Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search

The human serum N-linked glycoproteome has been determined through LC-MS/MS. The intact glycopeptides were identified through a spectral library search method embedded in the pMatchGlyco software. Four types of known N-glycosylation motifs, prevalent variable modifications and semi-tryptic digestion were considered during searching and the identified intact glycopeptides were validated through target-decoy and motif-specific false discovery rate (FDR) control. The results reveal site-specific glycosylation of serum glycoproteins and provide high-quality tandem mass spectra of 22,677 serum N-linked intact glycopeptides.


Introduction
Reactome is a curated database of pathways and reactions in human biology. Reactions can be considered as pathway 'steps'. Reactome defines a 'reaction' as any event in biology that changes the state of a biological molecule. Binding, activation, translocation, degradation and classical biochemical events involving a catalyst are all reactions. Information in the database is authored by expert biologists, entered and maintained by Reactome's team of curators and editorial staff. Reactome content frequently cross-references other resources e.g. NCBI, Ensembl, UniProt, KEGG (Gene and Compound), ChEBI, PubMed and GO. Orthologous reactions inferred from annotation for Homo sapiens are available for 17 non-human species including mouse, rat, chicken, puffer fish, worm, fly, yeast, rice, and Arabidopsis. Pathways are represented by simple diagrams following an SBGN-like format.
Reactome's annotated data describe reactions possible if all annotated proteins and small molecules were present and active simultaneously in a cell. By overlaying an experimental dataset on these annotations, a user can perform a pathway over-representation analysis. By overlaying quantitative expression data or time series, a user can visualize the extent of change in affected pathways and its progression. A binomial test is used to calculate the probability shown for each result, and the p-values are corrected for the multiple testing (Benjamini-Hochberg procedure) that arises from evaluating the submitted list of identifiers against every pathway.
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Properties
This is an overrepresentation analysis: A statistical (hypergeometric distribution) test that determines whether certain Reactome pathways are over-represented (enriched) in the submitted data. It answers the question 'Does my list contain more proteins for pathway X than would be expected by chance?' This test produces a probability score, which is corrected for false discovery rate using the Benjamani-Hochberg method.
• 404 out of 526 identifiers in the sample were found in Reactome, where 717 pathways were hit by at least one of them.
• All non-human identifiers have been converted to their human equivalent.
• This report is filtered to show only results for species 'Homo sapiens' and resource 'UniProt'.
• The unique ID for this analysis (token) is MjAyMDAyMDgxODM0MzJfMjQ4NjA%3D. This ID is valid for at least 7 days in Reactome's server. Use it to access Reactome services with your data.

Pathways details
For every pathway of the most significant pathways, we present its diagram, as well as a short summary, its bibliography and the list of inputs found in it.

References
Cellular compartments: plasma membrane, extracellular region.
Two inherent features of complement activation make its regulation very important: 1. There is an inherent positive feedback loop because the product of C3 activation forms part of an enzyme that causes more C3 activation.
2. There is continuous low-level activation of the alternative pathway (see Spontaneous hydrolysis of C3 thioester).
Complement cascade activation is regulated by a family of related proteins termed the regulators of complement activation (RCA). These are expressed on healthy host cells. Most pathogens do not express RCA proteins on their surface, but many have found ways to evade the complement system by stably binding the RCA that circulates in human plasma (Lambris et al. 2008); trapping RCA is by far the most widely employed strategy for avoiding the complement response. RCA recruitment is common in bacteria such as E. coli and streptococci (Kraiczy & Wurzner 2006) and has also been described for viruses, fungi and parasites. RCA deposition and the complement system also have an important role in tissue homeostasis, clearing dead cells and debris, and preventing damage from oxidative stress (Weismann et al. 2011).
RCA proteins control complement activation in two different ways; by promoting the irreversible dissociation (decay acceleration) of complement convertases and by acting as cofactors for Complement factor I (CFI)-mediated cleavage of C3b and C4b.
Decay accelerating factor (DAF, CD55), Complement factor H (FH), Membrane Cofactor Protein (MCP) and Complement receptor 1 (CR1) are composed of arrays of tandem globular domains termed CCPs (complement control protein repeats) or SCRs (short consensus repeats). CR1, MCP and FH are cofactors for the CFI-mediated cleavage of C3b, generating iC3b. CR1 and MCP are also cofactors for C4b cleavage.

