MOD Flashcards
What are the considerations for cell growth?
• Growth of a population of cells
- Distinguish between increase in cell numbers (hyperplasia) and increase in cell size (hypertrophy)
- Depends on integration of intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
¥ Growth at the cellular level (the cell cycle)
Ð Cell growth = increase in size (sometimes growth refers to this only) and cell division
Ð Cell cycle phases (G1, S, G2, and M)
Ð Progression controlled at three key checkpoints (restriction points)
¥ Loss of cells by programmed cell death (apoptosis)
Ð A coordinated program of cell dismantling ending in phagocytosis. Distinct from necrosis
Ð Occurs during normal development (e.g. separation of the digits, involution, immune and nervous system development)
Ð And in response to DNA damage and viral infection
What are growth factors, cytokines and interleukins?
¥ Proteins that:
Ð stimulate proliferation (called mitogens) and maintain survival
¥ usually named after originally identified target e.g. EGF, FGF, Interleukins (IL2 & IL4), NGF
¥ but see also PDGF (platelet-derived GF) and IGF1 (Insulin-like GF – the main effector of pituitary growth hormone)
Ð stimulate differentiation and inhibit proliferation e.g. TGF
Ð induce apoptosis e.g. TNFα and other members of the TNF family
What are the three broad classes of growth factors, cytokines and interleukins?
Ð Paracrine: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
Ð Autocrine: produced by a cell that also expresses the appropriate cell surface receptor
Ð Endocrine: like conventional hormones, released systemically for distant effects
What happens in DNA replication?
- DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand)
- New DNA is synthesized in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors at a replication fork by a multienzyme complex (a replication machine)
- Fidelity is determined by base pairing (A=T, G≡C) and presence of a proof reading enzyme in DNA polymerase
- Synthesis of the new DNA strand uses an RNA primer and occurs continuously on the leading strand and discontinuously on the trailing strand (giving rise to Okazaki fragments, which are ligated together after removal of the RNA primer)
What are the main stages of mitosis?
¥ Prophase (1) Ð Nucleus becomes less definite Ð Microtubular spindle apparatus assembles Ð Centrioles (yellow) migrate to poles ¥ Prometaphase Ð Nuclear membrane breaks down Ð Kinetochores attach to spindle in nuclear region ¥ Metaphase (2) Ð Chromosomes (blue) align in equatorial plane ¥ Anaphase (3) Ð Chromatids separate and migrate to opposite poles ¥ Telophase (4) Ð Daughter nuclei form ¥ Cytokinesis Ð Division of cytoplasm Ð Chromosomes decondense
What drugs act on S-phase of the cell cycle?
• S-Phase active
- 5-Fluorouracil (an analogue of thymidine blocks thymidylate synthesis).
- Bromodeoxyuridine (another analogue that may be incorporated into DNA and detected by antibodies to identify cells that have passed through the S-phase).
What drugs act on M-phase of the cell cycle?
Ð Colchicine (stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis – used in karyotype analysis)
Ð Vinca alkaloids (similar action to colchicine)
Ð Paclitaxel (Taxol, stabilizes microtubules, preventing de-polymerization)
5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer
What are cell cycle check points?
Controls (involving specific protein kinases and phosphatases) ensure the strict alternation of mitosis and DNA replication
What is the regulation of cyclin-CDK activity?
¥ Cyclical synthesis (gene expression) and destruction (by proteasome).
¥ Post translational modification by phosphorylation – depending on modification site may result in activation, inhibition or destruction
¥ Dephosphorylation
¥ Binding of cyclin-dependent kinase inhibitors
What is the retinoblastoma protein?
A key substrate of G1 and G1/S cyclin-dependent kinases
- Unphosphorylated RB binds E2F preventing its stimulation of S-phase protein expression
- Cyclin D-CDK4 and cyclin E-CDK2
- Released E2F stimulates expression of more cyclin E and S-phase proteins eg DNA polymerase, thymidine kinase, PCNA etc. DNA replication starts.
