Understanding the Alternative Pathway in Complement Activation
The complement system is part of the innate immunity and provides rapid protection against pathogens. Unlike other parts of the immune system, the complement system consists solely of proteins. These proteins participate in a tightly regulated series of proteolytic events that form an enzymatic pathway. It is driven by its three activation routes: the classical (CP), lectin (LP), and alternative pathway (AP). The AP stands out due to its spontaneous, antibody-independent activation. Unlike the CP, which requires antigen-antibody complexes, the AP is continuously active at a low level. This generates active C3 fragments that can rapidly respond to intruding by, for example, microorganisms. This allows the AP to serve as an immediate defence mechanism even before adaptive immunity is activated. In addition to protecting against pathogens the AP also plays a crucial role in clearing apoptotic cells, preventing biomaterial-induced inflammation, limiting thrombotic events, and participating in cancer immune surveillance. Given the broad involvement of the alternative pathway and its spontaneous activation in various physiological and pathological processes, it is important to examine its activation mechanism.
We are glad to support you!
Take advantage of our dedicated support team for any technical assistance you need while using our products or considering them for your research needs.
- Original supplier of innate immunity products since 1994.
- Our commitment to quality: Read more!
- Contact your local distributor for ordering.
- Contact our support team for more information.
Activation and Regulation Mechanisms of the Alternative Pathway
The AP is unique among the complement pathways in that it is always active at a very low level and, as such, is primed to respond rapidly to infection. The pathway is triggered by recognition of pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides from gram-negative bacteria, bacterial teichoic acids, fungal cell wall carbohydrates, and a variety of other structures. The low-level constitutive activation of the alternative pathway is known as ‘tickover’.
Activation of the AP begins with the spontaneous hydrolysis of the internal thioester bond in C3, producing C3(H2O) in the fluid phase. C3(H2O)binds to Factor B (FB), which is then cleaved by Factor D (FD), forming the fluid-phase C3 convertase (C3(H2O)Bb). This enzyme cleaves more C3 into C3b and C3a. Surface-bound C3b can bind additional Factor B, forming C3bBb complexes that amplify the response. Properdin (Factor P), the only known positive regulator of complement, binds to and stabilizes the C3bBb complexes, significantly prolonging its half-life. Unlike other pathways, the AP does not need specific recognition molecules but reacts to the general features of a surface. Activation of the complement system via the AP will therefore only proceed on surfaces that lack complement regulators. The alternative pathway functions as an amplification loop by continuously generating C3b once C3-convertases are formed on a surface. This loop can account for up to 80% of total complement activation, highlighting the central role of the AP, regardless of which pathway initiated the response. Eventually, the accumulation of C3b on the target surface leads to the formation of C5-convertases (either C4b2a3b or C3bBb3b), which cleave C5 into C5a and C5b, the latter initiating the terminal pathway.
The alternative pathway has to be tightly regulated because of its potential to rapidly amplify and target host tissue for C3b deposition and subsequent complement-mediated destruction. Factor H (FH) is one of the critical regulators of this pathway serving as a cofactor for the Factor I-mediated cleavage of C3(H2O) and C3b. FH is one of the most important negative regulators of the AP and a central member of the FH protein family, which includes its splice variant FHR-like 1 and six FH-related proteins (FHR-1, FHR-2, FHR-3, FHR-4A, FHR-4B and FHR-5). While the exact functions of the FHR proteins remain unclear, they are thought to act as natural antagonists of FH and modulate its activity. FH regulates the AP through three complementary mechanisms. First, it competes with FB for binding to C3(H2O) and C3b, thereby preventing the formation of the C3 convertases C3(H₂O)Bb and C3bBb. Second, FH exhibits decay-accelerating activity, destabilizing and disassembling existing convertases. Third, FH serves as a cofactor for factor I (FI), which cleaves C3b into its inactive form, iC3b, halting further activation. FI is a serine protease that requires cofactors such as FH or membrane-bound proteins to function. It plays a critical role in turning off complement activation once the threat has been contained.
There are also membrane-bound regulators. Membrane Cofactor Protein (MCP/CD46) acts as a cofactor for FI on the cell surface, while decay accelerating factor (DAF/CD55) disrupts C3 convertases to prevent excessive activation. Another key player in the AP is complement receptor 1 (CR1/CD35), it is found on red blood cells and phagocytes, combines both decay-accelerating and cofactor functions, contributing to immune complex clearance and self-protection.