References
In the complement cascade, a panel of soluble molecules rapidly and effectively senses a danger or damage and triggers reactions to provide a response that discriminates among foreign intruders, cellular debris, healthy and altered host cells (Ricklin D et al. 2010). Complement proteins circulate in the blood stream in functionally inactive states. When triggered the complement cascade generates enzymatically active molecules (such as C3/C5 convertases) and biological effectors: opsonins (C3b, C3d and C4b), anaphylatoxins (C3a and C5a), and C5b, which initiates assembly of the lytic membrane attack complex (MAC). Three branches lead to complement activation: the classical, lectin and alternative pathways (Kang YH et al. 2009;Ricklin D et al. 2010). The classical pathway is initiated by C1 complex binding to immune complexes, pentraxins or other targets such as apoptotic cells leading to cleavage of C4 and C2 components and formation of the classical C3 convertase, C4bC2a. The lectin pathway is activated by binding of mannan-binding lectin (MBL) to repetitive carbohydrate residues, or by binding of ficolins to carbohydrate or acetylated groups on target surfaces. MBL and ficolins interact with MBL-associated serine proteases (MASP) leading to cleavage of C4 and C2 and formation of the classical C3 convertase, C4bC2a. The alternative pathway is spontaneously activated by the hydrolysis of the internal thioester group of C3 to give C3(H2O). Alternative pathway activation involves interaction of C3(H2O) and/or previously generated C3b with factor B, which is cleaved by factor D to generate the alternative C3 convertases C3(H2O)Bb and/or C3bBb.
All three pathways merge at the proteolytic cleavage of component C3 by C3 convertases to form opsonin C3b and anaphylatoxin C3a. C3b covalently binds to glycoproteins scattered across the target cell surface. This is followed by an amplification reaction that generates additional C3 convertases and deposits more C3b at the local site. C3b can also bind to C3 convertases switching them to C5 convertases, which mediate C5 cleavage leading to MAC formation. Thus, the activation of the complement system leads to several important outcomes:<ul><li>opsonization of target cells to enhance phagocytosis,</li><li>lysis of target cells via membrane attack complex (MAC) assembly on the cell surface,</li><li>production of anaphylatoxins C3a/C5a involved in the host inflammatory response,</li><li>C5a-mediated leukocyte chemotaxis,</li><li>and clearance of antibody-antigen complexes.</li></ul> The complement system is able to distinguish between pathological and physiological challenges, i.e. the outcomes of complement activation are predetermined by the trigger and are tightly tuned by a combination of initiation events with several regulatory mechanisms. These regulatory mechanisms use soluble (e.g., C4BP, CFI and CFH) and membrane-bound regulators (e.g., CR1, CD46(MCP), CD55(DAF) and CD59) and are coordinated by complement receptors such as CR1, CR2, etc.
In response to microbial infection complement activation results in flagging microorganisms with opsonins for facilitated phagocytosis, formation of MAC on cells such as Gram-negative bacteria leading to cell lysis, and release of C3a and C5a to stimulate downstream immune responses and to attract leukocytes. Most pathogens can be eliminated by these complement-mediated host responses, though some pathogenic microorganisms have developed ways of avoiding complement recognition or blocking host complement attack resulting in greater virulence (Lambris JD et al. 2008;Serruto D et al. 2010).