What are the two families of cyclin-dependent kinase inhibitors (CKIs)?
- CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)
¥ Expression of members of this family stimulated weakly by TGF and strongly by DNA damage (involving TP53)
¥ Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
¥ Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs - Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
¥ Expression stimulated by TGF
¥ Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)
What is the sequence of events triggered by growth factors?
¥ Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)
¥ Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
¥ E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
¥ G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F
¥ E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.
What are the options after DNA damage is detected at checkpoints?
- stop the cycle (CDK inhibitors)
- attempt DNA repair (nucleotide or base excision enzymes)
- programmed cell death if repair impossible (BCL2 family, caspases)
What are the causes and mechanisms of cell damage/cell death?
- genetic
- inflammation
- physical
- traumatic damage
- infection
- chemical
What are the genetic causes of cell damage/death?
- Abnormal number chromosomes (aneuploidy)
- Abnormal chromosomes (deletions/translocations)
- Increased fragility (Fanconi’s anaemia)
- Failure of repair (Xeroderma pigmentosa)
- Inborn errors (storage disorders ie Tay Sachs disease)
What are the inflammation causes of cell damage/death?
- trauma
- thromboembolism
- atherosclerosis
- vasculitis
What are the physical causes of cell damage/death?
- irradiation
- heat
- cold
- barotrauma
What are the traumatic damage causes of cell damage/death?
- Interruption of blood supply
- Direct rupture of cells
- Entry of foreign agents
What are the infection causes of cell damage/death?
- Toxic agents
- Competition for nutrients
- Intracellular replication – viruses/mycobacteria provoking an immune response
What are the chemical causes of cell damage/death?
- Acids/corrosives
- Specific actions eg enzymes
- Interference with metabolism eg alcohol
What is necrosis?
most common cause of cell death. Occurs after stresses such as ischemia, trauma, chemical injury
- Whole groups of cells are affected
- Result of an injurious agent or event
- Reversible events proceed irreversible
- Energy deprivation causes changes. (e.g. cells unable to produce ATP because of oxygen deprivation)
- Cells swell due to influx of water (ATP is required for ion pumps to work).
- Haphazard destruction of organelles and nuclear material by enzymes from ruptured lysosomes.
- Cellular debris stimulates an inflammatory cell response
What is apoptosis?
programmed cell death. Designed to eliminate unwanted host cells through activation of a co-ordinated, internally programmed series of events effected by a dedicated set of gene products
What is autophagic cell death?
Autophagy is responsible for the degradation of normal proteins involved in cellular remodeling found during metamorphosis, aging and differentiation as well as for the digestion and removal of abnormal proteins that would otherwise accumulate following toxin exposure, cancer, or disease. An example is the death of breast cancer cells induced by Tamoxifen.
What are causes of necrosis?
- Usually caused by lack of blood supply to cells or tissues eg
- Injury
- Infection
- Cancer
- Infarction
- Inflammation
What are nuclear changes in necrosis?
- Pyknosis: Chromatin condensation/shrinkage.
- Karyorrhexis: Fragmentation of nucleus.
- Karyolysis: Dissolution of the chromatin by DNase causing a fading in
the basophillia of the chromatin.
What are cytoplasmic changes in necrosis?
- Opacification: denaturation of proteins with aggregation.
- Eosinophillia: exposure of basic amino groups.
- Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis).
What biochemical changes occur in necrosis?
- Release of enzymes such as creatine kinase or lactate dehydrogenase
- Release of proteins such as myoglobin
What clinical investigations are associated with cell death?
- Muscular dystrophy. Damaged muscles release creatine kinase and lactate dehydrogenase (M3 and M3H isoforms).
- Heart attack. Damaged muscle cells release lactate dehydrogenase (H3 and H3M isoforms).
- Bone and liver disease. Damaged tissues release alkaline phosphatase and lactate dehydrogenase isoforms (different isoforms specific to various tissues).
- Haemolytic anaemias. Damaged red cells release LDH1/2.
What are the types of necrosis?
