Ellinor I.B. Peerschke

Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets

ABSTRACT The adhesion of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Molecular mechanisms mediating S. aureus interactions with platelets, however, are incompletely understood. The present study describes the interaction between S. aureus protein A and gC1qR/p33, a multifunctional, ubiquitously distributed cellular protein, initially described as a binding site for the globular heads of C1q. Suspensions of fixed S. aureus or purified protein A, chemically cross-linked to agarose support beads, were found to capture native gC1qR from whole platelets. Moreover, biotinylated protein A bound specifically to fixed, adherent, human platelets. This interaction was inhibited by unlabeled protein A, soluble recombinant gC1qR (rgC1qR), or anti-gC1qR antibody F(ab′)2 fragments. The interaction between protein A and platelet gC1qR was underscored by studies illustrating preferential recognition of the protein A-bearing S. aureus Cowan I strain by gC1qR compared to recognition of the protein A-deficient Wood 46 strain, as well as inhibition of S. aureus Cowan I strain adhesion to immobilized platelets by soluble protein A. Further characterization of the protein A-gC1qR interaction by solid-phase enzyme-linked immunosorbent assay techniques measuring biotinylated gC1qR binding to immobilized protein A revealed specific binding that was inhibited by soluble protein A with a 50% inhibitory concentration of (3.3 ± 0.7) × 10−7 M (mean ± standard deviation; n = 3). Rabbit immunoglobulin G (IgG) also prevented gC1qR-protein A interactions, and inactivation of protein A tyrosil residues by hyperiodination, previously reported to prevent the binding of IgG Fc, but not Fab, domains to protein A, abrogated gC1qR binding. These results suggest similar protein A structural requirements for gC1qR and IgG Fc binding. Further studies of structure and function using a truncated gC1qR mutant lacking amino acids 74 to 95 demonstrated that the protein A binding domain lies outside of the gC1qR amino-terminal alpha helix, which contains binding sites for the globular heads of C1q. In conclusion, the data implicate the platelet gC1qR as a novel cellular binding site for staphylococcal protein A and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis.

gC1q-R/p33, a member of a new class of multifunctional and multicompartmental cellular proteins, is involved in inflammation and infection.

Human gC1q‐R (p33, p32, C1qBP, TAP) is a ubiquitously expressed, multiligand‐binding, multicompartmental cellular protein involved in various ligand‐mediated cellular responses. Although expressed on the surface of cells, an intriguing feature of the membrane‐associated form of gC1q‐R is that its translated amino acid sequence does not predict the presence of either a sequence motif compatible with a transmembrane segment or a consensus site for a glycosylphosphatidylinositol anchor. Moreover, the N‐terminal sequence of the pre‐pro‐protein of gC1q‐R contains a motif that targets the molecule to the mitochondria and as such was deemed unlikely to be expressed on the surface. However, several lines of experimental evidence clearly show that gC1q‐R is present in all compartments of the cell, including the extracellular cell surface. First, surface labeling of B lymphocytes with the membrane‐impermeable reagent sulfosuccinimidyl 6‐(biotinamido)hexanoate shows specific biotin incorporation into the surface‐expressed but not the intracellular form of gC1q‐R. Second, FACS and confocal laser scanning microscopic analyses using anti‐gC1q‐R IgG mAb 60.11 or 74.5.2, and the fluorophore Alexa 488‐conjugated F(ab′)2 goat anti‐mouse IgG as a probe, demonstrated specific staining of Raji cells (>95% viable). Three‐dimensional analyses of the same cells by confocal microscopy showed staining distribution that was consistent with surface expression. Third, endothelial gC1q‐R, which is associated with the urokinase plasminogen activator receptor, and cytokeratin 1 bind 125I‐high molecular weight kininogen in a specific manner, and the binding is inhibited dose‐dependently by mAb 74.5.2 recognizing gC1q‐R residues 204–218. Fourth, native gC1q‐R purified from Raji cell membranes but not intracellular gC1q‐R is glycosylated, as evidenced by a positive periodic acid Schiff stain as well as sensitivity to digestion with endoglycosidase H and F. Finally, cross‐linking experiments using C1q as a ligand indicate that both cC1q‐R and gC1q‐R are co‐immunoprecipitated with anti‐C1q. Taken together, the evidence accumulated to date supports the concept that in addition to its intracellular localization, gC1q‐R is expressed on the cell surface and can serve as a binding site for plasma and microbial proteins, but also challenges the existing paradigm that mitochondrial proteins never leave their designated compartment. It is therefore proposed that gC1q‐R belongs to a growing list of a class of proteins initially targeted to the mitochondria but then exported to different compartments of the cell through specific mechanisms which have yet to be identified. The designation ‘multifunctional and multicompartmental cellular proteins’ is proposed for this class of proteins.