All three complement pathways (classical, lectin and alternative) have been implicated in clearance of dying cells (Mevorach D et al. 1998;Ogden CA et al. 2001;Gullstrand B et al.2009;Kemper C et al. 2008). Altered surfaces of apoptotic cells are recognized by complement proteins leading to opsonization and subsequent phagocytosis. In contrast to pathogens, apoptotic cells are believed to induce only a limited complement activation by allowing opsonization of altered surfaces but restricting the terminal pathway of MAC formation (Gershov D et al. 2000;Braunschweig A and Jozsi M 2011). Thus, opsonization facilitates clearance of dying cells and cell debris without triggering danger signals and further inflammatory responses (Fraser DA et al. 2007, 2009Benoit ME et al. 2012). C1q-mediated complement activation by apoptotic cells has been shown in a variety of human cells: keratinocytes, human umbilical vein endothelial cells (HUVEC), Jurkat T lymphoblastoid cells, lung adenocarcinoma cells (Korb LC and Ahearn JM 1997;Mold C and Morris CA 2001;Navratil JS et al. 2001;Nauta AJ et al. 2004). In addition to C1q the opsonization of apoptotic Jurkat T cells with MBL also facilitated clearance of these cells by both dendritic cells (DC) and macrophages (Nauta AJ et al. 2004). Also C3b, iC3b and C4b deposition on apoptotic cells as a consequence of activation of the complement cascade may promote complement-mediated phagocytosis. C1q, MBL and cleavage fragments of C3/C4 can bind to several receptors expressed on macrophages (e.g. cC1qR (calreticulin), CR1, CR3, CR4) suggesting a potential clearance mechanism through this interaction (Mevorach D et al. 1998;Ogden CA et al. 2001). Apoptosis is also associated with an altered expression of complement regulators on the surface of apoptotic cells. CD46 (MCP) bound to the plasma membrane of a healthy cell protects it from complement-mediated attack by preventing deposition of C3b and C4b, and reduced expression of CD46 on dying cells may lead to enhanced opsonization (Elward K et al. 2005). Upregulation of CD55 (DAF) and CD59 on apoptotic cell surfaces may protect damaged cells against complement mediated lysis (Pedersen ED et al. 2007;Iborra A et al. 2003;Hensel F et al. 2001). In addition, fluid-phase complement regulators such as C4BP, CFH may also inhibit lysis of apoptotic cells by limiting complement activation (Trouw LA et al 2007;Braunschweig A and Jozsi M. 2011).
Complement facilitates the clearance of immune complexes (IC) from the circulation (Chevalier J and Kazatchkine MD 1989;Nielsen CH et al. 1997). Erythrocytes bear clusters of complement receptor 1 (CR1 or CD35), which serves as an immune adherence receptor for C3 and/or C4 fragments deposited on IC that are shuttled to liver and spleen, where IC are transferred and processed by tissue macrophages through an Fc receptor-mediated process.
Complement proteins are always present in the blood and a small percentage spontaneously activate. Inappropriate activation leads to host cell damage, so on healthy human cells any complement activation or amplification is strictly regulated by surface-bound regulators that accelerate decay of the convertases (CR1, CD55), act as a cofactor for the factor I (CFI)-mediated degradation of C3b and C4b (CR1, CD46), or prevent the formation of MAC (CD59). Soluble regulators such as C4BP, CFH and FHL1 recognize self surface pattern-like glycosaminoglycans and further impair activation.
Complement components interact with other biological systems. Upon microbial infection complement acts in cooperation with Toll-like receptors (TLRs) to amplify innate host defense. Anaphylatoxin C5a binds C5a receptor (C5aR) resulting in a synergistic enhancement of the TLR and C5aRmediated proinflammatory cytokine response to infection. This interplay is negatively modulated by co-ligation of TLR and the second C5a receptor, C5L2, suggesting the existence of complex immunomodulatory interactions (Kohl J 2006;Hajishengallis G and Lambris JD 2010). In addition to C5aR and C5L2, complement receptor 3 (CR3) facilitates TLR2 or TLR4 signaling pathways by promoting a recruitment of their sorting adaptor TIRAP (MAL) to the receptor complex (van Bruggen R et al. 2007;Kagan JC and Medzhitov R 2006). Complement may activate platelets or facilitate biochemical and morphological changes in the endothelium potentiating coagulation and contributing to homeostasis in response to injury (Oikonomopoulou K et al. 2012). The interplay of complement and coagulation also involves cleavage of C3 and C5 convertases by coagulation proteases, generating biologically active anaphylatoxins (Amara U et al. 2010). Complement is believed to link the innate response to both humoral and cell-mediated immunity (Toapanta FR and Ross TM 2006;Mongini PK et al. 1997). The majority of published data is based on experiments using mouse as a model organism. Further characterization of the influence of complement on B or T cell activation is required for the human system, since differences between murine models and the human system are not yet fully determined. Complement is also involved in regulation of mobilization and homing of hematopoietic stem/progenitor cells (HSPCs) from bone marrow to the circulation and peripheral tissue in order to accommodate blood cell replenishment (Reca R et al. 2006).
