Week 3B: Protein metabolism, amino acid catabolism and blood clotting Flashcards
-HC -WC -Chapter 7.4-7.5 -Chapter 23
HC19: Regulation of proteins?
-Transcriptional regulation
-Synthesize proteins as inactive pro-enzymes (zymogens)
-Isozymes
-Post translational modification like phosphorylation
-Allosteric regulation
Which two amino acids have a higher concntration in blood than a low standard level?
Alanine and glutamine
Proteins to energy
Degradation dietary or cellular proteins to amino acids. Degradation amino acids to carbon skeletons to acetyl-CoA (metabolic fuel) or TCA intermediates (gluconeogenesis). Amino group excreted through conversion to urea
The nitrogens of amino acids can also be used, for which molecules?
Purines, pyrimidines and nicotinic acid (NAD+) > bases
What happens with excess amino acids?
Breakdown for fuel (acetyl-CoA) or gluconeogenesis (TCA intermediates) and urea for amino group excretion.
Is there a storage of amino acids like for glycogen and TAGs?
No
Essential amino acids
-Histidine
-Isoleucine
-Leucine
-Lysine
-Methionine
-Phenylalanine
-Threonine
-Tryptophan
-Valine
Degradation dietary proteins
Proteolytic enzymes in the stomach and intestine
Danger proteolytic enzymes
Break down enzymes and cells when active in the cell freely
> synthesized and secreted as zymogens.
> majority of pancreas makes zymogens for peptidases to prevent degradation of the pancreas, liver and gallbladder.
Import proteins uptake
Proteins in intestine > amino acids and oligopeptides
> import amino acids, aminopeptidase on border intestinal cell breaks down oligopeptides to tri/dipeptides and amino acids, transported in by different transporters.
> In the cell, the tri/dipeptides are broken down by peptidases (in lysosome?) and all amino acids go to blood.
Where are the pro-peptidases activated?
In the lumen of the stomach and intestines
Zymogens for peptidases are secreted through
ER-Golgi excretion route with zymogen granules (vesicles)
Which duct connects the duodenum to the exocrine pancreas?
Ductus pancreaticus
Gastric zymogen?
Pepsinogen
> active enzyme: pepsin
Characteristic pepsinogen
Amino terminal part hangs in subtrate binding site as inhibitor.
> Low pH in stomach activates pepsinogen through conformational change
Enteropeptidase can cleave peptide bonds and thereby activate other zymogens. Explain.
Enteropeptidase activates trypsinogen to trypsin.
Trypsin induces:
-Trypsinogen > Trypsin (positive feedback)
-Chymotrypsinogen > Trypsin
-Proelastase > elastase
-Procarboxypeptidase > Carboxypeptidase
-Prolipase > Lipase
Where is the trypsin cascade taking place?
Duodenum lumen
Which layer protect the intestine lining from the proteolytic enzymes?
Thick lining of carbohydrates (mucin)
What happens to the dietary amino acids released to the blood?
Taken up by other tissues
Proteins have a … half-life
Variable
Relative half-life signaling proteins
Short, for quick inactivation of the signal
Short lived proteins
Regulation proteins like cyclins and metabolic regulation proteins
Long lived proteins
Hemoglobin (t1/2: 4 months), crystallin (eye)
What happens to misfolded and damaged proteins?
Mistakes in translation lead to misfolding and oxidative damage could also lead to damage. These are removed
Aggregation of proteins can lead to a neurodegenerative disease namely
Huntingtons Disease
Does every cell have the same systems for cellular proteolysis (used degradation systems always similar?)
No, depends on cell type, kind of macromolecules and specific cellular conditions
Two systems of cellular protein degradation
Autophagic-lysosomal system and ubiquitination proteasome system
When is the autophagic-lysosomal system active
Always, and upregulated in starvation or prolonged muscle labor
What does the autophagy system take up?
Whole proteins and organelles
Process autophagy
In an ER exit site, an omegasome is formed (Omega form bud), it grows to a phagophore and captures proteins and organelles.
> enclosing of the vesicle and release as autophagosome.
> Fusion with lysosome
> Breakdown of content
> Efflux of building blocks to cytosol
How are the autophagosome and lysosome fused?
