Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA.

The regulators of complement activation (RCA) gene cluster in 1q31-1q32 includes most of the genes encoding complement regulatory proteins. Genetic variability in the RCA gene cluster frequently involve copy number variations (CNVs), a type of chromosome structural variation causing alterations in the number of copies of specific regions of DNA. CNVs in the RCA gene cluster often relate with gene rearrangements that result in the generation of novel genes, carrying internal duplications or deletions, and hybrid genes, resulting from the fusion or exchange of genetic material between two different genes. These gene rearrangements are strongly associated with a number of rare and common diseases characterized by complement dysregulation. Identification of CNVs in the RCA gene cluster is critical in the molecular diagnostic of these diseases. It can be done by bioinformatics analysis of DNA sequence data generated by massive parallel sequencing techniques (NGS, next generation sequencing) but often requires special techniques like multiplex ligation-dependent probe amplification (MLPA). This is because the currently used massive parallel DNA sequencing approaches do not easily identify all the structural variations in the RCA gene cluster. We will describe here how to use the MLPA assays and two computational tools to analyze NGS data, NextGENe and ONCOCNV, to detect CNVs and gene rearrangements in the RCA gene cluster.

Therapeutic Targeting of the Complement System: From Rare Diseases to Pandemics.

The complement system was discovered at the end of the 19th century as a heat-labile plasma component that "complemented" the antibodies in killing microbes, hence the name "complement." Complement is also part of the innate immune system, protecting the host by recognition of pathogen-associated molecular patterns. However, complement is multifunctional far beyond infectious defense. It contributes to organ development, such as sculpting neuron synapses, promoting tissue regeneration and repair, and rapidly engaging and synergizing with a number of processes, including hemostasis leading to thromboinflammation. Complement is a double-edged sword. Although it usually protects the host, it may cause tissue damage when dysregulated or overactivated, such as in the systemic inflammatory reaction seen in trauma and sepsis and severe coronavirus disease 2019 (COVID-19). Damage-associated molecular patterns generated during ischemia-reperfusion injuries (myocardial infarction, stroke, and transplant dysfunction) and in chronic neurologic and rheumatic disease activate complement, thereby increasing damaging inflammation. Despite the long list of diseases with potential for ameliorating complement modulation, only a few rare diseases are approved for clinical treatment targeting complement. Those currently being efficiently treated include paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome, myasthenia gravis, and neuromyelitis optica spectrum disorders. Rare diseases, unfortunately, preclude robust clinical trials. The increasing evidence for complement as a pathogenetic driver in many more common diseases suggests an opportunity for future complement therapy, which, however, requires robust clinical trials; one ongoing example is COVID-19 disease. The current review aims to discuss complement in disease pathogenesis and discuss future pharmacological strategies to treat these diseases with complement-targeted therapies. SIGNIFICANCE STATEMENT: The complement system is the host's defense friend by protecting it from invading pathogens, promoting tissue repair, and maintaining homeostasis. Complement is a double-edged sword, since when dysregulated or overactivated it becomes the host's enemy, leading to tissue damage, organ failure, and, in worst case, death. A number of acute and chronic diseases are candidates for pharmacological treatment to avoid complement-dependent damage, ranging from the well established treatment for rare diseases to possible future treatment of large patient groups like the pandemic coronavirus disease 2019.

Creating functional sophistication from simple protein building blocks, exemplified by factor H and the regulators of complement activation.

Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a ß-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.

Overview of complement activation and regulation.

Complement is an important component of the innate immune system that is crucial for defense from microbial infections and for clearance of immune complexes and injured cells. In normal conditions complement is tightly controlled by a number of fluid-phase and cell surface proteins to avoid injury to autologous tissues. When complement is hyperactivated, as occurs in autoimmune diseases or in subjects with dysfunctional regulatory proteins, it drives a severe inflammatory response in numerous organs. The kidney appears to be particularly vulnerable to complement-mediated inflammatory injury. Injury may derive from deposition of circulating active complement fragments in glomeruli, but complement locally produced and activated in the kidney also may have a role. Many kidney disorders have been linked to abnormal complement activation, including immune-complex-mediated glomerulonephritis and rare genetic kidney diseases, but also tubulointerstitial injury associated with progressive proteinuric diseases or ischemia-reperfusion.

