WK08L1 - Fibrinolysis (Ben) Flashcards Preview

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Flashcards in WK08L1 - Fibrinolysis (Ben) Deck (23)
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1
Q

Describe a basic overview of fibrinolysis.

Include enzymes, their source, their activation + regulation.

A
  • 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
2
Q

How does plasminogen bind to fibrin before activation?

A

Via five kringle domains in its structure which bind positively charged Lys residues

3
Q

How does tPA bind to fibrin to form a complex with plasminogen?

What happens to it when it binds?

A

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
4
Q

Where is plasminogen cleaved for activation and what results from this cleavage?

A

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)

5
Q

What part of its structure blocks plasminogen from being cleaved by tPA?

How can this be removed?

A
  • 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)
6
Q

What endogenous activator of plasminogen exists, other than tPA?

Where is it made?

How does it function and how is it different from tPA?

A

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
7
Q

What must first happen to uPA before it is active?

Where does this happen and what other molecule acts on uPA to activate it?

A
  • must bind to uPA receptor on inflammatory cell membrane and be cleaved by either kallikrein or plasmin
8
Q

What exogenous plasminogen activators exist?

Where are they from and how do they act?

A

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)
9
Q

What other exogenous clot-degrading enzyme exists which works on a different aspect of clot structure?

A

Streptodornase

  • degrades DNA matrix of NETs (neutrophil extracellular traps)
10
Q

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?

A

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
11
Q

Plasmin’s specificity is similar to what other protease?

So it cleaves peptide bonds near which amino acids?

A

similar to trypsin

cleaves Lys and Arg peptide bonds

12
Q

Plasmin can cleave both fibrin and fibrinogen.

Where are the most susceptible bonds for cleavage by plasmin on both of these molecules?

A
  • 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)

13
Q

Describe the sequence of events in the cleavage of fibrinogen by plasmin.

A
  1. Plasmin cleaves the C-terminals of the alpha chains leaving fragment X, which lacks these C-terminals but can still polymerize
  2. Plasmin then cleaves btwn central E and peripheral D domains leaving fragment Y which can not polymerize
  3. A final cleavage btwn E and the other D leaves the smallest fragments D and E.
14
Q

Describe the sequence of events in the cleavage of fibrin by plasmin.

What must happen in order for the clot to dissolve?

A

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.

15
Q

Topographically, how must fibrin molecules be cleaved by plasmin in order for fibrinolysis to be effective?

Why?

A

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.

16
Q

Describe how plasmin moves over a fibrin molecule as it cleaves it.

A
  • 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
17
Q

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?

A

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

18
Q

What are the 2 main classes of plasmin inhibitors?

One class has 2 members… what are they?

Other class has only one… what is it?

A
  • SERPINs
    • α2-antiplasmin - same action as antithrombin… plasmin tries to cleave it but can’t complete the cleavage and becomes covalently bound to the inhibitor
      • similar concentration to plasminogen in the blood
    • PAI-1 - inhibits plasminogen activators (tPA and uPA)
      • lower conc. than α2-antiplasmin, but same conc. as tPA and uPA
  • α2-macroglobulin
    • same action as on thrombin… traps plasmin in its central cavity
19
Q

Which of the numbered plasma factors acts in a way that can block plasmin from dissolving fibrin clots?

What does it do?

A

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
20
Q

How well does XIIIa’s binding of α2-antiplasmin and fibrin inhibit fibrin dissolution?

How can this be overcome?

How is this used clinically?

A
  • it only delays dissolution
  • if tPA can produce enough plasmin, the α2-antiplasmin can be overtitrated
  • tPA is used clinically to dissolve fibrin clots in stroke patients within 3-4 hrs of the stroke
    • if not done within this time, clot is too stable and treatment is inefficient
21
Q

What enzyme acts on a positive feedback loop that drives fibrinolysis?

What kind of enyzme is it?

Where is it made?

How is it activated?

A

Thrombin-activatable Fibrinolysis Inhibitor (TAFI)

  • is a metalloprotease with carboxypeptidase activity
  • made by the liver and released to circulation
  • activated to TAFIa by thrombin-thrombomodulin
22
Q

Where do carboxypeptidases act on proteins?

A

they cleave peptide bonds formed by carboxy terminal AAs

(do NOT cleave carboxy terminal carboxylic group, which Kolev said was a false statement on the tests)

23
Q

How does TAFIa affect the positive feedback loop which perpetuates fibrinolysis?

Describe the loop and where TAFIa comes into play.

A

TAFIa inhibits the positive feedback loop and thus inhibits fibrinolysis

  • As plasmin cleaves fibrin at Lys residues, new C-terminal lysines are exposed
  • These lysines serve as binding sites for tPA and plasminogen, acting as positive feedback via further formation of plasmin
  • TAFIa has specificity for basic AAs, so it cleaves off the C-terminal Lys, thus removing these binding sites for tPA and plasminogen and inhibiting plasmin formation