Via a double membrane safe fusion so that no dangerous enzymes can leave
pH in cytosol and lysosome
Cytosol: 7.2
Lysosome 5.0: acidic
> active H+ influx into lysosome with H+ pump
Acid hydrolases in the lysosome
Nucleases, proteases, glycosidases, lipases etc
Function ubiquitin in proteolysis
Marking proteins for degradation
> Ub is a conseverd protein present in all eukaryotes
Connection Ub to target protein
Covalent attachment C-terminal glycine residue of Ub to epsilon-amino group of a lysine residue of target protein
Bond between Ub and target protein
Isopeptide bond
HN-C(=O)-Ub on the epsilon carbon of the side chain (alpha is the backbone central carbon)
Ubiquitination of a protein
1: Activation Ub by E1: Ub-activating enzyme
2: Transfer Ub to E1
3: Tranfer Ub to E2 (Ub conjugating enzyme)
4: E3 (Ub-ligase) binds E2-Ub complex and target protein, brings them in proximity and catalyzes the ligation.
Activation step ubiquitination
Adenylation of of Ub
> Adding AMP to the terminal glycine C-end
> Costs 2 ATP (regeneration AMP to ATP from ATP)
> Ub-adenylate intermediate (energy needed to make thioester bond)
> just like fatty acid activation for breakdown (by acyl-CoA synthetase)
After activation Ub >
Transfer Ub to E1 with a thioester bond (C(=O)-S-)
Which amino acid residue can be used for a thioester bond in ubiquitination?
Cysteine
Ub is transferred from E1 to E2. How is Ub connected to E2?
With a thioester bond to a cysteine of E2 as well
E3 binds both E2-Ub and the target protein. How does it induce the isopeptide bond and Ub transfer to the target protein.
It brings the two substrates into a conformation in which the C-terminal glycine carboxyl group and the epsilon-amino group from a lysine of the target protein are brought in close range.
How many approx. types of the ubiquitination enzymes
2 E1 types
40 E2 types
Over 700 E3 types
> one E3 for every (family) target protein
Proteins for degradation are ubiquitinated in a specific manner. How?
They are polyubiquitinated (chain of more than 4 Ub).
The Ub’s are linked through Lysine-48 specifically (other linkage for other fates)
What function has mono-ubiquitination (1 or 2 Ub added)?
Regulation of the protein.
How many lysines does Ub contain, and why does this matter?
7 different lysines for different linkages in polyubiquitin for different signals in regulation
> Poly-Ub linked with Lys-48 can bind to the regulatory cap of the proteasome
The protein must give a degradation signal to be targeted by the ubiquitination enzymes. Which ones?
-N-end rule: stability of cytoplasmic protein determined to large extent by the amino terminal amino acid
-Destruction box: marks proteins for destruction, present in cell cycle regulators (cyclins)
-PEST sequence: amino acid sequence rich in proline (P), glutamatic acid (E), serine (S) and threonine (T)
-Hydrophobic sequences: recognition misfolded or misassembled proteins: exposed hydrophobic character
Many proteins start with methionine as its amino terminal residue, this has a long half time. How does the N-terminal amino acid change?
-Methionine aminopeptidase can remove the N-terminal methionine > other aminopeptidases can remove newly generated N-terminal amino acids
- addition amino acids
> endopeptidases:
process proteins by proteolytic cleavage. (like zymogens).
Recognition degradation signals by:
E2-E3 complexes
> E3 has a substrate binding domain and E2 binding domain
> E3 are protein complex like APC and SCF
> Phosphorylation often regulates recognition of the substrate
N-end rule: instable amino acids and highly stabilizing amino acids
Low: Asn, Asp, Gln, Glu
High: Ala, Pro, Cys, Ser, Gly, Thr, Met, Val
E3-ligases are potential targets for intervention. Name E3-ligases associated with disease
-Mdm2
-SCF complex
-Parkin Protein
Mdm2
Binds and ubiquitinates tumor suppressor protein p53
> in tumors overexpressed
SCF complex
Ubiquitinates tumor suppressor protein p27
Parkin protein
Mutated E3 ligase in patients with familial Parkinsons, Ubiquitinates proteins in outer membrane of damaged mitochondria leading to selective degradation via mitophagy
Structure of the 26S proteasome
-19S regulatory caps
> Lid: recognition and binding of ubiquiinated proteins
> Base: 6 ATPases of AAA family, involved in unfolding of target proteins
-20S catalytic core
> 4 stacked rigns of 7 subunits each (alpha and beta rings)
> 3 of the beta subunits are proteases with different specificity
What happens to Ub in proteasome
Ub is cleaved off substrate and recycled
Where are proteins digested into in the proteasome?