- Coagulative necrosis - typically seen in hypoxic environments. Cell outlines remain after cell death and can be observed by light microscopy (e.g. myocardial infarction, infarct of the spleen)
- Liquefactive necrosis - is associated with cellular destruction and pus formation (e.g. pneumonia)
- Caseous necrosis - is a mix of coagulative necrosis and liquefactive necrosis (e.g. tuberculosis)
- Fatty necrosis - results from the action of lipases on fatty tissues (e.g. acute pancreatitis)
- Fibrinoid necrosis - caused by immune-mediated vascular damage. It is marked by deposition of fibrin-like proteinaceous material in arterial walls, which appears smudgy and acidophilic on light microscopy.
What are the functions of necrosis?
- Removes damaged cells from an organism
- Failure to do so may lead to chronic inflammation
What are the functions of apoptosis?
¥ Selective process for the deletion of superfluous, infected or transformed cells. ¥ Involved in:- ¥ Embryogenesis ¥ Metamorphosis ¥ Normal tissue turnover ¥ Endocrine-dependent tissue atrophy ¥ A variety of pathological conditions
What are examples of apoptosis?
- Cell death in embryonic hand to form individual fingers
- Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF)
- DNA damage-mediated apoptosis. If DNA is damaged due to radiation or chemo therapeutic agents, p53 (tumour suppressor gene product) accumulates. This arrests that cell cycle enabling the cell to repair the damage. If the repair process fails, p53 triggers apoptosis.
- Cell death in tumours causing regression
- Cell death in ciral disease (eg viral hepatitis)
- Cell death induced by cytotoxic T cells (ie cellular immune rejection or graft vs host disease)
- Death of neutrophils during an acute inflammatory response
- Death of immune cells (both T and B lymphocytes) after depletion of cytokines as well of death of autoreactive T cells in the developing thymus.
Apoptosis helps eliminate the tail during the metamorphosis of a tadpole into a frog.
What factors influence the balance of life and death at the cellular level?
- cell-cell and/or cell-metric contacts
- growth factors
- cytokines
- disruption of cell-cell and/or cell-matrix contacts
- lack of growth factors
- death domain ligands
- DNA damaging agents
What are the types of apoptosis?
- DNA damage - p53-dependent pathway
- interruption of the cell cycle
- inhibition of the protein synthesis
- viral infection
- change in redox state
- withdrawal of growth factors (e.g. IL-3)
- extracellular signals (eg TNF)
- T cell or NK (natural killer)
What are caspases?
Cysteine Aspartate-specific proteases
- Caspases are cysteine proteases that play a central role in the initiation of apoptosis
- Most proteases are synthesised as inactive precursors requiring activation (usually partial digestion by another protease)
What does caspase activation lead to?
Caspase activation leads to characteristic morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.
What happens in apoptosis?
- Single or few cells selected.
- Programmed cell death.
- Irreversible once initiated.
- Events are energy driven.
- Cells shrink as the cytoskeleton is disassembled.
- Orderly packaging of organelles and nuclear fragments in membrane bound vesicles.
- New molecules expressed on vesicle membranes stimulate phagocytosis, no inflammatory response
What nuclear changes occur in apoptosis?
- Nuclear chromatin condenses on nuclear membrane
2. DNA cleavage
What cytoplasmic changes occur in apoptosis?
- Shrinkage of cell. Organelles packaged into membrane of vesicles.
- Cell fragmentation, membrane bound vesicles bud off.
- Phagocytosis of cell fragments by macrophage and adjacent cell.
No leakage of cytosolic components
What biochemical changes occur in apoptosis?
- Expression of charged sugar molecules on outer and inner surface of membranes (recognised by macrophage and enhances phagocytosis)
- Expression of phosphatidylserine on extracellular leaflet of apoptotic cell
- Protein cleavage by proteases, caspases
How do we activate the initiator caspases?
By induced proximity.
For example:
- In response to receptor dimerization upon ligand binding or
- Cytochrome C release from the mitochondria
What is cytochrome C?
