Anticoagulation and fibrinolysis

Question 1

Mechanisms of anticoagulation

Answer 1

• Tissue factor pathway inhibitor (TFPI)
• Protein S and C system
• Antithrombin
• Heparin cofactor II
• Thrombomodulin
• Inhibition of plasmin: Plasminogen activating inhibitor (PAI), alpha-2 plasmin inhibitor (a2PI), thrombin activated fibrinolysis inhibitor (TAFI)

Question 2

Mechanisms of fibrinolysis

Answer 2

• Plasmin (and plasminogen)
• tPa (fibrin/annexin activated) activation of plasminogen
• uPA (uPAR activated) activation of plasminogen

Question 3

TFPI

Answer 3

• Prevents activation of X by TF/VII, forcing TF/VII activation of IX and coagulation to proceed through tenase complex
• TFPI is produced by endothelium and can be associated w/ heparin-like GAGs on endothelium surface
• TFPI can also be in solution, but this form of it requires protein S for stabilization (this form also allows TFPI to bind to platelets and inhibit their activation)
• Heparin also releases TFPI to inhibit thrombus formation

Question 4

Protein C and S

Answer 4

• Proteolytically inactive both factors Va and VIIIa (the cofactors)
• Activated protein C (APC) is responsible for the proteolysis
• Protein C is bound to its endothelial protein C receptor (EPCR) until activated by a complex of thrombin/thrombomodulin
• Once activated, APC binds to platelets and endothelium in complex with protein S to destroy endothelial/platelet bound factor VIIIa and Va
• Inhibition sequence is opposite of activation sequence: inhibits Va first, then inhibits VIIIa
• APC can also inhibit PAI1, thereby increasing fibrinolysis
• Both protein C and S are vit K dependent proteins

Question 5

Antithrombin 1

Answer 5

• Most important inhibitory protein, it is only biologically active when associated w/ heparin-like GAGs (GAGs are negatively charged and bind to lysine residues on antithrombin)
• Antithrombin/heparin complex exposes antithrombin’s active site, allowing it irreversibly inhibit thrombin
• Heparin GAGs are on endothelium and sub endothelium, thus they are present at sites of thrombin generation

Question 6

Antithrombin 2

Answer 6

• Antithrombin can also inactive factors IXa and Xa (indirectly inactivates Va, VIIIa, and XIa by inactivating thrombin)
• When bound to large MW heparin GAGs, antithrombin inhibits thrombin
• When bound to small MW heparin GAGs, antithrombin inactivates IXa and Xa (preferentially Xa)
• Small MW heparins are primarily used in Rx over large MW ones
• Heparin cofactor II can also inactivate thrombin
• Those w/ antithrombin deficiencies have a higher predisposition to thrombotic events

Question 7

Fibrinolytic system

Answer 7

• Fibrin is degraded down into fibrin degradation products (fdp) by plasmin (if it wasn’t cross-linked by XIII) or into D-dimers by plasmin (if it was cross-linked by XIII)
• Positive D-dimer test indicates that thrombin, XIII, and plasmin must all be active and generated (mostly a surrogate for thrombin)
• Plasmin can also degrade fibrinogen into fibrinogen degradation products (FDP)
• Plasmin formed from pronz plasminogen, which is activated with by tPA or uPA (both of which require receptors for activity)
• Plasmin is also central in tissue remodeling and angiogenesis (activates metalloproteinases, angiogenic GFs up regulate plasminogen expression on endothelium)

Question 8

Plasmin

Answer 8

• Plasmin binds to fibrin (or fibrinogen) at lysine residues to limit plug formation by degradation
• Free plasmin in blood can degrade many proteins including VIII and V, fibrinogen, and GPIIb/IIIa (thus limiting platelet aggregation)
• Plasmin is inhibited by many proteins, but most notably by alpha-2 plasmin inhibitor (a2PI) which is produced in the liver
• In a mature clot a2PI is covalently linked to the fibrin by XIII, resulting in resistance to degradation by plasmin
• a2PI in circulaiton rapidly inactivates free plasmin

Question 9

tPa

Answer 9

• Tissue plasminogen activator (tPA) is synthesized and secreted by endothelium activated by thrombin, cytokines, or increased venous pressure
• tPA requires cofactors fibrin or annexin (tPA/plasminogen receptor, on endothelium, expression induced by thrombin) to be activated and thus activate plasminogen to plasmin
• tPA bound to fibrin activates plasmin to degrade the fibrin, tPA bound to annexin activates plasmin to remain on endothelium (bound to annexin) and provide an anticoagulant barrier

Question 10

uPA

Answer 10

• uPA is also synthesized and secreted by endothelium, stimulated by thrombin, and requires its receptor uPAR in order to activate plasminogen
• uPAR (found on endothelium and monocytes) is closely linked to annexin-bound plasminogen, so when uPA binds to uPAR the two receptors interact and uPA converts plasminogen to plasmin
• uPAR expression induced by thrombin

Question 11

Plasminogen activator inhibitor 1 (PAI1)

Answer 11

• PAI1 is synthesized in the liver, by endothelium, and megakaryocytes
• It is only stable when bound to subendothelial matrix (to prevent fibrinolysis when there is endothelial damage)
• PAI1 inhibits both uPA and tPA to prevent palsmin activation
• PAI1 bound to subendothelial matrix protein vitronectin is active form
• Under circumstances when tPA is secreted by endothelium, active PAI1 is also secreted by endothelium or released by activated platelets
• PAI1 can be inhibited by APC, thus enhancing fibrinolysis

Question 12

Thrombin activated fibrinolysis inhibitor (TAFI)

Answer 12

• TAFI is converted to its active form by thrombin bound to thrombomodulin
• Activated TAFI removes terminal arginine and lysine residues from fibrin
• These positively charges residues are required for plasminogen to bind to fibrin, and for plasmin to cleave fibrin
• Thus TAFI prevents fibrinolysis by reducing plasmin generation (reducing plasminogen binding), and by reducing plasmin activity (plasmin cannot cleave fibrin)

Question 13

Thrombomodulin

Answer 13

• Thrombomodulin complexes w/ thrombin to achieve anticoagulation and procoagulation results
• The thrombomodulin/thrombin complex directly inhibits the bound thrombin
• The complex activates protein C to degrade Va and VIIIa, and to deactivate PAI1 (resulting in increased fibrinolysis)
• Thrombomodulin/thrombin complex also activates TAFI, preventing plasmin generation and activity (leading to inhibition of fibrinolysis)

Question 14

Rx of fibrinolysis

Answer 14

-EACA (Amicar) prevents plasminogen activators (tPA and uPA) and plasminogen from binding to fibrin