Into peptides of 7-9 amino acids
What happens to the peptide fragments released from the proteasome?
Digested to free amino acids by other proteases
Name a process regulated by protein degradation related to metabolism
Cholesterol metabolism
Catalytic residues of the beta subunits in proteasome
N-terminal threonine residues.
Name an important proteasome inhibitor
Bortezomib (Velcade)
Function Bortezomib
Inhibit the chymotrypsin activity of the proteasome
> used against multiple myeloma
> prevents Ub’ed proteins from degradation by proteasome
> non specific as degradation of all proteins that are targeted by the proteasome is inhibited
Bortezomib resembles the …
Isopeptide bond in Ub’ed proteins and has a reactive boron atom
NF-kB route
NF-kB is a TF (p65+p50) that initiates transcription of genes that prevent apoptosis.
> in healthy cells: inhibitor IkB binds to NF-kB to prevent NF-kB from translocating to the nucleus
In cancer cells, how is NFkB released from IkB?
Signals activate IkB kinase (IKK) to phosphorylate IkB for polyubiquitination and degradation IkB
Bortezomib against cancer
Inhibit IkB degradation and prevent NFkB activity in multiple myeloma tumor cells
H20 What part of AAs is useful and what part is dangerous
Amino groups are toxic by themselves (ammonium) > urea cycle
Carbon skeletons can be used as metabolic fuel or for gluconeogenesis
How is the alpha amino group removed in AAs?
Transamination and oxidative deamination to yield NH4+ and alpha-ketoglutarate
Name the general reaction and enzyme for transamination
alpha-amino acid + alpha-keto acid (alpha-ketoglutarate) <=> alpha-keto acid + alpha-amino acid (glutamate)
- By transaminases /aminotransferases
Alpha-ketoglutarate is an intermediate of the …
TCA cycle
Transamination of aspartate and alanine
Alanine + a-ketoglutarate <=> pyruvate+ glutamate (alanine aminotransferase)
Aspartate + a-ketoglutarate <=> oxaloacetate + glutamate (aspartate aminotransferase)
Transamination is ir/reversible and can be used for …
Reversible, used for synthesis of amino acids from alpha-keto acids like a-ketoglutarate.
Oxidative deamination yields …
NADPH
Oxidative deamination
Glutamate + NAD(P)+ <=> a-ketoglutarate + NH4+ + NAD(P)H + H+
(glutamate dehydrogenase)
Where does the oxidative deamination take place?
In the mitochondria (of the liver): enclosed because amino group is dangerous
Where is glutamate dehydrogenase predominantly present?
In the liver, in the mitochondria
> with some other enzymes of the urea cycle
> safe removal of toxic ammonium ion (NH4+)
High concentrations of NH4+ are harmful for…
the brain
Sum reaction transamination and deamination
alpha-AA + NAD(P)+ <=> alpha-keto acid + NH4+ (ammonium) + NAD(P)H + H+
difference ammonia and ammonium ion
Ammonia: NH3
Ammonium: NH4+
Where do the two amino groups from urea originate from?
One from the ammonium ion from the amino acid, one from aspartate (another AA, in urea cycle)
Which alpha-amino acids can convert their alpha-amino group directly to NH4+ (direct deamination)?
Serine and threonine
> catalyzed by dehydratases (dehydration precedes deamination)
> serine -> pyruvate + NH4+
> Threonine -> a-ketobutyrate + NH4+
The liver is responsible for deamination. What happens to amino acids in peripheral tissues?
Transport of amino groups to liver in the form of alanine and glutamine
When protein degradation in muscle?
Long exercise and starvation
Which amino acids are used as source of fuel for the muscle
Branched-chain AAs (the carbon skeletons): Leucine (Leu), Isoleucine (Ile) and Valine (Val)
> but muscle lacks enzymes for urea cycle, so NH4+ converted to alanine and glutamine
What happens to the glutamate from transamination in the muscle?
Glutamate + pyruvate > a-ketoglutarate + alanine (alanine aminotransferase)
> alanine is a non-toxic form of nitrogen transport between muscle and liver: glucose-alanine cycle.
