Regulatory mechanisms and fibrolysis
Regulatory Mechanisms and Fibrinolysis
· The Hemostatic System- Initiation & Propagation
· Regulator mechanisms
· Fibrinolysis
The Hemostatic System: Initiation and Propagation
• Pathways are not distinct, independent – actually interdependent.
• Normal coagulation requires
o Cells that express TF (usually extravascular)
o Platelets, usually intravascular
• 2 Phases
o Initiation______________________
§ Occurs on TF expressing cells
§ 3-5% thrombin
o Propagation_______________________
§ Occurs on platelets
§ 95% or more of thrombin
Initiation
• Begun by formation of extrinsic tenase complex (VIIa, TF, +Ca++, phospholipids)
• Generation of small amounts of thrombin
• Less efficient than intrinsic tenase complex
o But platelets, cofactors and procoagulants become activated
• Low level of thrombin generated in the initiation:
(1) activates platelets through cleavage of protease activated receptors PAR-1 and PAR-4
(2) activates factor V released from platelet a-granules
(3) activates factor VIII and dissociates it from VWF
(4) activates factor XI, the intrinsic accessory procoagulant that activates more factor IX
(5) Splits fibrinogen peptides A and B from fibrinogen and forms a preliminary fibrin network. The initial platelet plug is thus formed.
Propagation
• More than 95% of thrombin generation
• Occur on the surface of activate platelet
• Large number of platelets adhere to site of injury
o By low levels of thrombin______________ (initiation)
o Adhering to exposed collagen
o This creates COAT platelets (collagen and thrombin)
• COAT Platelets
o higher level of procoagulant activity than platelets exposed to collagen alone
o provide a surface for formation and amplification of complexes____________
o Both platelets and tissue factor-bearing cells are essential for physiologic coagulation
o Deficiencies of key proteins (VII, IX, VIII, X, V, or prothrombin) compromise thrombin generation and manifest as significant bleeding disorders
Coagulation Regulatory Mechanisms
• Balance between procoagulant & anticoagulant systems.
• Activated factors and/or platelets must be kept at the site of injury so fibrin formation is limited to the site of vascular injury
• Factors and platelets must be controlled so they are inactive when distant from a site of vessel damage and blood remains fluid in uninvolved vessels.
Principle Regulators
Tissue Factor Pathway Inhibitor (TFPI)
Antithrombin (AT)
Activated protein C (APC)
Tissue Factor Pathway Inhibitor (TFPI)
• TFPI is synthesized primarily by ECs and is also expressed on platelets
• Binds to and inhibits
o Factor Xa_____________
o VIIa:TF complex
• 2 Step Process
o First TFPI binds factor Xa and inactivates it
o then TFPI:Xa complex binds and inactivates TF: VIIa, preventing more activation of Xa.
• Alternatively, TFPI may _____bind directly________ to Xa and VIIa in the TF:VIIa:Xa complex
Protein C Regulatory System
• Thrombin cleaves fibrinogen generating a fibrin clot
o activates factors _____V, VIII, XI, XIII_________
o Propagating more thrombin generation
• In intact normal vessels
o thrombin avidly binds thrombomodulin
o triggers ____protein C regulatory system_________
§ revises thrombin’s function from a procoagulant enzyme to an anticoagulant.
• EC protein C receptor (ECPR)
o transmembrane protein
o binds protein C adjacent to the thrombomodulin-thrombin complex.
o augments the action of thrombin-thrombomodulin at least fivefold
• Activated protein C (APC)
o Dissociates from EPCR and binds its cofactor, free plasma protein S.
o stabilized APC-protein S complex hydrolyzes and inactivates factors
§ Va and VIIIa
§ slowing or blocking thrombin generation and coagulation.
• Protein S
o cofactor that binds and ___stabilizes APC_____________________
o synthesized the liver and circulates in the plasma in two forms:
§ 40% of protein S is free
§ 60% is covalently bound to complement control protein C4b-binding protein (C4bBP)
o bound protein S _____cannot participate_________ in the protein C anticoagulant
o free plasma protein S can serve as the APC
Antithrombin (AT)
• serine protease inhibitor (serpin)
• binds and neutralizes serine proteases
o thrombin
o Ixa
o Xa
o XIa
o XIIa
o Prekallikrein
o Plasmin
• requires ___heparin____________ for effective anticoagulant activity.
o available from endothelium-associated mast cell granules or as EC heparan sulfate.