A notable exception among AP regulators is properdin, which functions as a positive regulator. Properdin binds to the C3bBb complex and significantly prolongs its half-life, thereby enhancing the amplification of complement activation. Importantly, studies show that properdin does not initiate activation; instead, it binds only after initial C3b deposition. Properdin plays a supportive role in infection control. For example, β-glucans from Saccharomyces cerevisiae require properdin to achieve strong complement activation. Similarly, in models using zymosan or Escherichia coli, properdin enhances amplification but cannot independently trigger the pathway.
Together, these regulatory mechanisms ensure that the alternative pathway remains a rapid and powerful defence mechanism which is capable of eliminating threats efficiently, while maintaining control to protect host tissues. The dynamic balance between activation and inhibition is essential for effective immune function.
Consequences of Failed Alternative Pathway Regulation
Dysregulation of the AP leads to several pathologies. For example: Atypical hemolytic uremic syndrome (aHUS) results from uncontrolled complement activation on endothelial surfaces. In addition, C3 glomerulopathy (C3G) is driven by excessive C3 deposition in the kidney and Age-related macular degeneration (AMD) has also been linked to variations in complement genes, particularly those affecting regulation. For a more comprehensive list of alternative pathway-related diseases, please check the FAQ section. These diseases illustrate how delicate the balance is between protective immunity and pathological inflammation.
Defective function or regulation of Factor H (FH) is a critical contributor to these disorders. Mutations, deletions, or the presence of autoantibodies against FH disrupt its capacity to protect host tissues, leading to unrestrained complement activity. In aHUS, genetic mutations or anti-FH autoantibodies impair control over endothelial surfaces, triggering thrombotic microangiopathy. In C3G, abnormalities in the CFH-CFHR gene cluster, often involving hybrid gene formations or copy number variations, lead to persistent C3 convertase activity and renal injury.
Moreover, Factor H-related proteins (FHRs) can exacerbate disease by competing with FH for surface binding, tipping the balance toward complement activation. This competition is implicated not only in renal diseases but also in ocular disorders like AMD, where dysregulation of local complement control accelerates tissue degeneration.
Recent research has also shown the presence of anti-FH autoantibodies in broader autoimmune conditions and certain cancers, suggesting that complement dysregulation via FH disruption might have systemic consequences beyond the traditionally associated diseases. Understanding these mechanisms has highlighted FH and its family members as central players in complement-mediated pathologies and critical targets for emerging therapeutic strategies.
Dysregulation in Factor H, one of the most crucial complement regulators
FH is a key regulator of the complement system, having a crucial role in protecting human cells and tissues from complement-mediated damage. It controls the alternative pathway by inhibiting the formation and activity of the C3 convertase (C3bBb) through three main mechanisms. First, it accelerates the breakdown of existing C3bBb complexes (decay-accelerating activity). Second, it prevents the assembly of new C3bB complexes by competing with Factor B (FB) for binding to C3b. Third, and most arguably, FH acts as a cofactor for Factor I (FI), enabling the cleavage of C3b into its inactive form (iC3b), thereby halting further complement activation.
In addition to FH, there are also Factor H-related proteins (FHRs). They share structural similarities with FH, but generally lack cofactor or decay-accelerating activity. Instead, they can compete with FH for binding to C3b or cell surfaces, potentially reducing FH-mediated regulation. FH-related proteins also influence complement regulation, but in a more complex and sometimes opposing manner. Certain FHRs are considered to act as positive regulators of complement by promoting activation rather than inhibition. Their role is increasingly recognized in both host defence and the development of complement-driven diseases.
Dysregulation of FH function, whether by genetic mutation, autoantibodies, or altered FHR ratios, can have severe clinical consequences, leading to diseases such as atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and age-related macular degeneration (AMD). Given its pivotal role, FH is being explored both as a therapeutic target and a biomarker. Measuring FH and FHR levels in circulation is emerging as a powerful tool to improve diagnostics, predict disease progression, and monitor complement-targeted therapies. Thus, Factor H is not just a complement regulator; it is a critical checkpoint controlling the balance between protective immunity and pathological inflammation.
Emerging Clinical Applications and Associated Challenges in Measuring Alternative Pathway Markers
The increasing recognition of the AP in diverse pathological processes, from kidney diseases to Age-related macular degeneration and cardiovascular disorders, has growing interest in accurately measuring its activation. Clinical advances, such as C5 inhibitor eculizumab and the development of upstream AP-targeted drugs like FB and FD inhibitors in complement-targeted therapies underscore the importance of reliable detection of AP-specific markers, like Factor B, D and H, both for diagnostic and therapeutic monitoring purposes.
