The C1q Receptors: Focus on gC1qR/p33 (C1qBP, p32, HABP-1)1.

In the past several years, a number of C1q binding surface proteins or receptors have been described. This is not of course surprising considering the complexity of the C1q molecule and its ability to bind to a wide range of cellular and plasma proteins via both its collagen-like [cC1q] region and its heterotrimeric globular heads [gC1q] each of which in turn is capable of binding a specific ligand. However, while each of these "receptor" molecules undoubtedly plays a specific function within its restricted microenvironment, and therefore merits full attention, this review nonetheless, will singularly focus on the structure and function of gC1qR-a multi-functional and multi-compartmental protein, which plays an important role in inflammation, infection, and cancer. Although first identified as a receptor for C1q, gC1qR has been shown to bind to a plethora of proteins found in plasma, on the cell surface and on pathogenic microorganisms. The plasma proteins that bind to gC1qR are mostly blood coagulation proteins and include high molecular weight kininogen [HK], Factor XII [Hageman factor], fibrinogen, thrombin [FII], and multimeric vitronectin. This suggests that gC1qR can play an important role in modulating not only of fibrin formation, particularly at local sites of immune injury and/or inflammation, but by activating the kinin/kallikrein system, it is also able to generate, bradykinin, a powerful vasoactive peptide that is largely responsible for the swelling seen in angioedema. Another important function of gC1qR is in cancer, where it has been shown to play a role in tumor cell survival, growth and metastatic invasion by interacting with critical molecules in the tumor cell microenvironment including those of the complement system and kinin system. Finally, by virtue of its ability to interact with a growing list of pathogen-associated molecules, including bacterial and viral ligands, gC1qR is becoming recognized as an important pathogen recognition receptor [PRR]. Given the numerous roles it plays in a growing list of disease settings, gC1qR has now become a potential target for the development of monoclonal antibody-based and/or small molecule-based therapies.

The complement and contact activation systems: partnership in pathogenesis beyond angioedema.

The blood plasma contains four biologically important proteolytic cascades, which probably evolved from the same ancestral gene. This in part may explain why each cascade has very similar "initiating trigger" followed by sequential and cascade-like downstream enzymatic activation pattern. The four cascades are: the complement system, the blood clotting cascade, the fibrinolytic system, and the kallikrein-kinin system. Although much has been written about the interplay between all these enzymatic cascades, the cross-talk between the complement and the kinin generating systems has become particularly relevant as this interaction results in the generation of nascent molecules that have significant impact in various inflammatory diseases including angioedema and cancer. In this review, we will focus on the consequences of the interplay between the two systems by highlighting the role of a novel molecular link called gC1qR. Although this protein was first identified as a receptor for C1q, it is now recognized as a multiligand binding cellular protein, which serves not only as C1q receptor, but also as high affinity (KD  ≤ 0.8 nM) binding site for both high molecular weight kininogen (HK) and factor XII (FXII). At inflammatory sites, where atherogenic factors such as immune complexes and/or pathogens can activate the endothelial cell into a procoagulant and proinflammatory surface, the two pathways are activated to generate vasoactive peptides that contribute in various ways to the inflammatory processes associated with numerous diseases. More importantly, since recent observations strongly suggest an important role for both pathways in cancer, we will focus on how a growing tumor cluster can employ the byproducts derived from the two activation systems to ensure not only its survival and growth, but also its escape into distal sites of colonization.

Soluble gC1qR is an autocrine signal that induces B1R expression on endothelial cells.

