Regulatory Mechanisms and Fibrinolysis Student Outline.docx

MLS 340

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
    • Cells that express TF (usually extravascular)
    • Platelets, usually intravascular
  • 2 Phases
    • initiation
      • Occurs on TF expressing cells
      • 3-5% thrombin
    • 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
    • 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
    • By low levels of thrombin (initiation)
    • Adhering to exposed collagen
    • This creates COAT platelets (collagen and thrombin)
  • COAT Platelets
    • higher level of procoagulant activity than platelets exposed to collagen alone
    • provide a surface for formation and amplification of complexes
    • Both platelets and tissue factor-bearing cells are essential for physiologic coagulation
    • 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
      • factor xa
      • VIIa:TF complex
  • 2 Step Process
      • First TFPI binds factor Xa and inactivates it
      • 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
    • activates factors v, viii, xi, and xiii
    • Propagating more thrombin generation
  • In intact normal vessels
    • thrombin avidly binds thrombomodulin
    • triggers protein c regulatory system
      • revises thrombin’s function from a procoagulant enzyme to an anticoagulant.
  • EC protein C receptor (ECPR)
    • transmembrane protein
    • binds protein C adjacent to the thrombomodulin-thrombin complex.
    • augments the action of thrombin-thrombomodulin at least fivefold
  • Activated protein C (APC)
    • Dissociates from EPCR and binds its cofactor, free plasma protein S.
    • stabilized APC-protein S complex hydrolyzes and inactivates factors
      • Va and VIIIa
      • slowing or blocking thrombin generation and coagulation.
  • Protein S
    • cofactor that binds and stabilizes apc
    • 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)
    • bound protein S cannot participate in the protein C anticoagulant
    • free plasma protein S can serve as the APC

Antithrombin (AT)

  • serine protease inhibitor (serpin)
  • binds and neutralizes serine proteases
    • thrombin
    • Ixa
    • Xa
    • XIa
    • XIIa
    • Prekallikrein
    • Plasmin
  • Requires heparin for effective anticoagulant activity.
    • 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
    • allows binding of activated coagulation factors, causes inactivation
  • Inhibition of thrombin, factor X, and other serine proteases by AT is dependent on:
    • length of the heparin chain
    • 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
    • tissue plasminogen activator (TPA)
    • 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:
    • Factor XIa, XIIa fragments, Kallikrein, HMMK

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.
  • x, y, d, e d-dimers

Tissue Plasminogen Activator (TPA)

  • ECs secrete TPA
    • hydrolyzes fibrin-bound plasminogen
    • converting it to plasmin
    • initiating fibrinolysis
  • Circulating TPA is bound to inhibitors such as PAI-1
    • complexes are cleared from circulation
  • synthetic recombinant TPAs
    • mimic natural TPA
    • “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
    • Prekallekrein circulates complexed to HMWK
    • XIIa (+HMWK) activates prekallekrein to kallekrein and XI to XIa
    • Kallekrein and XIa then reciprocally activate XII to XIIa in a feedback system that amplifies the reaction
  • Kallekrein
    • An enzyme, also activates plasminogen to plasmin
    • Acts on HMWK to release bradykinin
      • Increase vascular permeability
      • Contract smooth muscle
      • Dilate small blood vessels
      • Induce inflammation and pain
      • Release prostaglandins from tissues