Disease-causing mutations in genes of the complement system.

Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of "conventional" complement deficiencies with these newly described developmental roles.

rhC1INH: a new drug for the treatment of attacks in hereditary angioedema caused by C1-inhibitor deficiency.

Recombinant human C1 esterase inhibitor (rhC1INH) (Ruconest(®), Pharming) is a new drug developed for the relief of symptoms occurring in patients with angioedema due to C1-inhibitor deficiency. Pertinent results have already been published elsewhere; this article summarizes the progress made since then. Similar to the purified C1-inhibitor derived from human plasma, the therapeutic efficacy of rhC1INH results from its ability to block the actions of enzymes belonging to the overactivated bradykinin-forming pathway, at multiple locations. During clinical trials into the management of acute edema, a total of 190 subjects received recombinant C1-inhibitor by intravenous infusion on 714 occasions altogether. Dose-ranging efficacy studies established 50 U/kg as the recommended dose, and demonstrated the effectiveness of this agent in all localizations of hereditary angioedema attacks. Studies into the safety of rhC1INH based on 300 administrations to healthy subjects or hereditary angioedema patients followed-up for 90 days have not detected the formation of autoantibodies against rhC1INH or IgE antibodies directed against rabbit proteins, even after repeated administration on multiple occasions. These findings met favorable appraisal by the EMA, which granted European marketing authorization for rhC1INH. Pharming is expected to file a biological licence with the US FDA by the end of 2010 to obtain marketing approval in the USA. The launch of rhC1INH onto the pharmaceutical market may represent an important progress in the management of hereditary angioedema patients.

Modulation of neurotransmitter receptors and synaptic differentiation by proteins containing complement-related domains.

Neurotransmitter receptors play central roles in basic neurotransmission and synaptic plasticity. Recent studies have revealed that some transmembrane and extracellular proteins bind to neurotransmitter receptors, forming protein complexes that are required for proper synaptic localization or gating of core receptor molecules. Consequently, the components of these complexes contribute to long-term potentiation, a process that is critical for learning and memory. Here, we review factors that regulate neurotransmitter receptors, with a focus on proteins containing CUB (complement C1r/C1s, Uegf, Bmp1) or CCP (complement control protein) domains, which are frequently found in complement system proteins. Proteins that contain these domains are structurally distinct from TARPs (transmembrane AMPA receptor regulatory proteins), and may constitute new protein families that modulate either the localization or function of neurotransmitter receptors. In addition, other CCP domain-containing proteins participate in dendritic patterning and/or synaptic differentiation, although current evidence has not identified any direct activities on neurotransmitter receptors. Some of these proteins are involved in pathologic conditions such as epileptic seizure and mental retardation. Together, these lines of information have shown that CUB and CCP domain-containing proteins contribute to a wide variety of neuronal events that ultimately establish neural circuits.

Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells.

In this study we demonstrate a new form of immunoregulation: engagement on CD4(+) T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (T(H)1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4(+) T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4(+) T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-gamma (IFN-gamma). Finally, gammadelta T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) gammadelta and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.

Inefficient complement system clearance of Trypanosoma cruzi metacyclic trypomastigotes enables resistant strains to invade eukaryotic cells.

The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1) which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2) the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3) whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia.

OBJECTIVE: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. DESIGN: Prospective cohort study. SETTING: Denver complement study (June 2005-June 2008). POPULATION: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. METHODS: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. MAIN OUTCOME MEASURE: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. RESULTS: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. CONCLUSIONS: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Smallpox inhibitor of complement enzymes (SPICE): dissecting functional sites and abrogating activity.

Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host's immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity approximately 200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.

Functional basis of protection against age-related macular degeneration conferred by a common polymorphism in complement factor B.

Mutations and polymorphisms in complement genes have been linked with numerous rare and prevalent disorders, implicating dysregulation of complement in pathogenesis. The 3 common alleles of factor B (fB) encode Arg (fB(32R)), Gln (fB(32Q)), or Trp (fB(32W)) at position 32 in the Ba domain. The fB(32Q) allele is protective for age-related macular degeneration, the commonest cause of blindness in developed countries. Factor B variants were purified from plasma of homozygous individuals and were tested in hemolysis assays. The protective variant fB(32Q) had decreased activity compared with fB(32R). Biacore comparison revealed markedly different proenzyme formation; fB(32R) bound C3b with 4-fold higher affinity, and formation of activated convertase was enhanced. Binding and functional differences were confirmed with recombinant fB(32R) and fB(32Q); an intermediate affinity was revealed for fB(32W). To confirm contribution of Ba to binding, affinity of Ba for C3b was determined. Ba-fB(32R) had 3-fold higher affinity compared with Ba-fB(32Q). We demonstrate that the disease-protective effect of fB(32Q) is consequent on decreased potential to form convertase and amplify complement activation. Knowledge of the functional consequences of polymorphisms in complement activators and regulators will aid disease prediction and inform targeting of diagnostics and therapeutics.

Smallpox inhibitor of complement enzymes (SPICE): regulation of complement activation on cells and mechanism of its cellular attachment.

Despite eradication of smallpox three decades ago, public health concerns remain due to its potential use as a bioterrorist weapon. Smallpox and other orthopoxviruses express virulence factors that inhibit the host's complement system. In this study, our goals were to characterize the ability of the smallpox inhibitor of complement enzymes, SPICE, to regulate human complement on the cell surface. We demonstrate that SPICE binds to a variety of cell types and that the heparan sulfate and chondroitin sulfate glycosaminoglycans serve as attachment sites. A transmembrane-engineered version as well as soluble recombinant SPICE inhibited complement activation at the C3 convertase step with equal or greater efficiency than that of the related host regulators. Moreover, SPICE attached to glycosaminoglycans was more efficient than transmembrane SPICE. We also demonstrate that this virulence activity of SPICE on cells could be blocked by a mAb to SPICE. These results provide insights related to the complement inhibitory activities of poxviral inhibitors of complement and describe a mAb with therapeutic potential.

Productos de activación del complemento (C3a y C5b-9) como marcadores de actividad de la dermatomiositis. Comparación con parámetros bioquímicos tradicionales / Complement activation products (C3a and C5b-9) as markers of activity of dermatomyositis. Comparison with usual biochemical parameters

Introducción. La dermatomiositis (DM) es una enfermedad de origen autoinmune, incluida en el grupo de las miopatías inflamatorias idiopáticas. En el control clínico de este proceso se precisan marcadores que permitan determinar el grado de actividad de la enfermedad, facilitando así el ajuste a la terapia inmunomoduladora. Se analiza la relación entre los productos de activación del complemento (PAC) y la actividad de la DM y su utilidad en el seguimiento de la enfermedad y en la predicción de las reagudizaciones en relación a los parámetros bioquímicos habituales. Material y métodos. Se estudiaron 16 pacientes con DM, que fueron seguidos periódicamente. En cada revisión se estableció el grado de actividad cutánea y muscular del proceso, y se correlacionó dicha actividad con los niveles plasmáticos de C3a y C5b-9, determinados mediante técnica de ELISA. Resultados. Si bien se obtuvo cierta correlación entre la actividad del proceso y los niveles plasmáticos de C3a y C5b-9, la intensidad de dicha correlación no superó la obtenida por los marcadores bioquímicos tradicionales. En la capacidad de predicción de reagudizaciones, C3a se mostró como el marcador más sensible (100 %), con una especificidad suficiente (83,3 %). Conclusiones. C3a y en menor medida C5b-9 serían de utilidad en la identificación de pacientes con DM especialmente activas, así como en la predicción de reagudizaciones del proceso. Sin embargo, no tienen una utilidad superior al resto de marcadores bioquímicos como marcadores de actividad actual