Glucose alanine cycle
In muscle:
Glucose to pyruvate
Pyruvate + glutamate > alanine
In blood: alanine transport
In liver:
Alanine to glutamate and pyruvate
Glutamate deamination in mitochondria and urea cycle
Pyruvate to glucose
Glucose to blood to go back to muscle
Amino group reaction to glutamine in muscle and peripheral tissues
NH4+ + Glutamate + ATP > Glutamine + ADP + Pi (glutamine synthetase)
What happens to the glutamine when created in periphery?
Transport via blood to kidney, intestine and immune cells
> Fuel (energy)
> Nitrogen donor for purine biosynthesis
> pH regulation (kidney)
Why do immune cells need amino acids when you are sick?
They need proteins to proliferate (fuel), and they need proteins instead of other fuels.
Different organs need different …
amino acids
What happens to glutamine after being used by kidney or gut?
Conversion to alanine and used in the liver for deamination
What happens to the alanines which enter the liver for breakdown
First: transamination and oxidative deamination
Urea cycle characteristics
-Cyclic process
-In liver, compartmentalization
-amino groups in urea from NH4+ and aspartate
-Carbon atom from hydrogen carbonate (HCO3-, hydration of CO2)
-oxygen from water (H2O)
Which step in inside the mitochondrial matrix (urea cycle)
Making carbamoyl-phosphate and making citrulline by condensing it with ornithine
First (activation and committed) step of urea cycle
NH4+ + HCO3- + 2 ATP > Carbamoyl-phosphate + 2 ADP + Pi (carbamoyl-phosphate synthetase)
Other reaction in mitochondrial matrix of urea cycle (step 2)
Ornithine (amino acid) + carbamoyl phosphate > Citrulline + Pi (ornithine transcarbamoylase)
Urea cycle in cytosol
-Citrulline + aspartate + ATP > argininosuccinate + AMP + PPi (argininosuccinate synthase)
- Argininosuccinate > arginine + fumarate (argininosuccinase)
-Arginine + H2O > ornithine + urea (arginase)
-Ornithine to mitochondrion
Sum reaction urea cycle
NH4+ + HCO3- + 3 ATP _ aspartate + H2O > urea + 2 ADP + 2 Pi + AMP + PPi + fumarate
Regulation urea cycle
In committed step: Carbamoyl-phosphate synthetase needs allosteric activator: N-acetylglutamate
How is N-acetylglutamate made?
Acetyl-CoA + Glutamate > … + CoA
Which other amino acid is needed to make N-acetylglutamate and make the urea cycle work?
Arginine: activates the enzyme to make N-acetylglutamate.
> feed forward activation: arginine is product protein degradation (and intermediate urea cycle) > signals that nitrogen is produced.
Link urea cycle and gluconeogenesis
Argininosuccinate gets converted to arginine and fumarate
> Fumarate can be converted into oxaloacetate via malate shuttle, and oxaloacetate can be used for gluconeogenesis
> oxaloacetate can also be transaminated to form aspartate (using the conversion of glutamate to a-ketoglutarate) (enzyme aspartate aminotransferase), for the urea cycle.
Fates carbon skeletons of amino acids
-Glucose or glycogen
-Cellular respiration
-FA synthesis
20 amino acids can be converted into seven molecules for ketogenic and glucogenic breakdown. Name them
-Ketogenic: acetyl-CoA, acetoacetyl-CoA (for ketone bodies, fatty acid synthesis)
-Glucogenic: pyruvate, a-ketoglutarate, succinyl-CoA, fumarate, oxaloacetate (most amino acids are glucogenic, some both ketogenic and glucogenic) (TCA intermediates or pyruvate)
Which amino acids are ketogenic, which both keto and gluco and the rest only gluco
Keto: leucine and lysine
Gluco+keto: isoleucine, phenylalanine, tryptophan, tyrosine
Which amino acids lead to TCA intermediates?
C4 and C5 amino acid carbon skeletons
> some C4’s are ketogenic
C3 amino acids are converted to pyruvate. Name conversion alanine
Alanine + a-ketoglutarate <=> pyruvate + glutamate (transamination by alanine aminotransferase)
How are the C3 amino acids serine and cystein converted to pyruvate
-Serine is deaminated (> pyruvate + NH4+)
- Cysteine to pyruvates creates sulfur atom emergence in H2S
> also, glycine, threonine, tryptophan
Which C4 amino acids are converted to oxaloacetate?