However, despite advances, several challenges remain. Activation of the complement pathway is highly sensitive and prone to dysregulation. In clinical practice, this sensitivity can be both beneficial and a challenge. Complement reacts quickly to infections, tissue injury, or immune dysregulation, making it a valuable tool for early diagnosis. Its rapid changes during disease progression or recovery also enable physicians to closely monitor treatment effects. Given its involvement in a wide spectrum of diseases, it is broadly applicable as a clinical indicator. However, complement can also activate artificially and therefore requires strict control of pre-analytical conditions to ensure accurate measurement. Clinicians must be able to rely on consistent and robust measurements to make the right conclusions, whether for diagnostic evaluation, monitoring treatment efficacy or assessing disease severity.
Three critical factors for accurate complement analysis are: correct sample handling, standardized methodologies, and high analytical sensitivity and specificity.
Correct sample handling is essential, as the complement system is easily triggered. Blood drawn or processed incorrectly or inaccurate procedures, such as incorrect anticoagulants, may lead to spurious activation or degradation of components. Concerning, as Brandwijk et al. showed, nearly half of published studies did not use the proper sample type or handling method. [1] That is a serious problem if clinical decisions depend on the outcome.
Optimal sample handling guidelines:
- Use EDTA-plasma when measuring activation products like C3a, C5a, or sTCC;
- Keep samples on ice;
- Process within 1 hour;
- Store at -80°C;
- Avoid freeze-thaw cycles.
Moreover, standardization is still lacking. Currently, there are no universally applied protocols for complement measurement. Differences in antibodies, reagents, calibration standards, and assay platforms result in significant variability between labs, limiting the comparability of data. For diagnostics to be clinically meaningful and comparable, standardized assays and handling protocols must become the norm. This is especially urgent as more complement-targeted therapies move into clinical use.
Sensitivity and specificity are also very critical for reliable complement measurement. Complement activation is complex, involving multiple overlapping pathways that can trigger a range of downstream effects. To be clinically useful, assays must have both high sensitivity and high specificity. Sensitivity refers to the ability of a test to detect even very low levels of a specific complement protein or activation fragment. Specificity refers to the ability to detect the correct complement component of a fragment without cross-reactivity to other related proteins.
Without sufficient sensitivity and specificity, complement measurements can easily be misleading, especially given the structural similarities between full protein and their activation products. At Hycult Biotech, we specifically address these challenges. Our assays and antibodies are developed to deliver reliable and reproducible complement protein measurement. We ensure low batch-to-batch variability, have thoroughly validated the specificity of our antibodies and assays, and provide standardized protocols for assay performance. In several cases, we use antibodies that recognize neo-epitopes. Neo-epitopes are specific sites that become exposed only after cleavage of the complement protein. This approach maximizes both sensitivity and specificity, ensuring that our assays detect exactly the right complement molecule.
The expanding role of complement in human pathology continues to innovative therapeutic development across diverse clinical areas. While early successes have validated complement as a druggable system, continued efforts are needed to address the challenges.
Related products
Find out our relevant products for alternative pathway research.
C3d, Human, ELISA kit
Properdin, Human, ELISA kit
Complement factor D, Human, ELISA kit
C3c, Human, ELISA kit
C3, Mouse, mAb 11H9
C3b/iC3b/C3c, Mouse, mAb 2/11
Contact us for more information
At Hycult Biotech, we recognize the growing demand for advanced complement research tools that facilitate accurate analysis and targeted intervention in immune-related diseases. Our portfolio includes high-quality complement pathway inhibitors (how to inactivate complement), complement assay kits, and monoclonal antibodies designed to support drug discovery and translational research. On our website, you can search for products that match your research needs. Additionally, we are happy to provide expert advice. Feel free to contact us via the contact form.
Frequently asked questions
The AP is activated spontaneously through a mechanism known as “C3 tick-over.” In this process, native C3 undergoes continuous low-level hydrolysis, forming C3(H₂O), which can bind FB. Once bound, FB is cleaved by FD into Factor Bb and Ba, generating the fluid-phase C3 convertase C3(H₂O)Bb. This enzyme cleaves additional C3 into C3a and C3b. C3b can then bind to microbial or host cells, forming the surface-bound C3 convertase C3bBb. In addition, Properdin stabilizes this convertase, enhancing its activity.