- Mitochondrial matrix protein
- Known for many years to be released in response to oxidative stress by a ‘permeability transition’
- Any inducers of the permeability transition also eventually induce apoptosis
How is the release of cytochrome C regulated?
- Bcl-2 is a member a multi-gene family in mammals
- anti-apoptotic bcl-2, bcl-XL, others
- pro-apoptotic Bax, Bid, Bad, others
The bcl-2 family members form dimers
What is the link between p53 and apoptosis?
Mutations in the p53 gene are the most common mutations in cancer. Some mutations destroy the ability of p53 to induce apoptosis
What is asymptomatic carriage?
carrying an organism that can be passed on to others, but without experiencing any symptoms.
What is microbial antagonism?
- Balanced ecological niche – good health
- Bacterial overgrowth eg caused by antibiotics
The removal of the normal flora of gut through antibiotics which can cause Pseudomembraneous colitis by the overgrowth as C.difficile. Damages and disrupts the defence mechanism.
What are the general concepts of bacterial pathogenicity?
- host susceptibility and defences
- bacterial virulence
- host-mediated pathogenesis
- intracellular growth
- asymptomatic infections and carriage
What is host susceptibility and defences?
- Innate and adaptive immunity; immune-statu – young, old, HIV positive genetics
- Barriers; antimicrobial peptides; turnover; iron binding proteins
- Signalling, recruitment and inflammation
What is bacterial virulence?
- Capacity to cause disease – virulent strains eg of E.coli
- Genes: chromosomal, plasmids, transposons, bacteriophages, genetic exchange, evolution
- Capacity to cause disease has to be encoded in its genome – virulence factors
What are Koch’s postulates?
Discovered that microorganisms are responsible for causing disease.
A microorganism has to:
• Be present in every case of the infection
• Be cultured from cases in vitro
• Reproduce disease in an animal
• Be isolated from the infected animal
• Not possible for non-culturable organisms eg leprosy, syphilis
• Need molecular tests eg PCR – eg HepC
• What about food poisoning – B.cereus and toxins – no infection with organism, but disease
What types of infection are there?
- Local: surface infection eg from a small wound on the skin – V.cholera, N.gonorrhoeae
- Invasive: penetrate barriers spread, wound – shigella, staph aureus
- Systemic: via blood, lymph to other sites – S.typhi, N.meningitidis
Effects at different site from colonisation – toxins, endotoxins
What are the stages of infection?
- Acquisition
- Colonisation – adherence
- Penetration
- Multiplication and spread
- Immune evasion
- Damage
- Transmission – shedding
- Resolution
Not all microbes need all stages
Disease not always required for transmission – asymptomatic shedding.
What are virulence factors?
- adherence factors
- invasion factors
- capsules
- endotoxins
- exotoxins
- siderophores
What are adherence factors?
colonise mucosal sites by using pills (fimbrae) to adhere to cells
What are invasion factors?
surface components and secreted effector proteins
What are capsules?
polysaccharides - protect from opsonisation and phagocytosis
What are endotoxins?
lipopolysaccharide on gram negatives - cause fever, changes in blood pressure, inflammation, lethal shock
What are exotoxins?
protein toxins and enzymes produced and/or secreted e.g. cytotoxins, neurotoxins, and enterotoxins
What are siderophores?
iron-binding factors to compete with the host for iron haemoglobin, transferring and lactoferrin
How do bacteria attach to host surfaces?
- Complex molecular interactions
- projecting fibrils on surface
- other membrane surface molecules expressed by bacteria
What are bacterial toxins?
‘cause damage to cells, tissues or the whole host organisms, thereby contributing to disease’
Exotoxin – released proteins found in Gram positive and gram negative
Endotoxins – powerful immunostimulants, portion of LPS found in gram negative cell walls, (also lipotheicoic acids of gram positive)
Toxins – often described by resultant action on different cells
- Cytotoxins
- Enterotoxins
- Neurotoxins
- Leukocidins
- Ciliostatic toxins
What is the functions of toxins in infection?