Aspartate and asparagine
> Aspartate transamination
> Asparagine + H2O > aspartate + NH4+
(then transamination afterwards)
C5 AA conversion to a-ketoglutarate
Histidine, glutamine, proline, arginine
> via glutamate and conversion to a-ketoglutarate by glutamate dehydrogenase
Degradation steps branched chain amino acids in muscle
Leucine, isoleucine and valine
-Transamination with a-ketoglutarate to a-ketoacid
-oxidative decarboxylation in CoA dependent reaction catalyzed by branched chain a-keto acid dehydrogenase complex (analogous to pyruvate dehydrogenase complex)
-CoA derivative of a-keto acid resembles short chain FA and is degraded via beta oxidation into acetyl-CoA and possibly proprionyl if uneven carbons.
Which branched chain amino acids yield proprionyl-CoA and how is it converted?
Valine and isoleucine
> converted to succinyl-CoA
Which amino acid yields acetoacetatyl-CoA in beta oxidation to yield two acetyl-CoA?
Leucine
Degradation aromatic amino acids
Phenylalanine, tyrosine and tryptophan are converted into acetoacetate, fumarate and pyruvate
> O2 required to break open aromatic ring
> catalysis by oxygenases
Types of oxygenases
-Monooxygenases: one oxygen atom of O2 appears in product, other to H2O
-Dioxygenases: both oxygen atoms of O2 appear in product
Degradation phenylalanine to tyrosine
Phenylalanine (+ O2 + tetrahydrobiopterin) hydroxylation to tyrosine (and H2O and quinonoid dihydrobiopterin which is regenerated to tetra…)(phenylalanine hydroxylase, a monooxygenase)
Tyrosine breakdown
Tyrosine is transaminated, then four steps to acetoacetate andfumarate (two dioxygenases are involved)
Phenylalanine can be converted to tyrosine, but also to phenylpyruvate using a transaminase (which uses a-ketoacid > a-AA), explain the system
Km transaminase»_space; Km hydroxylase, so this pathway through transaminase becomes important when phenylalanine accumulates.
PKU
-Defect phenylalanine hydroxylase
-Accumulation of the substrate leads to metabolic disease and conversion to phenylpyruvate
-Not because of absence of the product, the phenylpyruvate is unwanted.
-Inhibition development CNS by phenylalanine is probably cause of mental retardation in patients
Diagnosis and treatment PKU
Heel prick
Treament: low-phenylalanine diet supplemented with tyrosine
> phenylalanine is an essential AA, tyrosine not, but with PKU, you cannot make it so it becomes essential
HC21: Reactions to vascular injury
-VWF/collagen: induces platelet activation
-Tissue factor: induces coagulation together with activated platelets
-Vasocontriction
Initiators hemostasis
Collagen > primary hemostasis
Tissue factor > secondary hemostasis
Functions endothelial cells in hemostasis
-Barrier function
-Stores organelles with the platelet binding Von Willebrand Factor (VWF): crucial for primary hemostasis in adhesion process of platelets
How are the storage tubule organelles for VWF in endothelial cells called?
Weibet-Palade bodies
VWF structure and characteristics
Large multimeric protein synthesized by endothelial cells and megakaryotes (precursors platelets)
> form long size proteins
> released into circulation upon activation of the cells
What happens to VWF in blood?
The normally in helix condensed protein unfolds and adheres to platelets in blood
Primary vs secondary hemostasis
Primary: rapid formation of platelet plug
Secondary: stabilization of platelet plug by fibrin network
UL-VWF multimers are on the surface of which cells upon release of Weibel-Palade bodies
Endothelial cells
Adhesion blood platelets by VWF methods
-VWF dependent adhesion of platelets on endothelium
-Also: adhesion platelets via collagen bound VWF in subendothelial matrix
How do we get clots after platelet adhesion?
Activation of platelets: aggregation, rapid process!
Morphology platelets
-Anucleate cell fragments
-Production in bone marrow
-Short lifespan of 10 days
-Tightly regulated processed
Megakaryotes to platelets
Megakaryotes in bone marrow form cytoplasmic protrusions which cross the endothelial cell barrier > regulated pinch off. > proplatelets which develop into platelets
Platelet activation through collagen receptors which react to exposed collagen in subendothelial matrix
-Initial binding: Glycoprotein Ib (GPIb) binds to VWF attached to collagen. (tethering)
-Binding exposed collagen to GPVI and alpha2beta1
-GPVI causes signalling to activate bindings by a2B1 and alphaIIb-beta3 (aIIbB3)
-a2B1 causes stable adhesion by binding collagen > then binding GPVI for signalling and stable adhesion.