Guideline for Reversal of Antithrombotics in Intracranial Hemorrhage : A Statement for Healthcare Professionals from the Neurocritical Care Society and Society of Critical Care Medicine.

BackgroundThe use of antithrombotic agents, including anticoagulants, antiplatelet agents, and thrombolytics has increased over the last decade and is expected to continue to rise. Although antithrombotic-associated intracranial hemorrhage can be devastating, rapid reversal of coagulopathy may help limit hematoma expansion and improve outcomes.MethodsThe Neurocritical Care Society, in conjunction with the Society of Critical Care Medicine, organized an international, multi-institutional committee with expertise in neurocritical care, neurology, neurosurgery, stroke, hematology, hemato-pathology, emergency medicine, pharmacy, nursing, and guideline development to evaluate the literature and develop an evidence-based practice guideline. Formalized literature searches were conducted, and studies meeting the criteria established by the committee were evaluated.ResultsUtilizing the GRADE methodology, the committee developed recommendations for reversal of vitamin K antagonists, direct factor Xa antagonists, direct thrombin inhibitors, unfractionated heparin, low-molecular weight heparin, heparinoids, pentasaccharides, thrombolytics, and antiplatelet agents in the setting of intracranial hemorrhage.ConclusionsThis guideline provides timely, evidence-based reversal strategies to assist practitioners in the care of patients with antithrombotic-associated intracranial hemorrhage.

Complement activation on platelets: implications for vascular inflammation and thrombosis.

Platelets participate in a variety of responses of the blood to injury. An emerging body of evidence suggests that these cells express an intrinsic capacity to interact with and trigger both classical and alternative pathways of complement. This activity requires cell activation with biochemical agonists and/or shear stress, and is associated with the expression of P-selectin, gC1qR, and chondroitin sulfate. Platelet mediated complement activation measurably increases soluble inflammatory mediators (C3a and C5a). Platelets may also serve as targets of classical complement activation in autoimmune conditions such as antiphospholipid syndromes (APS) and immune thrombocytopenia purpura (ITP). Retrospective correlation with clinical data suggests that enhanced platelet associated complement activation correlates with increased arterial thrombotic events in patients with lupus erythematosus and APS, and evidence of enhanced platelet clearance from the circulation in patients with ITP. Taken together, these data support a role for platelet mediated complement activation in vascular inflammation and thrombosis.

Blood platelets activate the classical pathway of human complement

Summary.  Objective: Activation of the complement system plays a key role in inflammation associated with vascular injury. Recently, platelet P‐selectin was shown to activate C3 via the alternative pathway of human complement. As platelets also posses binding sites for C1q, the recognition unit of the classical complement pathway, the present study examined classical pathway activation on platelets. Methods: Complement activation was assessed by either a solid phase enzyme‐linked immunosorbent assay (ELISA) or flow cytometry. Results: Using the ELISA approach, 2‐ to 10‐fold increases (P < 0.001) in C1q and C4d deposition were demonstrated on adherent platelets following exposure (60 min 37 °C) to diluted (1/10) human plasma or serum. Similar results were obtained by flow cytometry using activated platelets in suspension. C1q and C4d deposition on platelets was accompanied by an approximately 4‐fold increase in fluid phase C4d and C3a generation. Consistent with activation of the classical complement pathway, C4 cleavage failed to occur in serum depleted of C1q but was unchanged in factor B deficient serum. C4 activation was enhanced by platelet stimulation using chemical (SFLLRN peptide) or mechanical (shear) means, and decreased following platelet exposure to plasmin. These treatments were accompanied by changes in platelet surface gC1qR/p33 expression, a cellular C1q binding protein. In purified systems, recombinant gC1qR/p33 supported C4 activation, in a C1q dependent manner. Conclusion: These data provide the first evidence for C1q dependent classical complement pathway activation on platelets, and support a role for gC1qR/p33 in this process. However, monoclonal antibodies (mAb) to gC1qR/p33 produced only modest (20% ± 8%, mean ± SD, n = 5) reductions in C4 activation on platelets. Thus, further studies are required to investigate the involvement of additional platelet membrane constituents in classical complement pathway activation.