Bradykinin (BK) is one of the most potent vasodilator agonists known and belongs to the kinin family of proinflammatory peptides. BK induces its activity via two G protein-coupled receptors: BK receptor 1 (B1R) and BK receptor 2. Although BK receptor 2 is constitutively expressed on endothelial cells (ECs), B1R is induced by IL-1ß. The C1q receptor, receptor for the globular heads of C1q (gC1qR), which plays a role in BK generation, is expressed on activated ECs and is also secreted as soluble gC1qR (sgC1qR). Because sgC1qR can bind to ECs, we hypothesized that it may also serve as an autocrine/paracrine signal for the induction of B1R expression. In this study, we show that gC1qR binds to ECs via a highly conserved domain consisting of residues 174-180, as assessed by solid-phase binding assay and deconvolution fluorescence microscopy. Incubation of ECs (24 h, 37 °C) with sgC1qR resulted in enhancement of B1R expression, whereas incubation with gC1qR lacking aa 174-180 and 154-162 had a diminished effect. Binding of sgC1qR to ECs was through surface-bound fibrinogen and was inhibited by anti-fibrinogen. In summary, our data suggest that, at sites of inflammation, sgC1qR can enhance vascular permeability by upregulation of B1R expression through de novo synthesis, as well as rapid translocation of preformed B1R.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: Utilizing 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. CONCLUSIONS: This guideline provides timely, evidence-based reversal strategies to assist practitioners in the care of patients with antithrombotic-associated intracranial hemorrhage.

DC-SIGN, C1q, and gC1qR form a trimolecular receptor complex on the surface of monocyte-derived immature dendritic cells.

C1q modulates the differentiation and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because the 2 C1q receptors found on the DC surface-gC1qR and cC1qR-lack a direct conduit into intracellular elements, we postulated that the receptors must form complexes with transmembrane partners. In the present study, we show that DC-SIGN, a C-type lectin expressed on DCs, binds directly to C1q, as assessed by ELISA, flow cytometry, and immunoprecipitation experiments. Surface plasmon resonance analysis revealed that the interaction was specific, and both intact C1q and the globular portion of C1q bound to DC-SIGN. Whereas IgG reduced this binding significantly, the Arg residues (162-163) of the C1q-A chain, which are thought to contribute to the C1q-IgG interaction, were not required for C1q binding to DC-SIGN. Binding was reduced significantly in the absence of Ca(2+) and by preincubation of DC-SIGN with mannan, suggesting that C1q binds to DC-SIGN at its principal Ca(2+)-binding pocket, which has increased affinity for mannose residues. Antigen-capture ELISA and immunofluorescence microscopy revealed that C1q and gC1qR associate with DC-SIGN on blood DC precursors and immature DCs. The results of the present study suggest that C1q/gC1qR may regulate DC differentiation and function through the DC-SIGN-mediated induction of cell-signaling pathways.

The C1q and gC1qR axis as a novel checkpoint inhibitor in cancer.

Understanding at the molecular level of the cell biology of tumors has led to significant treatment advances in the past. Despite such advances however, development of therapy resistance and tumor recurrence are still unresolved major challenges. This therefore underscores the need to identify novel tumor targets and develop corresponding therapies to supplement existing biologic and cytotoxic approaches so that a deeper and more sustained treatment responses could be achieved. The complement system is emerging as a potential novel target for cancer therapy. Data accumulated to date show that complement proteins, and in particular C1q and its receptors cC1qR/CR and gC1qR/p33/HABP1, are overexpressed in most cancer cells and together are involved not only in shaping the inflammatory tumor microenvironment, but also in the regulation of angiogenesis, metastasis, and cell proliferation. In addition to the soluble form of C1q that is found in plasma, the C1q molecule is also found anchored on the cell membrane of monocytes, macrophages, dendritic cells, and cancer cells, via a 22aa long leader peptide found only in the A-chain. This orientation leaves its 6 globular heads exposed outwardly and thus available for high affinity binding to a wide range of molecular ligands that enhance tumor cell survival, migration, and proliferation. Similarly, the gC1qR molecule is not only overexpressed in most cancer types but is also released into the microenvironment where it has been shown to be associated with cancer cell proliferation and metastasis by activation of the complement and kinin systems. Co-culture of either T cells or cancer cells with purified C1q or anti-gC1qR has been shown to induce an anti-proliferative response. It is therefore postulated that in the tumor microenvironment, the interaction between C1q expressing cancer cells and gC1qR bearing cytotoxic T cells results in T cell suppression in a manner akin to the PD-L1 and PD-1 interaction.