> collagen binding initiates inside out signalling and stable adhesion of blood platelets
Effect Binding GPVI signalling and stable adhesion
Release ADP from dense granules and release thromboxane A2 (TXA2) to promote further activation of aggregation of platelets
Inhibitors of platelet adhesion, secreted by healthy endothelium?
NO and PGI2
Inside out signalling promotes exposure of binding sites on glycoprotein aIIbB3: explain
The transmembrane dimeric protein aIIbB3 is closed when stable adhesion and no binding of GPVI to collagen.
When GPVI is bound to collagen, the signalling induces open conformation and binding site exposure which binds the ligand fibrinogen.
Fibrinogen crosslinks platelets whcih finally results in platelet aggregation.
aIIbB3 is also called
GPIIb/IIIa
Deficiency in platelet function leads to
blood loss, bleeding disorder
> disfunctional GPIb, GPIIb/IIIa and VWF (defect VWF: Von Willebrand disease)
Von Willebrand disease
Polymers cannot be formed, platelets cannot be recruited efficiently
Regulation primary hemostasis: size and activity of VWF polymers control
By VWF cleaving protease ADAMTS13
Hemostatic balance is maintained in hemostasis through the..
coordinated activity of pro- and anti-coagulant proteins
> too much clotting > thrombosis
Polymers of VWF need to be exactly the right size for homeostasis, how?
Exact cleavage of VWF by ADAMTS13 for right size maintenance
ADAMTS13 deficiency effect on VWF polymers
Prolongs polymers of VWF
> too much platelet aggregation
What happens to cleaved VWF
Released as plasma VWF
How is the disease of deficient ADAMTS13 called?
Thrombotic thrombocytopenic (shortage platelets) purpura (purple, small bleeds in microvessels) > TTP
TTP effects
Thrombotic microangiopathy: platelet rich thrombi in microcirculation
> hemolytic anemia: fragmented red blood cells, schistocytes (sikkel)
> fever, kidney failure, neurological symptoms
Congenital vs acquired TTP
Congenital: genetic defect in gene for ADAMTS13
Acquired: autoantibodies
When treatment if TTP
Direct treatment needed, risk for heart attack and brain damage
HC22: Secondary hemostasis requires activation of the … cascade
coagulation
How is fibrinogen converted into fibrin
Thrombin cleaves and releases fibrinopeptide A and B
> fibrin can crosslink: gamma subunits bind to the released tail which was blocked by fibrinopeptide A.
Which factor crosslinks soluble fibrin into insoluble fibrin clots?
Factor XIIIa
Pathway fibrinogen to crosslinked fibrin mesh
Fibrinogen > Fibrin mesh (thrombin)
Fibrin mesh > crosslinked fibrin mesh (Factor XIIIa)
One factor Xa leads to how many fibrin?
1000 fibrin (amplification in the cascade is huge)
Initiation coagulation cascade
Tissue factor, containing a transmembrane domain, binds to factor VIIa (FVII activated). The complex activates factor X and factor IX (to FXa and FIXa)
Blood coagulation involves the sequential activation of ..
serine proteases
> these are inactive zymogens
> a cofactor and a activated serine protease (bound to membrane) activate the next serine protease zymogen by cleaving a peptide domain.
How is FVIIa bound to the membrane (it has to bind tissue factor with the transmembrane domain)
GIa domain of FVIIa contains modified glutamic acids that bind to phospholipids
How can FVII bind to the membrane
gamma-carboxylation of glutamic acids in FVII GIa domain
> gamma-carboxyglutamate residue with a lot of carboxylic groups
> binding calcium ions with carboxylic groups > binding to phospholipids/membranes (the heads)
Which blood coagulation factors have gla domains to bind membranes?
FVII, FIX, FX and prothrombin
> prothrombin brough in proximity of FXa (serine protease) and FVa (stimulatory protein). FXa can activate prothrombin to thrombin
Amplification and propagation steps in coagulation
-Tissue factor/FVIIa (FVIIa is the serine protease, Tissue factor the coactivator) activate FX to FXa and FIX to FIXa
> FXa promotes activation of FVII
> FIXa binds the membrane and binds coactivator FVIIIa. FXa activates FX to FXa as well.