Novel pathogenic mechanism and therapeutic approaches to angioedema associated with C1 inhibitor deficiency.

BACKGROUND Activation of bradykinin-mediated B2 receptor has been shown to play an important role in the onset of angioedema associated with C1 inhibitor deficiency. This finding has led to the development of novel therapeutic drugs such as the B2 receptor antagonist icatibant. However, it is unclear whether other receptors expressed on endothelial cells contribute to the release of kinins and vascular leakage in these patients. The recognition of their role may have obvious therapeutic implications. OBJECTIVE Our aim was to investigate the involvement of B1 and gC1q receptors in in vitro and in vivo models of vascular leakage induced by plasma samples obtained from patients with C1 inhibitor deficiency. METHODS The vascular leakage was evaluated in vitro on endothelial cells by a transwell model system and in vivo on rat mesentery microvessels by intravital microscopy. RESULTS We observed that the attack phase plasma from C1 inhibitor-deficient patients caused a delayed fluorescein-labeled albumin leakage as opposed to the rapid effect of bradykinin, whereas remission plasma elicited a modest effect compared with control plasma. The plasma permeabilizing effect was prevented by blocking the gC1q receptor-high-molecular-weight kininogen interaction, was partially inhibited by B2 receptor or B1 receptor antagonists, and was totally prevented by the mixture of the 2 antagonists. Involvement of B1 receptor was supported by the finding that albumin leakage caused by attack phase plasma was enhanced by IL-1beta and was markedly reduced by brefeldin A. CONCLUSION Our data suggest that both B1 receptor and gC1q receptor are involved in the vascular leakage induced by hereditary and acquired angioedema plasma.

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

Combinatorial Ig libraries with phage display allow in vitro generation of human Ig fragments without the need to maintain hybridomas in ongoing cell culture or to select circulating Ig from human serum. Identifying tumor‐associated antigens on the surface of intact tumor cells, as opposed to purified proteins, presents a challenge due to the difficulty of preserving complex 3‐D epitopic sites on the cell surface, the variable expression of antigens on different malignant cell types and the stereotactic interference of closely associated proteins on the intact membrane surface limiting accessibility to antigenic sites. A combinatorial Ig library of 1010 clones was generated from the cDNA of PBMCs derived from patients with breast adenocarcinoma. Following subtractive panning, the library was enriched for Ig (Fab fragment) binding to intact adenocarcinoma cells and the resultant Fabs were screened against a cDNA expression library, itself generated from breast cancer cells. Using this approach, we isolated clones from the cDNA library expressing gC1q‐R, a glycoprotein comprising the major structure of C1, the first component of the complement system. gC1q‐R is a 33 kDa glycoprotein expressed not only on the cell surface but also intracellularly, with motifs that target it to mitochondria and complete homology with HABP and human HeLa cell protein p32, which is copurified with pre‐mRNA SF2. Sequencing of the gene encoding tumor‐associated gC1q‐R did not reveal any consistent tumor‐specific mutations. However, histochemical staining with anti‐gC1q‐R MAb demonstrated marked differential expression of gC1q‐R in thyroid, colon, pancreatic, gastric, esophageal and lung adenocarcinomas compared to their nonmalignant histologic counterparts. In contrast, differential expression was not seen in endometrial, renal and prostate carcinomas. Despite high expression in breast carcinoma, gC1q‐R was also expressed in nonmalignant breast tissue. Although the precise relation of gC1q‐R to carcinogenesis remains unclear, our finding of tumor overexpression and the known multivalent binding of gC1q‐R to not only C1q itself but also a variety of circulating plasma proteins as well as its involvement in cell‐to‐cell interactions suggest that gC1q‐R may have a role in tumor metastases and potentially serve in molecule‐specific targeting of malignant cells.