Targeting gC1qR domains for therapy against infection and inflammation.

Abstract The receptor for the globular heads of C1q, gC1qR/p33, is a widely expressed cellular protein, which binds to diverse ligands including plasma proteins, cellular proteins, and microbial ligands. In addition to C1q, gC1qR also binds high molecular weight kininogen (HK), which also has two other cell surface sites, namely, cytokeratin 1 and urokinase plasminogen activator receptor (uPAR). On endothelial cells (ECs), the three molecules form two closely associated bimolecular complexes of gC1qR/cytokeratin 1 and uPAR/cytokeratin 1. However, by virtue of its high affinity for HK, gC1qR plays a central role in the assembly of the kallikrein-kinin system, leading to the generation of bradykinin (BK). BK in turn is largely responsible for the vascular leakage and associated inflammation seen in angioedema patients. Therefore, blockade of gC1qR by inhibitory peptides or antibodies may not only prevent the generation of BK but also reduce Clq-induced or microbial-ligand-induced inflammatory responses. Employing synthetic peptides and gClqR deletion mutants, we confirmed previously predicted sites for C1q (residues 75-96) and HK (residues 204-218) and identified additional sites for both C1q and HK (residues 190-202), for C1q (residues 144-162), and for HIV-1 gp41 (residues 174-180). With the exception of residues 75-96, which is located in the alphaA coiled-coil N-terminal segment, most of the identified residues form part of the highly charged loops connecting the various beta-strands in the crystal structure. Taken together, the data support the notion that gC1qR could serve as a novel molecular target for the design of antibody-based and/or peptide-based therapy to attenuate acute and/or chronic inflammation associated with vascular leakage and infection.

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 approximately 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.

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 x 10(9)/l) (P = 0.027) and thrombocytopenia (platelet count < 100 x 10(9)/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.

Screening coagulation testing using the APTT: which reagent to choose?

APTT testing is integral to hemostasis testing. A prolonged result, however, can be difficult to interpret, depending on the APTT reagent's sensitivity to the lupus anticoagulant. This often generates additional laboratory testing for both factor deficiencies and the presence of a lupus anticoagulant, and in so doing, delays patient management. We have found it useful to provide APTT testing with both a lupus anticoagulant sensitive and insensitive reagent, to facilitate the rapid exclusion of significant factor deficiencies. The following case report illustrates the utility of this approach and provides a backdrop for necessary discussions between laboratories and clinicians regarding which APTT reagent best meets their clinical need for screening hemostasis testing.

Globular C1q Receptor (gC1qR/p32/HABP1) Is Overexpressed in Malignant Pleural Mesothelioma and Is Associated With Increased Survival in Surgical Patients Treated With Chemotherapy.

Introduction: Globular C1q receptor (gC1qR/p32/HABP1) is overexpressed in a variety of cancers, particularly adenocarcinomas. This study investigated gC1qR expression in malignant pleural mesothelioma (MPM) and its pathophysiologic correlates in a surgical patient cohort. Methods: Tissue microarrays comprising 6 tumoral and 3 stromal cores from 265 patients with MPM (216 epithelioid, 26 biphasic, and 23 sarcomatoid; 1989-2010) were investigated by immunohistochemistry for gC1qR expression (intensity and distribution by H-score, range 0-300), and immune cell infiltration. Overall survival (OS) was analyzed by the Kaplan-Meier method (high vs. low gC1qR expression delineated by median score) in the whole cohort and by neoadjuvant chemotherapy (NAC) status. Multivariable Cox analysis included stage, chemotherapy, and immune cell infiltration. Results: gC1qR was overexpressed in all histological types of MPMs (263/265, 99.2%) compared to normal pleura. In epithelioid MPM, high gC1qR expression was associated with better OS (median 25 vs. 11 months; p = 0.020) among NAC patients, and among patients without NAC (No-NAC) but who received post-operative chemotherapy (median OS 38 vs. 19 months; p = 0.0007). In multivariable analysis, high gC1qR expression was an independent factor for improved OS in patients treated with NAC. In the No-NAC cohort, high gC1qR expression correlated with lower tumor stage. Moreover, the influence of Ki67 and CD4 T-cell infiltration on OS were more pronounced among patients with high gC1qR expression. Conclusion: This is the first description of gC1qR expression in MPM. The data identify gC1qR as a potential new prognostic factor in patients treated with surgery and chemotherapy.