The AP plays a dual role within the complement system both as an initiator and an amplifier of complement activation. It is continuously active at a low level via spontaneous hydrolysis of C3, allowing for immediate innate immune response in the absence of specific recognition molecules. Once triggered, the AP amplifies complement activation by forming the C3bBb convertase, which rapidly generates large amounts of C3b. Once C3b is generated, either via tick-over or through activation by the CP or LP, C3b can bind to target surfaces and initiate the formation of the AP-specific C3 convertase (C3bBb). This marks the transition to the amplification loop, in which the AP enhances complement activation by generating large amounts of additional C3b. Thereby, the AP not only initiates its own activation but also amplifies responses triggered by the CP and LP.
The AP differs from the classical and lectin pathways predominantly in how it is activated. While the classical pathway needs immune complexes and the lectin pathway depends on recognition of microbial carbohydrates, the AP activates spontaneously through a process called “C3 tick-over.” This involves continuous low-level hydrolysis of C3, leading to the formation of C3(H₂O), which can initiate complement activation without engagement of a recognition molecule.
Unlike the classical and lectin pathways, the AP is at a basal level active at a low level, serving as a constant surveillance mechanism of the innate immune system. This baseline activity allows the immune system to respond rapidly to pathogens or altered self-surfaces. The AP does not need recognition molecules, making it part of the innate immune system’s immediate defence.
Properdin functions as a positive regulator in the AP by stabilizing the C3 convertase complexes C3bBb and thereby enhancing the amplification of the complement response. Properdin binds to surface-bound C3bBb and extends the half-life of the convertase, supporting continued C3 activation. It also helps direct complement activation to pathogen surfaces by selectively binding to them, contributing to immune defence.
FH is one of the most important regulators of the AP, essential for protecting host cells from unintended complement attack. It inhibits the AP at multiple levels: by accelerating the decay of the C3 convertase (C3bBb), by competing with FB for C3b binding, and by acting as a cofactor for FI to cleave C3b into inactive iC3b. These actions help prevent excessive complement activation in the fluid phase and on host surfaces.
In contrast, the FHRs lack regulatory functions like decay acceleration or cofactor activity but can compete with FH for binding sites on C3b and cellular surfaces. This competition can reduce FH-mediated control and promote complement activation, which is why FHRs are sometimes referred to as FH deregulators. Their function appears to support a more positive regulation of the AP, and they are implicated in modulating immune responses and contributing to complement-related diseases.
The AP contributes to disease pathogenesis by providing a constant state of low-level spontaneous activation, which, if not properly regulated, can lead to uncontrolled complement activation. This dysregulation plays a key role in many diseases not driven by autoantibodies, including inflammatory, autoimmune, and kidney disorders. Genetic variants in AP-related genes have been found in patients with these diseases, highlighting the pathway’s role in both health and pathology. The AP does not require a recognition molecule, making its activation dependent on the balance of local regulators and surface context.
Dysfunction of the alternative pathway is linked to several diseases, where its dysregulated activation drives or amplifies pathology. The following diseases are associated with the dysfunction of the alternative pathway:
- C3 glomerulopathy (C3G): Characterized by excessive deposition of C3 fragments in the glomeruli due to impaired regulation of the alternative pathway. This is often driven by mutations or autoantibodies that stabilize the C3 convertase or inhibit regulatory proteins like factor H. [2]
- Atypical hemolytic uremic syndrome (aHUS): Condition where defective control of the alternative pathway leads to complement-mediated injury of the microvascular endothelium, especially in the kidneys. Mutations in complement regulators (e.g., factor H, factor I, MCP) or presence of anti-factor H autoantibodies are often involved. [2][3]
- IgA nephropathy: In this common glomerulonephritis, secondary activation of the alternative pathway contributes to disease progression. Elevated FHR1 and FHR5 proteins promote C3 activation, enhancing glomerular injury. [2]
- Lupus nephritis: Although classically linked to the classical pathway, studies show that the alternative pathway amplifies complement activation and tissue damage in lupus, particularly in mouse models deficient in factor B or D, which show protection from renal injury. [3]
- Cardiovascular disease (CVD): In patients with chronic kidney disease (CKD), increased alternative pathway activation markers like factor D and Factor Ba correlate with endothelial dysfunction and elevated CVD risk. This suggests the pathway contributes to vascular inflammation and atherosclerosis. [2]
Age-related macular degeneration (AMD): Strongly associated with polymorphisms in the CFH gene, which impair regulation of the alternative pathway at the retinal pigment epithelium, leading to complement-mediated retinal damage. [2][3]