated protein kinases A and C, which phosphorylate the receptor and effect a change in receptor conformation such that interaction with the G protein is impaired (5). An important consequence of agonistmediated receptor phosphorylation and desensitization by GRKs is the subsequent internalization of phosphorylated receptors into the cell (9). This process is mediated by ␤-arrestin, which binds to components of the clathrin-mediated endocytic machinery and targets the ligand-bound receptor to clathrincoated pits for endocytosis (10, 11). Interestingly, PKA phosphorylation, although an important mechanism for desensitization (4 -8), appears to play only a small role in ␤ 2 AR

Platelet factor 4 (PF4)/heparin antibody, typically associated with heparin therapy, is reported in some heparin-naive people. Seroprevalence in the general population, however, remains unclear. We prospectively evaluated PF4/heparin antibody in approximately 4,000 blood bank donors using a commercial enzyme-linked immunosorbent assay for initial and then repeated (confirmatory) testing. Antibody was detected initially in 249 (6.6%; 95% confidence interval [CI], 5.8%-7.4%) of 3,795 donors and repeatedly in 163 (4.3%; 95% CI, 3.7%-5.0%) of 3,789 evaluable donors. “Unconfirmed” positives were mostly (93%) low positives (optical density [OD] = 0.40-0.59). Of 163 repeatedly positive samples, 116 (71.2%) were low positives, and 124 (76.1%) exhibited heparin-dependent binding. Predominant isotypes of intermediate to high seropositive samples (OD >0.6) were IgG (20/39 [51%]), IgM (9/39 [23%]), and indeterminate (10/39 [26%]). The marked background seroprevalence of PF4/heparin antibody (4.3%-6.6%) with the preponderance of low (and frequently nonreproducible) positives in blood donors suggests the need for further assay calibration, categorization of antibody level, and studies evaluating clinical relevance of “naturally occurring” PF4/heparin antibodies.

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