WK08L1 - Fibrinolysis (Ben) Flashcards
Describe a basic overview of fibrinolysis.
Include enzymes, their source, their activation + regulation.
- tissue plasminogen activator (tPA) made by endothelial cells and plasminogen made by the liver complex w/ each other on surface of fibrin clot
- tPA cleaves/activates plasminogen, forming plasmin which can break down fibrin
- SERPINs (specifically alpha-2 antiplasmin) can bind and inhibit plasmin
How does plasminogen bind to fibrin before activation?
Via five kringle domains in its structure which bind positively charged Lys residues
How does tPA bind to fibrin to form a complex with plasminogen?
What happens to it when it binds?
Via both kringle and finger domains
- doesn’t require Lys residues like plasminogen, but needs protofibril assembly of fibrin monomers, which increases binding sites
- unbound tPA has low activity, binding changes conformation of its active site and increases activity 100x
Where is plasminogen cleaved for activation and what results from this cleavage?
tPA cleaves it at an arginine residue (R561, marked PA in img)
- results in the assembly of a Ser, Asp and His residue in the catalytic domain to form the active site
(shown by blue circles)
What part of its structure blocks plasminogen from being cleaved by tPA?
How can this be removed?
- an N-terminal “activation” peptide of plasminogen blocks the cleavage site for tPA
- Can be removed 2 ways:
- conformational - plasminogen binding of fibrin via Kringle domains moves activation peptide away
- proteolytic - active plasmin can cleave the activation peptide (even if the plasminogen is not fibrin-bound)
What endogenous activator of plasminogen exists, other than tPA?
Where is it made?
How does it function and how is it different from tPA?
Urokinase (AKA uPA)
- made by inflammatory cells + tumor cells
- cleaves same bond as tPA, but does not require fibrin as co-factor
- instead uses receptor uPAR on surface of inflam./tumor cell as co-factor
- necessary for resolution of inflammation due to fibrin
- used by tumor cells for EC matrix invasion
What must first happen to uPA before it is active?
Where does this happen and what other molecule acts on uPA to activate it?
- must bind to uPA receptor on inflammatory cell membrane and be cleaved by either kallikrein or plasmin
What exogenous plasminogen activators exist?
Where are they from and how do they act?
Streptokinase + Staphylokinase
- from bacteria
-
bind + convert plasminogen to a plasminogen activator which contribute to plasmin activation and clot breakdown
- (normally clots can restrict movement of pathogens, this works against that)
What other exogenous clot-degrading enzyme exists which works on a different aspect of clot structure?
Streptodornase
- degrades DNA matrix of NETs (neutrophil extracellular traps)
Which of the numbered plasma factors also acts as a plasminogen activator?
How does its activity compare to other activators?
What pathway other than direct plasminogen activation can it use to stimulate fibrinolysis?
Factor XII
(a coag. factor activated in relation to inflammation via polyphosphate)
- activates plasminogen with lower efficiency than tPA or uPA, but has 1000x higher concentration than either of these
- can also activate prekallikrein –> kallikrein which will then activate uPA
Plasmin’s specificity is similar to what other protease?
So it cleaves peptide bonds near which amino acids?
similar to trypsin
cleaves Lys and Arg peptide bonds
Plasmin can cleave both fibrin and fibrinogen.
Where are the most susceptible bonds for cleavage by plasmin on both of these molecules?
- most susceptible bonds are at C-terminals of alpha chains
(the C terminals of each of the two alpha chains which dimerize with each other in the center of the molecule)
Describe the sequence of events in the cleavage of fibrinogen by plasmin.
- Plasmin cleaves the C-terminals of the alpha chains leaving fragment X, which lacks these C-terminals but can still polymerize
- Plasmin then cleaves btwn central E and peripheral D domains leaving fragment Y which can not polymerize
- A final cleavage btwn E and the other D leaves the smallest fragments D and E.
Describe the sequence of events in the cleavage of fibrin by plasmin.
What must happen in order for the clot to dissolve?
The sequence is essentially the same as with fibrin.
Only 1/4 of D-E domain connections must be cleaved in order to dissolve the clot, because at normal blood shear rates dissolution can occur even without all bonds cleaved.
Topographically, how must fibrin molecules be cleaved by plasmin in order for fibrinolysis to be effective?
Why?
All 3 chains (alpha, beta and gamma) must be cleaved in the same cross-section.
Because if not cleaved this way, chains will overlap and remain connected via non-covalent interactions.
Describe how plasmin moves over a fibrin molecule as it cleaves it.
- Kringle 1 domain binds to a lysine residue on fibrin in such a way that positions the catalytic site right at the susceptible peptide bond btwn E and D domains
- After this bond is cleaved, plasmin does not need to dissociate to cleave the 2 other chains (b/c it is a large enough molecule to reach them)
- After a whole monomer is cleaved, Kringle 2 can bind to the next monomer (plasmin = 14 nm, btwn monomers = only 6 nm) and continue cleavage
What happens to plasmin activity when all but one of its kringle domains are removed?
When all kringle domains are removed?
What other molecule has similar action to this kringle-less plasmin?
miniplasmin - experimentally produced plasmin with only one K domain, half the efficiency of normal plasmin
microplasmin - no K domains, only catalytic site, about 1/8 activity of plasmin
elastase - released from granules by activated neutrophils, can digest fibrin with abt same effic. as microplasmin, also only has catalytic domain, no kringles
Which of the numbered plasma factors acts in a way that can block plasmin from dissolving fibrin clots?
What does it do?
factor XIIIa
- can form Lys-Gln isopeptide bonds (similar to its action on fibrin) between α2-antiplasmin and fibrin
- α2-antiplasmin will thend “shield” fibrin from plasmin