Regulated complement deposition on the surface of human endothelial cells: effect of tobacco smoke and shear stress.

Cigarette smoke and hemodynamic stress both contribute to vascular inflammation and associated atherosclerosis. We recently demonstrated direct activation of complement components C4 and C3 on human endothelial cells (EC). The present study was designed to explore complement activation on bone marrow microvascular endothelial cells (BMEC) and human umbilical vein endothelial cells (HUVEC) in response to endothelial cell injury by tobacco smoke extract, shear stress, or other known inflammatory and atherogenic mediators, lipopolysaccharide (LPS) and INF-gamma. Following treatment, confluent EC monolayers were exposed to plasma (60 min, 37 degrees C), and cell surface deposition of stable complement derivatives C4d, iC3b and SC5b-9 was measured in situ using an ELISA approach. Consistent with previous results, moderate levels of C4d, iC3b and SC5b-9 deposition were observed on native EC monolayers exposed to human plasma. Tobacco smoke and shear stress enhanced EC C4d deposition. In contrast, LPS and INF-gamma failed to affect EC mediated complement activation, despite evidence of EC activation illustrated by ICAM-1 expression. The combination of tobacco smoke and shear stress nearly doubled EC C4d expression. No increases in iC3b or SC5b-9 were noted, suggesting inhibition of classical and alternative pathway C3 convertase assembly or activity. Indeed, concomitantly increased surface expression of complement regulatory proteins CD35 (CR1) and CD55 was observed following EC exposure to tobacco smoke and shear stress. These results suggest that a balance between complement activation and regulation exists at the EC surface, and may impact vascular injury leading to thrombosis, arteriosclerosis, and atherogenesis.

Expression of complement components and inhibitors on platelet microparticles.

Platelet microparticles (PMP) are released from activated platelets and play an important role in hemostasis, thrombosis and inflammation. Since platelets were recently found to demonstrate an intrinsic capacity for activating both classical and alternative pathways of the complement system, the present study extended these observations to PMP. PMP were generated by treating platelets with 10 microM A23187 (37 degrees C, 5 min). PMP were identified by flow cytometry, based on size, Annexin V binding, and expression of P-selectin and GPIIb (CD41). PMP expressed gC1qR/p33, a multifunctional cellular protein that was recently described to activate the classical complement cascade. PMP also expressed the classical pathway and contact system regulator, C1 inhibitor (C1-INH), as well as CD55 and CD59. Despite C1-INH expression, PMP supported classical pathway C4 activation in the presence of purified C1 and C4. Moreover, statistically significant deposition of C3b and C5b-9 was detected on PMP exposed to plasma, concurrently with expression of CD55 and CD59. These data provide the first evidence for the ability of PMP to support in situ complement activation. Complement activation contributes to a variety of vascular and inflammatory disease states including atherosclerosis and ischemia/reperfusion injury.

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).

HITTING the Diagnosis: Testing for Heparin-Induced Thrombocytopenia in Cancer Patients.

OBJECTIVES: To evaluate the use of a pretest probability score (4Ts score) in cancer patients to guide ordering of laboratory screening tests for heparin-induced thrombocytopenia (HIT). METHODS: A retrospective chart review was conducted for patients (n = 140) in whom laboratory testing for HIT was requested. 4Ts scores were calculated and correlated with heparin-endogenous platelet factor 4 antibody enzyme-linked immunosorbent assay (ELISA) test results. RESULTS: All patients with a high pretest probability of HIT (4Ts score = 6-7) had positive ELISA results, compared to 26.1% of patients with intermediate (4Ts score = 4-5) and 4.3% of patients with low (4Ts score ≤3) pretest probability. No patients with 4Ts scores of 2 or less had positive ELISA results. CONCLUSIONS: HIT can be ruled out in cancer patients (negative predictive value and sensitivity = 100%) with low pretest probability, defined by 4Ts scores of 2 or less, significantly reducing the need for laboratory testing in this patient population.

