Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by immune complexes containing platelet factor 4 (PF4), antibodies to PF4 and heparin or cellular glycosaminoglycans (GAGs). Here we solve the crystal structures of the: (1) PF4 tetramer/fondaparinux complex, (2) PF4 tetramer/KKO-Fab complex (a murine monoclonal HIT-like antibody) and (3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4 monoclonal antibody). Fondaparinux binds to the ‘closed' end of the PF4 tetramer and stabilizes its conformation. This interaction in turn stabilizes the epitope for KKO on the ‘open' end of the tetramer. Fondaparinux and KKO thereby collaborate to ‘stabilize' the ternary pathogenic immune complex. Binding of RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation and platelet aggregation in vitro, and thrombus progression in vivo. The atomic structures provide a basis to develop new diagnostics and non-anticoagulant therapeutics for HIT.

The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and͞or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves. T he nucleolus long has been known as a dense subnuclear structure at which ribosomal genes are clustered and ribosomal transcription, rRNA processing, and ribosomal subunit assembly occurs (1). More recently, it has become clear that the RNA components of other ribonucleoproteins also may localize to the nucleolus (2, 3), including the signal recognition particle (SRP) (4). Protein components of both RNase P and RNase MRP also recently have been shown to be localized in the nucleolus (5). The hypothesis has been advanced that the nucleolus not only has evolved to carry out ribosome synthesis but, in fact, is the site of assembly of and͞or interaction between multiple ribonucleoprotein machines, many of which are involved in protein synthesis (6). However, no information regarding the potential nucleolar localization of the other components of the SRP has been available.The SRP is a ribonucleoprotein that arrests translation of secretory or membrane proteins and docks the nascent polypeptide-ribosome complex at receptors on the endoplasmic reticulum (ER), whereupon translation is resumed to direct the protein into the membrane assembly or secretory pathways (7-9). In mammalian cells, the SRP contains six proteins (7) and an Ϸ300-nt RNA (10) that previously had been known as 7S RNA or 7SL RNA (11-13). In an early study it was found that ...

Key Points Closed head trauma sequentially releases tPA followed by uPA from injured brain. Increased uPA is responsible for delayed intracerebral hemorrhage, which is prevented by a tPA variant that inhibits uPA activity.

Rapid laboratory assessment of heparininduced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4 K50E . Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4 K50E dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4 K50E . This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT. (Blood. 2012;120(5): 1137-1142) IntroductionHeparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin or glycosaminoglycans (GAGs). [1][2][3] However, antibodies to PF4/heparin are detected by ELISA far more frequently than antibodies that activate platelets or than clinical disease. [4][5][6] For example, anti-PF4/heparin antibodies are detected in 25% to 60% of patients who receive unfractionated heparin after cardiopulmonary bypass surgery and a high proportion of hospitalized patients in other medical settings, 4,7-9 an incidence that far exceeds the prevalence of HIT. 1,10 The reason why only a fraction of patients with anti-PF4 antibodies detected by ELISA develop HIT is unclear and is only partially explained by antibody titer and IgG isotype. 5,9,[11][12][13][14] One clue to the differences in the pathogenic potential of anti-PF4/heparin antibodies may begin with the finding that heparin and PF4 form complexes of diverse size that depend on the molar ratio of the reactants. [15][16][17] HIT antibodies and the HIT-like monoclonal antibody KKO bind and activate platelets and monocytes and promote thrombosis in an animal model over a narrow molar ratio of reactants. 18,19 At these molar ratios, ultralarge complexes (ULCs) form in solution between heparin and multiple PF4 tetramers capable of binding multiple antibody molecules 16 that in the case of platelets may lead to sustained engagement of FcR␥IIA, which initiates aggregation. [20][21][22] Molecular replacement stud...

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle ␣-actin (␣-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression. (Blood. 2008;112: 100-110) IntroductionUrokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes, including cell proliferation and migration during angiogenesis, tissue regeneration, inflammatory responses, and tumor growth/metastases. These complex processes all involve intracellular signal transduction and regulation of gene transcription in addition to proteolysis (see Alfano et al 1 for review). uPA is secreted as a single-chain protein (scuPA) that consists of an N-terminal EGF-like domain (GFD), a kringle domain (KD), and a serine protease domain. Binding of uPA to its high-affinity receptor CD87 (uPAR) is mediated by the GFD. 2 Plasmin converts scuPA into a proteolytically active 2-chain enzyme (tcuPA) 3 that is rapidly inhibited primarily by plasminogen activator inhibitor-1 (PAI-1). tcuPA-PAI-1 complexes are internalized with the aid of lipoprotein receptor-related protein (LRP) 4 by clathrin-mediated endocytosis. The tcuPA-PAl-1 complexes traffic to lysosomes and are degraded, while unoccupied uPAR and LRP recycle back to the cell surface. 5 uPA-induced signal transduction occurs via uPAR-dependent and uPAR-independent pathways (reviewed in Alfano et al 1 ; Kjoller 6 ; Blasi and Carmeliet 7 ). Among the latter, we have shown that cleavage of scuPA by plasmin releases the GFD fragment, generating a form of uPA unable to bind to uPAR, 8 but that stimulates migration of smooth muscle cells (SMCs). 9 Signal transduction by this scuPA fragment may be mediated in part by LRP 10 and certain integrins. 11 However, there is limited information as to the mechanism by which uPA modifies gene transcription, [12][13][14][15] and our previous studies have provided reason to hypothesize that cells express additional uPA-binding proteins that possess distinct signal-transducing activities involved in cell contractility, migration, an...