Thus, the complement system orchestrates the host defense by sensing a danger signal and transmitting it into specific cellular responses while extensively communicating with associated biological pathways ranging from immunity and inflammation to homeostasis and development.
N.B. Originally the larger fragment of Complement Factor 2 (C2) was designated C2a. However, complement scientists decided that the smaller of all C fragments should be designated with an 'a', the larger with a 'b', changing the nomenclature for C2. Recent literature may use the updated nomenclature and refer to the larger C2 fragment as C2b, and refer to the classical C3 convertase as C4bC2b. Throughout this pathway Reactome adheres to the original convention to agree with the current (Sep 2013) Uniprot names for C2 fragments.

Date
Action Author The formation of a fibrin clot at the site of an injury to the wall of a normal blood vessel is an essential part of the process to stop blood loss after vascular injury. The reactions that lead to fibrin clot formation are commonly described as a cascade, in which the product of each step is an enzyme or cofactor needed for following reactions to proceed efficiently. The entire clotting cascade can be divided into three portions, the extrinsic pathway, the intrinsic pathway, and the common pathway. The extrinsic pathway begins with the release of tissue factor at the site of vascular injury and leads to the activation of factor X. The intrinsic pathway provides an alternative mechanism for activation of factor X, starting from the activation of factor XII. The common pathway consists of the steps linking the activation of factor X to the formation of a multimeric, cross-linked fibrin clot.
Each of these pathways includes not only a cascade of events that generate the catalytic activities needed for clot formation, but also numerous positive and negative regulatory events. Davie EW, Fujikawa K & Kisiel W (1991
Platelets function as exocytotic cells, secreting a plethora of effector molecules at sites of vascular injury. Platelets contain a number of distinguishable storage granules including alpha granules, dense granules and lysosomes. On activation platelets release a variety of proteins, largely from storage granules but also as the result of apparent cell lysis. These act in an autocrine or paracrine fashion to modulate cell signaling.
Alpha granules contain mainly polypeptides such as fibrinogen, von Willebrand factor, growth factors and protease inhibitors that that supplement thrombin generation at the site of injury.
Dense granules contain small molecules, particularly adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin and calcium, all recruit platelets to the site of injury.
The molecular mechanism which facilitates granule release involves soluble NSF attachment protein receptors (SNAREs), which assemble into complexes to form a universal membrane fusion ap-
The extracellular matrix is a component of all mammalian tissues, a network consisting largely of the fibrous proteins collagen, elastin and associated-microfibrils, fibronectin and laminins embed- ECM remodeling is involved in the regulation of cell differentiation processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. Redundant mechanisms modulate the expression and function of ECM modifying enzymes. Abnormal ECM dynamics can lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer.
Collagen is the most abundant fibrous protein within the ECM constituting up to 30% of total protein in multicellular animals. Collagen provides tensile strength. It associates with elastic fibres, composed of elastin and fibrillin microfibrils, which give tissues the ability to recover after stretching. Other ECM proteins such as fibronectin, laminins, and matricellular proteins participate as Response to elevated platelet cytosolic Ca2+ (R-HSA-76005) 8.
Activation of phospholipase C enzymes results in the generation of second messengers of the phosphatidylinositol pathway. The events resulting from this pathway are a rise in intracellular calcium and activation of Protein Kinase C (PKC). Phospholipase C cleaves the phosphodiester bond in PIP2 to form 1,2 Diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3). IP3 opens Ca2+ channels in the platelet dense tubular system, raising intracellular Ca2+ levels. DAG is a second messenger that regulates a family of Ser/Thr kinases consisting of PKC isozymes (Nishizuka 1995). DAG achieves activation of PKC isozymes by increasing their affinity for phospholipid. Most PKC enzymes are also calcium-dependent, so their activation is in synergy with the rise in intracellular Ca2+. Platelets contain several PKC isoforms that can be activated by DAG and/or Ca2+ (Chang 1997). Walker TR & Watson SP (1993). Synergy between Ca2+ and protein kinase C is the major factor in determining the level of secretion from human platelets. Biochem J, 289, 277-82.