• AT’s amplified 2000-fold by binding to heparin
• Heparin induces a conformational change in the AT molecule
o allows binding of activated coagulation factors, causes inactivation
• Inhibition of thrombin, factor X, and other serine proteases by AT is dependent on:
o _____length______________ of the heparin chain
o Longer heparin chains are able to bind both molecules to produce inhibition of thrombin
Heparin Cofactor II (HCII)
• 2nd line inhibitor of thrombin
• primarily targets thrombin
• requires heparin
Other SERPINS
• ZPI
• protein C inhibitor
• a1-protease inhibitor (a1-antitrypsin)
• a2-macroglobulin
• a2-antiplasmin
• PAI-1
Fibrinolytic System
• Activation of coagulation also activates ________fibrin lysis_____________.
• Removal of unwanted fibrin deposits by bound plasmin.
• Re-establishes blood flow and facilitates the healing process
• Two activators of fibrinolysis
o ___tissue plasminogen activator_______ (TPA)
o Urokinase plasminogen activator (UPA),
§ Convert fibrin-bound plasminogen into plasmin
· the principle enzyme of the fibrinolytic system
• delicate balance between the activators and inhibitors in this system.
Plasminogen
• Plasmin is a serine protease that systematically digests fibrin polymer
• Synthesized by the liver__________.
• Circulates as a zymogen.
• Plasminogen is activated to plasmin by tissue plasminogen activator (tPA), urokinase, or other endogenous activators:
o Factor XIa, XIIa fragments, Kallikrein, HMMK
• FDPs: fibrin degradation products – x, y, d, e, D-dimer
•
Plasmin
• Bound plasmin digests clots and restores blood vessel patency.
• free plasmin can be found in the circulation
• capable of digesting plasma fibrinogen, factor V, factor VIII, and fibronectin.
Tissue Plasminogen Activator (TPA)
• ECs secrete TPA
o hydrolyzes fibrin-bound plasminogen
o converting it to ___plasmin____________
o initiating fibrinolysis
• Circulating TPA is bound to inhibitors such as PAI-1
o complexes are cleared from circulation
• synthetic recombinant TPAs
o mimic natural TPA
o “clot-busting”
Urokinase Plasminogen Activator
• Secreted in urinary tract epithelial cells, monocytes, and macrophages
• Doesn’t bind to fibrin
• Becomes incorporated into the mix of fibrin-bound plasminogen and TPA at the time of thrombus formation
• Intrinsic plasminogen activator
• Small amounts circulate in the plasma and thus it plays a minor role in in-vivo fibrinolysis.
• Purified urokinase preparations are widely used to dissolve clots
Plasminogen Inhibitors
Plasminogen activator inhibitor-1 (PAI-1)
• Principle inhibitor of plasminogen
§ Inactivate ____TPA and UPA_________________
• is produced by ECs, megakaryocytes, smooth muscle cells, fibroblasts, monocytes, adipocytes, hepatocytes, and other cell types
• Behaves as an acute phase reactant protein.
§ Increased after major surgery, MI and severe trauma.
• Increased PAI-1 levels correlate with reduced fibrinolytic activity and increased risk of thrombosis
Plasmin inhibitors
Alpha 2 antiplasmin
• Main inhibitor of plasmin and therefore the fibrinolytic system.
• Interferes with absorption of plasminogen to fibrin.
• Same binding site as fibrin therefore the plasmin absorbed onto fibrin is protected from the action. Only free, circulating plasmin is attacked
Fibrinolysis by plasmin
• Proteolytically degrades both ____fibrin and native fibrinogen_____ in circulation.
• Products termed fibrin/fibrinogen degradation products (FDPs)
§ Fragment X (still capable of clotting).
§ Fragment X into Fragment Y + Fragment D
§ Fragment Y into Fragment D + Fragment E
§ Result = 2 D’s and 1 E
• D-Dimer specific to fibrin degradation (to rule out DIC, DVT, PE)
Fibrin Degradation Products FDPs
• Interfere with further thrombin–induced fibrin formation and increase vascular permeability
• Degrade V, VIII, XIIa, fibrinogen, fibrin, and GP1b
• Cleave C3 into fragments and activate complement
Kinin System
• Important in inflammation, ______vascular permeability__________, and chemotaxis
• Involved in contact activation of intrinsic pathway
o Prekallekrein circulates complexed to HMWK
o XIIa (+HMWK) activates prekallekrein to kallekrein and XI to XIa
o Kallekrein and XIa then reciprocally activate XII to XIIa in a feedback system that amplifies the reaction
• Kallekrein
o An enzyme, also activates plasminogen to plasmin
o Acts on HMWK to release ____bradykinin________________
§ Increase vascular permeability
§ Contract smooth muscle
§ Dilate small blood vessels
§ Induce inflammation and pain
§ Release prostaglandins from tissues