- Promote adhesion, survival or spread of bacterial – hyaluronidase, collagenases
- Damage or destroy cells cell membranes – phospholipases; pores
- Interfere with cell metabolism – cholera; diphtheria
- Affects nerves – neurotoxins – botulism and tetanus
Survival, growth and transmission?
How can toxins be classified by site of action?
- TYPE I – at cell membrane, not transported in
- TYPE II – on cell membrane, membrane damage
- TYPE III – intracellular effect after translocation
- Extracellular – cellular matrix or connective tissue
Exotoxins – make pores in molecules, stop signalling, bind to receptors being internalised and undermined metabolic potential of the cell, direct entry of toxin via a receptor to be released inside cell and then has an effect on a target inside the cell, or can be injected into the cell via injectosomes and the toxin then has a pharmacological action. A range of pathogens have a range of toxins that can do one or more of these mechanisms.
How do type I toxins act?
stimulates signalling proteins, guanyl cyclase (GC) changes intracellular cGMP
eg E.coli ST enterotoxin – Travellers diarhhoea
Short polypeptides of about 19 aa – it is heat stable because it cant be destroyed through cooking etc. Binds to and inhibiting a GC protein which normally responds to other signals, but modifying cGMP causes disruption of chloride channels, causing water efflux and acute diarrhoea.
What are lysins?
Lysins are toxins that cause membrane damage by forming pores etc. Pneumonia, food poisoning, infants, etc – there is a whole range of pathogens with virulence factors that work like this.
How can the same pathogen cause different types of diseases?
because they can bind to different types of cell
How can neurotoxins work?
Clostridium tetani and tetanus
Blocks synaptic transmission of things such as glycine, causing muscular spasm.
The tetanus toxin targets synaptobrevin which is part of the presynaptic vesicles. If you release the toxin it blocks the release of inhibitory neurones of the neurotransmitters, causing over excitation giving spastic paralysis.
Clostridium botulinum and botulism
Also used in botox. The toxin produced is also a zinc dependent endopeptidase, the binding component takes it to the neuromuscular junction synapse, blocking the release of acetyl choline. You get flaccid paralysis because of where it has gone to, even though it is a similar metabolic action as previous example – but the binding activity affects the results.
About Whooping cough…
Bordetella pertussis
- Invasive adenylate cyclase – injects its own AC to disrupt cAMP levels inside cells.
- Lethal toxin (dermonecrotic toxin) – superantigen – tracheal cytotoxin
- Pertussis toxin, PTx – changes cAMP but also acts as an adhesive molecule – multifunctional
Allows bacteria to adhere to the cilia allowing it to grow and bring more pathogens.
What is the complement system?
Originally described as a heat-sensitive component of serum that could augment the ability of antibiotics to inactivate antigen.
A group of plasma proteins; normally inactive
- More than 30 plasma and cell surface proteins
- 9 central components of the complement cascade
- complement component 1 (C1) to C9
Triggers activation of complement pathways
- part of both innate immunity and humoural immunity
Complement activation – purpose
- generation of products that mediate effector functions of complement
What are the rules of the complement system?
Complement activation involves a cascade of enzymatic cleavage of complement proteins (proteolytic cascade).
Amplification (each activated protease can generate multiple activated proteases in the following step)
Products of complement proteolysis attach covalently to microbial surfaces (or Abs bound to microbes/other Ags)
Stabilisation (complement proteins are inactive/transiently active when in fluid form; stable activation when deposited on microbes)
Regulatory proteins inhibit complement activation on healthy host cells; absent from microbes.
‘Specificity’ (complement activation on microbial surfaces whilst minimising complement-mediated damage to host cells)
What are the roles of the complement system?
To eliminate microbes
- opsonisation of pathogens (facilitates phagocytosis)
- inflammation (recruitment and activation of leucocytes)
- Lysis of microbes (Membrane Attack Complex, MAC)
- To eliminate apoptotic cells/debris
- To promote clearance of Ag:Ab (immune) complexes
- To promote B cell activation
Complement is involved in disease pathogens.