Hemostasis is a physiological response that culminates in the arrest of bleeding from an injured vessel. Under normal conditions the vascular endothelium supports vasodilation, inhibits platelet adhesion and activation, suppresses coagulation, enhances fibrin cleavage and is anti-inflammatory in character. Under acute vascular trauma, vasoconstrictor mechanisms predominate and the endothelium becomes prothrombotic, procoagulatory and proinflammatory in nature. This is achieved by a reduction of endothelial dilating agents: adenosine, NO and prostacyclin; and by the direct action of ADP, serotonin and thromboxane on vascular smooth muscle cells to elicit their contraction (Becker et al. 2000).
The chief trigger for the change in endothelial function that leads to the formation of a haemostatic thrombus is the loss of the endothelial cell barrier between blood and extracellular matrix components (Ruggeri 2002). Circulating platelets identify and discriminate areas of endothelial lesions; here, they adhere to the exposed sub endothelium. Their interaction with the various thrombogenic substrates and locally generated or released agonists results in platelet activation. This process is described as possessing two stages, firstly, adhesion -the initial tethering to a surface, and secondly aggregation -the platelet-platelet cohesion (Savage & Cattaneo et al. 2001).
Platelet activation begins with the initial binding of adhesive ligands and of the excitatory platelet agonists (released or generated at the sites of vascular trauma) to cognate receptors on the platelet membrane (Ruggeri 2002). Intracellular signaling reactions then enhance the adhesive and procoagulant properties of tethered platelets or of platelets circulating in the proximity. Once platelets have adhered they degranulate, releasing stored secondary agents such as ADP, ATP, and synthesize thromboxane A2. These amplify the response, activating and recruiting further platelets to the area and promoting platelet aggregation. These amplify the response, activating and recruiting further platelets to the area and promoting platelet aggregation. Adenosine nucleotides signal through P2 purinergic receptors on the platelet membrane. ADP activates P2Y1 and P2Y12, which signal via both the alpha and gamma:beta components of the heterotrimeric G-protein (Hirsch et al. 2001(Hirsch et al. , 2006, while ATP activates the ionotropic P2X1 receptor (Kunapuli et al. 2003). Activation of these receptors initiates a complex signaling cascade that ultimately results in platelet activation, aggregation and thrombus formation (Kahner et al. 2006).
Integrin AlphaIIbBeta3 is the most abundant platelet receptor, with 40 000 to 80 000 copies per resting platelet, acting as a major receptor for fibrinogen and other adhesive molecules (Wagner et al. 1996). Activation of AlphaIIbBeta3 enhances adhesion and leads to platelet-platelet interactions, and thus aggregation (Philips et al. 1991). GP VI is the most potent collagen receptor initiating signal generation, an ability derived from its interaction with the FcRI gamma chain. This results in the phosphorylation of the gamma-chain by non-receptor tyrosine kinases of the Src family (1). The phosphotyrosine motif is recognized by the SH2 domains of Syk, a tyrosine kinase. This association activates the Syk enzyme, leading to activation (by tyrosine phosphorylation) of PLC gamma2 (2).
Thrombin is an important platelet agonist generated on the membrane of stimulated platelets.
Thrombin acts via cell surface Protease Activated Receptors (PARs). PARs are G-protein coupled receptors activated by a proteolytic cleavage in an extracellular loop (Vu, 1991) (3). Activated PARs signal via G alpha q (4) and via the beta:gamma component of the G-protein (5). Both stimulate PLC giving rise to PIP2 hydrolysis and consequent activation of PI3K (6). PLCgamma2 activation also gives rise to IP3 (7) which stimulates the IP3 receptor (8) leading to increased intracellular calcium.
Platelet activation further results in the scramblase-mediated transport of negatively-charged phospholipids to the platelet surface. These phospholipids provide a catalytic surface (with the charge provided by phosphatidylserine and phosphatidylethanolamine) for the tenase complex (formed by the activated forms of the blood coagulation factors factor VIII and factor I).