Introduction. Dermatomyositis (DM) is an autoimmune disease included in the group of idiopathic inflammatory myopathies. Markers of disease activity are needed for clinical control in order to facilitate adjustment of immunomodulatory therapy. We analyzed the relationship between complement activation products (CAP) and the activity of dermatomyositis and its usefulness in the follow-up of the disease and the prediction of recrudescences related to usual biochemical parameters. Material and methods. We studied 16 patients with DM that were followed periodically. In each appointment the degree of cutaneous and muscular activity was assessed and such disease activity was correlated with plasma levels of C3a and C5b-9, measured by ELISA. Results. Though we obtained certain correlation between disease activity and plasma levels of C3a and C5b-9, the strength of such correlation was not superior to that obtained by usual biochemical markers. C3a was shown to be the most sensitive marker (100 %) with a sufficient specificity (83.3 %) in the capability to predict recrudescences. Conclusions. C3a and, to a lesser extent C5b-9, would be useful in the identification of patients with especially active DM as well as in predicting disease recrudescences. Nevertheless they are not superior to the rest of biochemical markers as indicators of current activity

Structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation.

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.

The complement system in the pathophysiology of pregnancy.

A fully active complement system deriving from the maternal circulation as well as from local production by various cell source is present in the placenta. The role of this system at the placental level, as in any other tissue in the body, is to protect both the fetus and the mother against infectious and other toxic agents. As fetal tissues are semi-allogeneic and alloantibodies commonly develop in the mother, the placenta is potentially subject to complement-mediated immune attack at the feto-maternal interface with the potential risk of fetal loss. Uncontrolled complement activation is prevented in successful pregnancy by the three regulatory proteins DAF, MCP and CD59 positioned on the surface of trophoblasts. The critical role played by these complement regulators is supported by the embryonic lethality observed in mice deficient in the complement regulator Crry. Excessive complement activation in the placenta places the fetus at risk for growth restriction or death. The role played by the complement system in the fetal damage induced by anti-phospholipid antibodies in a mouse model will be examined.

Modulated interaction of the ERM protein, moesin, with CD93.

CD93 is a cell-surface glycoprotein that has been shown to influence defence collagen-enhanced Fc-receptor or CR1-mediated phagocytosis of suboptimally opsonized targets in vitro, and CD93-deficient mice are defective in the clearance of apoptotic cells in vivo. To investigate the mechanism of CD93 modulation of phagocytic activity, GST fusion proteins containing the 47 amino acid intracellular domain (GST-Cyto), or various mutants of the intracellular domain of CD93, were constructed and used to identify intracellular CD93-binding molecules. The intracellular protein moesin, well characterized for its role in linking transmembrane proteins to the cytoskeleton and in cytoskeletal remodelling, bound to GST-Cyto when either cell lysates or recombinant moesin were used as a source of interacting molecules. An association of moesin with CD93 within intact cells was confirmed by co-capping moesin with CD93 in human monocytes. The moesin-binding site on CD93 mapped to the first four positively charged amino acids in the juxtamembrane region of the CD93 cytoplasmic tail. Interestingly, deletion of the last 11 amino acids from the C terminus of CD93 (GST-Cyto-C11) dramatically increased moesin binding to the cytoplasmic tail of CD93 in the cell lysate assay, but not when the binding of purified recombinant moesin was assessed. Furthermore, moesin binding to CD93 was enhanced by the addition of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Taken together, these data suggest that the interaction of moesin with the CD93 cytoplasmic domain is modulated by binding of other intracellular molecules to the C11 region and implies that a PIP(2) signalling pathway is involved in CD93 function.