What is the classical pathway of complement?
Classical pathway:
- Discovered first
- C1 interacts with antibodies (IgM, IgG) bound to microbes
- Effector mechanism of humoural (adaptive) immunity
- Initiated by binding of C1 to antigen-bound IgG or IgM
- C1 – multimeric protein complex of C1q, C1r or C1s
- C1q binds to antibodies (IgM, IgG1, IgG3)
- C1r and C1s are proteases
- C1 does not bind to soluble (free) antibody molecues
- C1 binds only to antibodies that are bound to Ag
What is the alternative pathway of complement?
Alternative pathway:
- Discovered later (but phylogenetically older)
- Direct recognition of microbial structures (innate immunity)
• Spontaneous cleavage of C3 in plasma (C3 tickover)
• C3 contains a reactive thioester bond (hidden)
• C3 cleavage induces conformational change in C3b, which exposes the thioester bond
• Exposed thioester bond reacts with amino or hydroxyl groups on surface of microbes to form ester bonds covalent attachment of C3b to surface of microbes/cells
In the absence of covalent attachment C3b remains in fluid phase, rapidly inactivated by hydrolysis further complement activation is stopped
What is the lectin pathway of complement?
Lectin pathway:
- MBL (mannose binding lectin) recognise terminal mannose residues on microbes (innate immunity)
- Ficolin recognises residues on microbes (innate)
What are the similar consequences for the different complement pathways?
Similar consequences:
- Generation of proteolytic enzymatic complexes
- C3 convertase: cleaves C3 in C3a and C3b
- C5 convertase: cleaves C5 in C5a and C5b
Proteolytic products of complement proteins identifies by lower case letter suffixes, ‘a’ for the smaller product; ‘b’ for the larger product.
C3 cleavage is crucial for all complement functions.
What does C3 converts do in the alternative pathway?
- Covalently tethered C3b binds Factor B
- Bound Factor B is cleaved by Factor D (plasma protease)
- Generates Bb (large) and Ba (small) fragments
- Bb remains attached to C3b
- C3bBb complex is the alternative pathway C3 convertase
C3 convertase has the main function to cleave more C3 molecules amplification of complement activation
• Newly generated C3b deposits on microbial surface
• C3a is a soluble fragment; mediates biological activities
What are the late steps of the alternative pathway?
- C5 convertase cleaves C5 in C5b (large) and C5a (small)
- C5a is a soluble fragment; has several biological activities
- C5b remains bound to C5 convertase
- C5v recruits C6 and C7
- C7 is hydrophobic inserts into lipid membranes
- C7 recruits C8 (three chains; one inserts into membrane)
- C5b-C8 complex recruits C9 C9 polymerases pores
- C5b-C9 is called MAC (Membrane Attack Complex)
- Lysis of microbe by MAC; entry of H2O via C9 pores
What is the C1 complement component?
- C1q hexamer: 6 globular heads connected via collagen-like arms to central stalk (‘brunch of tulips’)
- C1r (x2) and C1s (x2) form a tetramer
What happens with C3 convertase in the classical pathway?
- C3 convertase cleaves more C3 molecules
- Newly generated C3b deposits on microbial surface
- C3a is a soluble fragment; mediates biological activities
- Some C3b binds to C3 convertase complex C4b2a3b
- C4b2a3b is the classical pathway C5 convertase
- C5 convertase cleaves C5 and initiates late steps of complement activation
What is the complement activation in the lectin pathway?
- Initiated in absence of antibodies
- Triggered by microbial carbohydrate recognition by PRRs
- PRRs involved: MBL (mannose/mannan binding lectin), ficolins, similar structure to C1q
- MBL binds to mannose residues on microorganisms
- Ficolin binds to N-acetylglucosamine
- binding of MBL to mannose residues on microorganisms
- binding of ficolin to N-acetylglucosamine residues on microbes
- activates of MBL-Associated Serine Proteases (MASP1 and MASP2)
- MASPs cleave C4 and then C2
- Then proceeds similarly to classical pathway