Edit history Date
Action Author Integrin cell surface interactions (R-HSA-216083) 11.
The extracellular matrix (ECM) is a network of macro-molecules that underlies all epithelia and endothelia and that surrounds all connective tissue cells. This matrix provides the mechanical strength and also influences the behavior and differentiation state of cells in contact with it. The ECM are diverse in composition, but they generally comprise a mixture of fibrillar proteins, polysaccharides synthesized, secreted and organized by neighboring cells. Collagens, fibronectin, and laminins are the principal components involved in cell matrix interactions; other components, such as vitronectin, thrombospondin, and osteopontin, although less abundant, are also important adhesive molecules.
Integrins are the receptors that mediate cell adhesion to ECM. Integrins consists of one alpha and one beta subunit forming a noncovalently bound heterodimer. 18 alpha and 8 beta subunits have been identified in humans that combine to form 24 different receptors.
The integrin dimers can be broadly divided into three families consisting of the beta1, beta2/beta7, and beta3/alphaV integrins. beta1 associates with 12 alpha-subunits and can be further divided into RGD-, collagen-, or laminin binding and the related alpha4/alpha9 integrins that recognise both matrix and vascular ligands. beta2/beta7 integrins are restricted to leukocytes and mediate cell-cell rather than cell-matrix interactions, although some recognize fibrinogen. The beta3/alphaV family members are all RGD receptors and comprise aIIbb3, an important receptor on platelets, and the remaining b-subunits, which all associate with alphaV. It is the collagen receptors and leukocytespecific integrins that contain alpha A-domains. ECM proteoglycans (R-HSA-3000178) 12.
Proteoglycans are major components of the extracellular matrix. In cartilage the matrix constitutes more than 90% of tissue dry weight. Proteoglycans are proteins substituted with glycosaminoglycans (GAGs), linear polysaccharides consisting of a repeating disaccharide, generally of an acetylated amino sugar alternating with a uronic acid. Most proteoglycans are located in the extracellular space. Proteoglycans are highly diverse, both in terms of the core proteins and the subtypes of GAG chains, namely chondroitin sulfate (CS), keratan sulfate (KS), dermatan sulfate (DS) and heparan sulfate (HS). Hyaluronan is a non-sulfated GAG whose molecular weight runs into millions of Dalton; in articular cartilage, a single hyaluronan molecule can hold upto 100 aggrecan molecules and these aggregates are stabilized by a link protein.
Neutrophils are the most abundant leukocytes (white blood cells), indispensable in defending the body against invading microorganisms. In response to infection, neutrophils leave the circulation and migrate towards the inflammatory focus. They contain several subsets of granules that are mobilized to fuse with the cell membrane or phagosomal membrane, resulting in the exocytosis or exposure of membrane proteins. Traditionally, neutrophil granule constituents are described as antimicrobial or proteolytic, but granules also introduce membrane proteins to the cell surface, changing how the neutrophil responds to its environment (Borregaard et al. 2007). Primed neutrophils actively secrete cytokines and other inflammatory mediators and can present antigens via MHC II, stimulating T-cells (Wright et al. 2010).
Granules form during neutrophil differentiation. Granule subtypes can be distinguished by their content but overlap in structure and composition. The differences are believed to be a consequence of changing protein expression and differential timing of granule formation during the terminal processes of neutrophil differentiation, rather than sorting (Le Cabec et al. 1996).
The intrinsic pathway of blood clotting connects interactions among kininogen (high molecular weight kininogen, HK), prekallikrein (PK), and factor XII to the activation of clotting factor X by a series of reactions that is independent of the extrinsic pathway and that is not subject to inhibition by TFPI. It is thus essential for the prolongation of the clotting cascade: while the reactions of the extrinsic pathway appear to be sufficient to initiate clot formation, those of the intrinsic pathway are required to maintain it (Broze 1995;Davie et al. 1991;Monroe et al. 2002). The intrinsic pathway can be divided into three parts: 1) reactions involving interactions of kininogen, prekallikrein, and factor XII, leading to the activation of factor XII, 2) reactions involving factor XI, factor IX, factor VIII, and von Willebrand factor (vWF) leading to the activation of factors VIII and IX, and 3) reactions that inactivate factor XIIa and kallikrein.
