Fibrinolysis. Plasminogen activation

Question 1

Fibrinolysis

Answer 1

• process that prevents blood clots from gown and becoming problematic
• 2 processes: primary or secondary fibrinolysis
• main enzyme is plasmin
• fragments cleared by proteases or kidney/liver

Question 2

Plasmin

Answer 2

• produced in inactive form: plasminogen
• plasminogen has affinity for fibrin
• serine-protease, 2 chains, disulfide-bonds
• hydrolyses peptide bonds
• D and E degradents of fibrin, or D-dimers

Question 3

Plasminogen

Answer 3

• produced in liver
• 7 domains
• C-terminal chymotrypsin-like serine protease domain
• N-terminal Pan Apple domain PAp + 5 Kringle domains

Question 4

Pan Apple domain

Answer 4

important determinants for maintaining plasminogen in closed form

Question 5

Kringle Domain

Answer 5

• responsible for binding to lysine residues present in receptors and substrates
• autonomous protein domains
• fold into large loop, 3 disulfide bonds
• important in protein-protein interaction with blood coagulation factors

Question 6

Tissue plasmin activator tPA

Answer 6

• produced by health vascular endothelia near injury and by tumor cells
• requires fibrin as cofactor
• does not require proteolytic activation
• inhibited by plasminogen activator inhibitors 1 and 2
• induced by bradykinin (kininogen)
• expensive

Question 7

Urokinase plasmin activator uPA

Answer 7

• produced by endothelia lining excretory ducts
• can be cleaved and activated by kallikrein or by positive feedback of plasmin
• can activate plasmin whenever
• action important in dissolving clots in excretory tracts
• inhibited by plasminogen activator inhibitors 1 or 2

Question 8

Streptokinase

Answer 8

• protein produced by bacteria
• no enzymatic activity
• forms a complex with plasminogen ⇒ SK-plasminogen ⇒ plasminogen’s protease domain take up active conformation ⇒ convert plasminogen to plasmin
• cheaper, more used

Question 9

Plasminogen activation

Answer 9

1. tissue plasmin activator
2. urokinase plasmin activator
3. streptokinase
4. positive feedback by plasmin
   - depends on conformation
   - activators present in plasma in different concentrations
   - activators regulated by inhibitory mechanism
   - activors’s co-factors present in different concentrations
   - co-Factor: FIBRIN

Question 10

Inhibitors of fibrinolytic enzymes

Answer 10

1. Alpha 2 plasmin inhibitor

2. Alpha 2 macroglobulin

Question 11

Alpha 2 plasmin inhibitor / alpha 2 antiplasmin

Answer 11

• serine protease inhibitor
• inactivates plasmin by forming covalent complex
• concentration 1 uM
• formes a plasmin-plasmin inhibitor complex
• plasmin is protected if bound to fibrin- but TAFI helps decreasing binding
• FXIII form cross-links between fibrin + a2PI⇒ maintain action of inhibitor

Question 12

Alpha 2 macroglobulin

Answer 12

• homotetramer plasma inhibitor, each sib-unit has active part
• produced by liver, but also in macrophages, fibroblasts and adrenocortical cells
• largest major non-immunoglobulin protein in plasma
• acts as an anti-protease
• inhibits plasmin and kallikrein and thrombin
• carrier protein

Question 13

Rate constant of plasmin inhibitors

Answer 13

1. alpha 2 plasmin inhibitor: 4X10 6
2. alpha 2 macroglobulin: 3x10 5
3. antithrombin: 1,6x10 5
4. alpha 1 protease inhibitor: 1,5x10 5

Question 14

Plasminogen activator inhibitor 1 PAI1

Answer 14

• serpin mechanism

- forms a complex with uPA/tPA

Question 15

Thrombin activated fibrinolysis inhibitor TAFI

Answer 15

• metalloprotease + carboxypeptidase
• local action
• cleaves basic AA: lysine, arginine from C-terminus
• cleaves lysine during fibrinolysis from degradation products ⇒ decreases efficiency of fibrin as co-factor to tPA
• in blood as proenzyme, activated by thrombin-TM complex
• not specific for fibrin (others e.g. bradykinin)
• unstable molecule, denature easily in body