Is the A-Chain the Engine That Drives the Diversity of C1q Functions? Revisiting Its Unique Structure.

The immunopathological functions associated with human C1q are still growing in terms of novelty, diversity, and pathologic relevance. It is, therefore, not surprising that C1q is being recognized as an important molecular bridge between innate and adaptive immunity. The secret of this functional diversity, in turn, resides in the elegant but complex structure of the C1q molecule, which is assembled from three distinct gene products: A, B, and C, each of which has evolved from a separate and unique ancestral gene template. The C1q molecule is made up of 6A, 6B, and 6C polypeptide chains, which are held together through strong covalent and non-covalent bonds to form the 18-chain, bouquet-of-flower-like protein that we know today. The assembled C1q protein displays at least two distinct structural and functional regions: the collagen-like region (cC1q) and the globular head region (gC1q), each being capable of driving a diverse range of ligand- or receptor-mediated biological functions. What is most intriguing, however, is the observation that most of the functions appear to be predominantly driven by the A-chain of the molecule, which begs the question: what are the evolutionary modifications or rearrangements that singularly shaped the primordial A-chain gene to become a pluripotent and versatile component of the intact C1q molecule? Here, we revisit and discuss some of the known unique structural and functional features of the A-chain, which may have contributed to its versatility.

The contribution of gC1qR/p33 in infection and inflammation.

Human gC1qR/p33 is a multi-compartmental and multi-functional cellular protein expressed on a wide range of tissues and cell types including lymphocytes, endothelial cells, dendritic cells, and platelets. Although originally isolated as a receptor for C1q by virtue of its affinity (K(d)=15-50 nM), and specificity for the globular heads of this molecule, a large body of evidence has now been accumulated which shows that in addition to C1q, gC1qR can serve as a receptor for diverse proinflammatory ligands including proteins of the plasma kinin-forming system, most notably high molecular weight kininogen (HK; K(d)=9 nM). In addition, gC1qR has been reported to recognize and bind a number of functional antigens of viral and bacterial origin. It is its ability to interact with microbial antigens and its potential to serve as a cellular protein for bacterial attachment and/or entry that has been the focus of our laboratory in the past few years. On the surface of activated platelets, gC1qR has been shown to serve as a binding site for Staphylococcus aureus and this binding is mediated by protein A. Since the binding of S. aureus to platelets is postulated to play a major role in the pathogenesis of endocarditis, gC1qR may provide a suitable surface for the initial adhesion of the bacterium. Recent data also demonstrate that the exosporium of Bacillus cereus, a member of a genus of aerobic, Gram-positive, spore-forming rod-like bacilli, which includes the deadly Bacillus anthracis, contains a binding site for gC1qR. Therefore, by virtue of its ability to recognize plasma proteins such as C1q and HK, as well as bacterial and viral antigens, cell-surface gC1qR not only is able to generate proinflammatory byproducts from the complement and kinin/kallikrein systems, but also can be an efficient vehicle and platform for a plethora of pathogenic microorganisms.

Proposed research training guidelines for residents in laboratory medicine.

It is expected that the role of the clinical pathologist will evolve from the more passive role of managing testing facilities to one of active service provider, using powerful molecular, cell biologic, and biochemical tools. The scope of knowledge required to be an effective physician scientist or an accomplished practicing clinical pathologist, however, cannot be acquired through clinical training alone and requires dedicated, structured research learning time. The goal of this article is to consider mechanisms that effectively integrate research training and scholarly activity into residency education in laboratory medicine/clinical pathology. The proposed curricula are purposely unstructured to allow maximum flexibility for training programs to meet the needs and career goals of individual residents.