Kininogen, prekallikrein, and factor XII were first identified as proteins needed for the rapid formation of clots when whole blood is exposed to negatively charged surfaces in vitro. Studies in vitro have identified several possible sets of interactions, in which small quantities of one or more of these proteins 'autoactivate' and then catalyze the formation of larger quantities of activated factors. Recent work, however, suggests that these factors form complexes on endothelial cell surfaces mediated by C1q binding protein (C1q bp), that the first activation event is the cleavage of prekallikrein by prolylcarboxypeptidase, and that the resulting kallikrein catalyzes the activation of factor XII (Schmaier 2004).
The second group of events, occurs in vivo on the surfaces of activated platelets (although most biochemical characterization of the reactions was originally done with purified proteins in solution).
Factor XI binds to the platelet glycoprotein (GP) Ib:IX:V complex, where it can be activated by cleavage either by thrombin (generated by reactions of the common pathway) or by activated factor XII (generated in the first part of the intrinsic pathway). Activated factor XI in turn catalyzes the activation of factor IX. Simultaneously, factor VIII, complexed with vWF, is cleaved by thrombin, activating it and causing its release from vWF. Activated factors VIII and IX form a complex on the platelet surface that very efficiently converts factor X to activated factor X. (Activated factors X and V then form a complex that efficiently activates thrombin.) While these two groups of events can be viewed as forming a single functional pathway (e.g., Davie et al. 1991), human clinical genetic data cast doubt on this view. Individuals deficient in kininogen, prekallikrein, or factor XII proteins exhibit normal blood clot formation in vivo. In contrast, deficiencies of factor XI can be associated with failure of blood clotting under some conditions, and deficiencies of vWF, factor VIII, or factor IX cause severe abnormalities -von Willebrand disease, hemophilia A, and hemophilia B, respectively. These data suggest that while the second group of events is essential for normal clot formation in vivo, the first group has a different function (e.g.,

Schmaier 2004).
Finally, reactions neutralize proteins activated in the first part of the intrinsic pathway. Kallikrein forms stable complexes with either C1 inhibitor (C1Inh) or with alpha2-macroglobulin, and factor XIIa forms stable complexes with C1Inh. The relevance of these neutralization events to the regulation of blood clotting is unclear, however. The physiological abnormalities observed in individuals who lack C1Inh appear to be due entirely to abnormalities of complement activation; blood clotting appears to proceed normally. This observation is consistent with the hypothesis, above, that factor XIIa plays a limited role in normal blood clotting under physiological conditions. These events are outlined in the drawing: black arrows connect the substrates (inputs) and products (outputs) of individual reactions; blue lines connect activated enzymes to the reactions they catalyze.
The common pathway consists of the cascade of activation events leading from the formation of activated factor X to the formation of active thrombin, the cleavage of fibrinogen by thrombin, and the formation of cleaved fibrin into a stable multimeric, cross-linked complex. Thrombin also efficiently catalyzes the activation of several factors required earlier in the clotting cascade, thus acting in effect as a positive regulator of clotting. At the same time, thrombin activates protein C, which in turn catalyzes the inactivation of several of these upstream factors, thereby limiting the clotting process. Thrombin can be trapped in stable, inactive complexes with: antithrombin-III (SERPINC1), a circulating blood protein; heparin cofactor II (SERPIND1) which inhibits thrombin in a dermatan sulfate-dependent manner in the arterial vasculature; protein C inhibitor (SER-PINA5) that inhibits thrombin in complex with thrombomodulin; and Protease nexin-1 (SERPINE2) that inhibits thrombin at the vessel wall and platelet surface.
The quantitative interplay among these positive and negative modulators is critical to the normal regulation of clotting, facilitating the rapid formation of a protective clot at the site of injury, while limiting and physically confining the process.