Platelet Mediated Complement Activation

The complement system comprises a series of proteases and inhibitors that are activated in cascade-like fashion during host defense (Makrides 1998). A growing body of evidence supports the hypothesis that immune mechanisms, including complement activation, are involved in inflammatory conditions associated with vascular injury (Acostan et al. 2004; Giannakopoulos et al. 2007), and disseminated intravascular coagulation associated with massive trauma (Huber-Lang, this volume). We propose that platelets and platelet derived microparticles focus complement to sites of vascular injury where regulated complement activation participates in clearing terminally activated platelets and microparticles from the circulation, and dysregulated complement activation contributes to inflammation and thrombosis. Given the central role of platelets in hemostasis and thrombosis, it is not surprising that activated complement components have been demonstrated in many types of atherosclerotic and thrombotic vascular lesions (Torzewsjki et al. 2007; Niculescu et al. 2004).

Complement activation on platelets correlates with a decrease in circulating immature platelets in patients with immune thrombocytopenic purpura

The role of the complement system in immune thrombocytopenic purpura (ITP) is not well defined. We examined plasma from 79 patients with ITP, 50 healthy volunteers, and 25 patients with non‐immune mediated thrombocytopenia, to investigate their complement activation/fixation capacity (CAC) on immobilized heterologous platelets. Enhanced CAC was found in 46 plasma samples (59%) from patients with ITP, but no samples from patients with non‐immune mediated thrombocytopenia. Plasma from healthy volunteers was used for comparison. In patients with ITP, an enhanced plasma CAC was associated with a decreased circulating absolute immature platelet fraction (A‐IPF) (<15 × 109/l) (P = 0·027) and thrombocytopenia (platelet count < 100 × 109/l) (P = 0·024). The positive predictive value of an enhanced CAC for a low A‐IPF was 93%, with a specificity of 77%. The specificity and positive predictive values increased to 100% when plasma CAC was defined strictly by enhanced C1q and/or C4d deposition on test platelets. Although no statistically significant correlation emerged between CAC and response to different pharmacological therapies, an enhanced response to splenectomy was noted (P < 0·063). Thus, complement fixation may contribute to the thrombocytopenia of ITP by enhancing clearance of opsonized platelets from the circulation, and/or directly damaging platelets and megakaryocytes.

Role of the Receptor for the Globular Domain of C1q Protein in the Pathogenesis of Hepatitis C Virus-Related Cryoglobulin Vascular Damage

Mixed cryoglobulinemia (MC) is a lymphoproliferative disorder observed in ∼10 to 15% of hepatitis C virus (HCV)-infected patients. Circulating, nonenveloped HCV core protein, which has been detected in cryoprecipitable immune complexes, interacts with immunocytes through the receptor for the globular domain of C1q protein (gC1q-R). In this study, we have evaluated circulating gC1q-R levels in chronically HCV-infected patients, with and without MC. These levels were significantly higher in MC patients than in those without MC and in healthy controls and paralleled specific mRNA expression in PBL. Soluble gC1q-R circulates as a complexed form containing both C1q and HCV core proteins. Higher serum gC1q-R levels negatively correlated with circulating concentrations of the C4d fragment. The presence of sequestered C4d in the vascular bed of skin biopsies from MC patients was indicative of in situ complement activation. In vitro studies showed that release of soluble gC1q-R is regulated by HCV core-mediated inhibition of cell proliferation. Our results indicate that up-regulation of gC1q-R expression is a distinctive feature of MC, and that dysregulated shedding of C1q-R molecules contributes to vascular cryoglobulin-induced damage via the classic complement-mediated pathway.