> FXa binds membrane and coactivator FVa. FXa activates prothrombin to thrombin (thrombin is released whereas prothrombin is membrane bound)
Effects active thrombin
Amplification
> Activate FV, FVIII and FXI
> FXIa activates FIX to FIXa
Fibrinogen > fibrin
Thrombin is released after activation from prothrombin. Is this also the case for FXa?
No, it stays membrane bound.
First FX as substrate for VIIIa/FIXa > then FXa to FVa to work as complex and activate prothrombin
How are FVIII and FV bound to phospholipid membranes?
Through hydrophobic protusions of C domains
Hemophilia: what is it
A bleeding disorder
> X-linked, only in men
- causes bleeding in joints and muscle > severe damage
Treatment hemophilia A and B by supplements
Hemophilia A: defect FVIII: supplementation
Hemphilia B: defect FIX: supplementation
> 2-3 times per week, intravenous administration
Long term effect hemophilia
Joint swells because of bleeding: bone wearing, permanent damage and destroyed joint
Other treatments hemophilia beside supplements
-Disruptive treatments, bispecific antibody
-gene therapy
BIVV001 treatment for hemophilia
Novel FVIII-VWF fusion protein with extended half life because of added groups
> against hemophilia A
FVIII bypassing agent for hemophilia A: emicizumab
Bispecific anti-FIX-FX antibody which mimics the function of FVIII: bringing together FIXa (the protease, working in hemophilia A) and FX.
> modified: not immunogenic
> can be injected in the belly instead of vene: subcutaneous administration
> long half-life: once per week infusion
> breakthrough bleeding may occur: requiring additional treatment with FVIII/FVIIa
Gene therapy for hemophilia
AAV directed gene therapy
> deliver vector with adeno-associated virus (AAV, naturally occuring harmless virus)
> wild type gene in format that can be used: an expression cassete containing liver specific promotor and FVII cDNA.
> in hepatocytes
> AAV capsid broken down in hepatocytes but vector deliver to nucleus
> permanent one time use cure (intravenous)
Disadvantages AAV gene therapy
-permanent cure for hemophilia B but decrease FVIII for hemophilia A (after 5-7 years)
-Possible immune response against AAV or the own FIX or FVIII
-Cancer risk: the vector may (partly) integrate in a proto-oncogene of the own DNA.
Venous thrombosis types and prevalence
1/3: pulmonary embolism
2/3: deep vein thrombosis
> thrombotic events occur in leg, arm or pelvis as well
> blood clotting behind flaps of veins, create embolus which releases (blood clot in bloodstream which can block arteries)
Name serine protease inhibitors and other proteins which control blood coagulation
-AT inhibits FIXa, FXa, IIa (thrombin)
-Activated Protein C (on endothelium) activates Protein S. Prot S/APC complex inhibits FVIIIa and FVa
-TFPI inhibits
Risk factors venous thrombosis
-AT (antithrombin) deficiency
-Prot C deficiency
-Prot S deficiency
-Factor V Leiden
-Prothrombin gene mutation
Anticoagulant pathway mediated by protein C
Thrombin binds thrombomodulin on endothelial cell
> Protein C is converted to APC by thrombomodulin
> APC binds Prot S and complex inhibits FVIIIa and FVa
Activated protein C (APC) together wit protein S inactivate … and thereby …
FV and FVIII and thereby thrombin formation
> Prothrombin and FXa inhibit APC in breakdown FVa to FVi
> Prot S and FV activate breakdown FVa to FVi
> FIXa and FX inhibit APC in breakdown FVIIIa to FVIIIi, while FV and Prot S promote it.
Factor V Leiden
Mutation at cleavage site for APC in Factor V
> Normal factor V has cleavage sites at Arg506 and subsequently Arg306 and Arg679
> In Factor V Leiden: Arg506> Gln, thus only cleavage at Arg306 and Arg679.
(less inhibition by APC, still a functional domain)
Treatment venous thrombosis
Inhibit blood coagulation
> enhance activity of natural inhibitors: heparin promoted activity of anthrombin: reactive loop targeting active site for FXa, FIXa and thrombin becomes exposed after binding heparin.
> direct inhibition of serine proteases by small molecule inhibitors of thrombin or FXa
> inhibition of serine proteases by interfering with membrane binding: vitamin K antagonists