These events are outlined in the drawing: black arrows connect the substrates (inputs) and products (outputs) of individual reactions, and blue lines connect output activated enzymes to the other reactions that they catalyze. Davie EW, Fujikawa K & Kisiel W (1991 Immune System (R-HSA-168256) 17. Humans are exposed to millions of potential pathogens daily, through contact, ingestion, and inhalation. Our ability to avoid infection depends on the adaptive immune system and during the first critical hours and days of exposure to a new pathogen, our innate immune system. Non-integrin membrane-ECM interactions (R-HSA-3000171) 18.

References
Several non-integrin membrane proteins interact with extracellular matrix proteins. Transmembrane proteoglycans may associate with integrins and growth factor receptors to influence their function, or they can signal independently, often influencing the actin cytoskeleton. ( Cell surface interactions at the vascular wall (R-HSA-202733) 19.

Cellular compartments: plasma membrane.
Leukocyte extravasation is a rigorously controlled process that guides white cell movement from the vascular lumen to sites of tissue inflammation. The powerful adhesive interactions that are required for leukocytes to withstand local flow at the vessel wall is a multistep process mediated by different adhesion molecules. Platelets adhered to injured vessel walls form strong adhesive substrates for leukocytes. For instance, the initial tethering and rolling of leukocytes over the site of injury are mediated by reversible binding of selectins to their cognate cell-surface glycoconjugates.
Endothelial cells are tightly connected through various proteins, which regulate the organization of the junctional complex and bind to cytoskeletal proteins or cytoplasmic interaction partners that allow the transfer of intracellular signals. An important role for these junctional proteins in governing the transendothelial migration of leukocytes under normal or inflammatory conditions has been established.
This pathway describes some of the key interactions that assist in the process of platelet and leukocyte interaction with the endothelium, in response to injury. Activation of C3 and C5 (R-HSA-174577) 20.

Entities found in this pathway (34)
Cellular compartments: extracellular region, plasma membrane.
The 3 pathways of complement activation converge on the cleavage of C3 by C3 convertases. C3 convertase cleaves C3 into C3a and C3b -a central step of complement activation. C3a remains in the fluid phase and acts as an anaphylatoxin, whereas C3b can form additional C3 convertases hastening the production of C3b. Besides, C3b binds to C3 convertases to form C5 convertase, which can act as an opsonin, or is degraded into fragments which cannot form an active convertase. Terminal pathway of complement (R-HSA-166665) 21.

Entities found in this pathway (6)
After cleavage of C5, C5b undergoes conformational changes and exposes a binding site for C6.
C5b6 binds C7 resulting in the exposure of membrane binding sites and incorporation into target membranes. The membrane-bound C5b-7 complex can then bind C8. C5b-8 acts as a polymerizing agent for C9. The first C9 bound to C5b-8 undergoes major structural changes enabling formation of an elongated molecule and allows binding of additional C9 molecules and insertion of C9 cylinders into the target membrane. The number of C9 molecules varies from 1-12 in the membrane, although polymers containing up to fifteen C9 molecules are also possible. Post-translational modification: synthesis of GPI-anchored proteins (R-HSA-163125)

22.
Glycosylphosphatidyl inositol (GPI) acts as a membrane anchor for many cell surface proteins. GPI is synthesized in the endoplasmic reticulum. In humans, a single pathway consisting of eleven reactions appears to be responsible for the synthesis of the major GPI species involved in membrane protein anchoring.
As a nascent protein fated to become GPI-anchored moves into the lumen of the endoplasmic reticulum, it is attacked by a transamidase complex that cleaves it near its carboxy terminus and attaches an acylated GPI moiety. The GPI moiety is deacylated, yielding a protein-GPI conjugate that can be efficiently transported to the Golgi apparatus. Other semaphorin interactions (R-HSA-416700) 23.  Initial triggering of complement (R-HSA-166663) 24. Complement activation is due to a cascade of proteolytic steps, performed by serine protease domains in some of the components. Three different pathways of activation are distinguished triggered by target-bound antibody (the classical pathway); microbial polysaccharide structures (the lectin pathway); or recognition of other "foreign" surface structures (the alternative pathway) by C3b. All three merge in the pivotal activation of C3 and, subsequently, of C5 by highly specific enzymatic complexes, the so-called C3/C5 convertases. A complement system with three C3 activation pathways and a common lytic pathway is found only in jawed vertebrates.

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