The  Coagula+on  Cascade  

Lecture  2  

2

Hemostasis/Coagula+on  

•  Hemostasis  •  Coagula+on  

•  Components  –  Blood  vessels  –  Platelets  –  Coagula+on  proteins  –  Fibrinolysis  –  Serine  protease  inhibitors  

–  Kinin  system  –  Complement  system    

•  Hemostasis  –  Primary  

•  Forma+on  of  the  “platelet  plug”  •  Ini$ally  halts  the  loss  of  blood  

–  Secondary  •  Reinforcement  of  the  platelet  plug  by  fibrin  forma/on  

Balance  between  bleeding  and  cloEng  

Major  components  

Minor  components  

3  

Secondary  Hemostasis  •  Purpose—to  reinforce  the  primary  platelet  plug  (primary  hemostasis)  •  Products  of  secondary  hemostasis  

1.  Thrombin  2.  Fibrin  

•  Defects  lead  to  1.  Decreased  fibrin  produc+on  2.  Reduces  the  stability  of  the  formed  clot  

•  Mediated  by  proteins—coagula+on  factors  •  Coagula+on  factors  are  produced  in  the  liver  

–  Factor  VIII  and  vWF  probably  excep+ons  •  Coagula+on  cascade  describes  the  ac+vity  of  the  coagula+on  proteins  •  Consists  of:  

1.  Substrates—substances  acted  upon  by  an  enzyme  2.  Cofactors—components  that  takes  part  in  a  catalyzed  reac+on  3.  Enzymes  (mostly  serine  proteases)—components  that  speeds  up  a  chemical  

reac+on  

7

Coagula+on  Cascade  

•  Paul  Morawitz—1905  

8  

Tissue  damage  

Thrombokinase    

Prothrombin  +  Ca2+  

FIBRIN  CLOT  

Thrombin  

Fibrinogen  

1.  MacFarlaine—cascade  concept  2.   Davie  and  Ratnoff—waterfall  concept  3.  Modern  Concepts  

9

Surface, XII Prekallikrein HMWK

Simplified  Coagula+on  Cascade  

IX IXa

Tissue Factor VII(VIIa)

VIII VIIIa

Xa X

II Thrombin V Va

Fibrinogen Fibrin

PC

aPC

Platelet Aggregation

Stable Clot

XIII

XIIIa

Central  Player  •  Converts  Fibrinogen  to  Fibrin  •  Platelet  ac+va+on  •  FV,  VIII,  XI  •  FXIII    XI XIa

Coagula+

on  Factors  

Factor   Name   Pathway  I   Fibrinogen   Common  

II   Prothrombin   Common  

III   Tissue  Factor   Extrinsic  

V   Labile  Factor   Common  

VII   Proconver+n  (Stabile  Factor)   Extrinsic  

VIII   An+hemophilic  Factor   Intrinsic  

IX   Plasma  thromboplas+c  component  (PTC)  

Intrinsic  

X   Stuart-­‐Prower  factor   Common  

XI   Plasma  thromboplas+n  antecedent  (PTA)  

Intrinsic  

XII   Hageman  Factor   Intrinsic  

XIII   Fibrin-­‐stabilizing  factor   ?Common  

PK   Prekallikrein  (Fletcher)   Intrinsic  

HMWK   High  molecular  weight  kininogen  (Fitzgerald)  

Intrinsic  

IV   Ionized  calcium  

VI   Not  assigned    

10  

Extrinsic  Pathway  •  Endothelial  damage  occurs  à  allows  TF  to  enter  the  

vessel  lumen  

•  FVII  floa+ng  by  in  the  circula+on  binds  to  TF                          [+  (Ca2+)]  forming  a  TF:VIIa  complex  

•  TF:VIIa  complex  converts  FX  to  FXa  

•  Therefore  the  [TF:VIIa  +Ca  +  PF3]  complex  is  also  known  as  the  “EXTRINSIC  TENASE”  

•  FXa  +  Va  converts  prothrombin  (FII)  to  thrombin  (IIa)    

–  This  results  in  the  ini+al  burst  of  thrombin  (IIa)  

•  Thrombin  feeds  back  up  to  ac+vate  FV,  FVIII,  FXI,  FXIII  

•  TF:VIIa  complex  also  converts  FIXα  to  FIXa  

•  FIXa  +  FVIIIa  also  converts  FX  to  FXa  –  [FIXa  +  FVIIIa  +  Ca2+  +  PF3]  is  also  known  as  

the  “INTRINSIC  TENASE”  

•  Fig  24-­‐14  

11

Intrinsic  Pathway  •  Consist  of  

1.  PK  –  prekallikrein  (Fletcher)  2.  HMWK  –  high  molecular  weight  kininogen  (Fitzgerald)  3.  FXII  –  Hageman  factor  4.  FXI  –  plasma  thromboplas+n  antecedent    

 •  Characteris+cs  

1.  Ini+ated    in  vivo    by  exposure  to  subendothelial  collagen  which  has  a  nega+ve  charge  2.  In  the    in  vitro    by  glass,  kaolin,  celite,  ellagic  acid,  silica  3.  Ini+ated  under  pathologic  condi+ons  by  exposure  to  lipopolysaccharide  (bacteria)  4.  PK,  HMWK,  FXII  do  NOT  play  a  significant  role  in  in  vivo  hemostasis    

a.  Pa+ents  with  PK,  HMWK,  FXII  à  do  NOT  bleed      (but  tend  to  thrombose)  b.  Increased  interac+on  of  these  factors  with  

–  Complement  system  –  Kinin    system  –  Fibrinoly+c  system    

12  

Intrinsic  Pathway  1.  Exposure  to  nega+vely  charged  collagen  

(subendothelium  or  phospholipids)  

2.  Ini+ally  FXII  is  auto-­‐ac+vated  to  XIIa  and  binds  to  high  molecular  weight  kininogen  (HMWK)    

–  Forms  a  complex  that  binds  to  the  subendothelial  collagen  layer  

3.  FXIIa  in  the  presence  of  prekallikrein  (PK)  and  HMWK  converts  FXI  to  FXIa  

4.  FXIa  +  Ca2+  converts  FIX  to  FIXa    

5.  [FIXa  +  FVIIIa  +  Ca2+  +  PF3]  complex    à  “INTRINSIC  TENASE  complex”  converts  FX  to  FXa  

6.  FVIIIa  –  a  cofactor  orients  FIXa  and  FXa  in  the  proper  orienta+on  to  alach  to  the  platelet  phospholipid  surface  (PF3)  

•  Figure  24-­‐15  

13

Complexes  

Figure  24-­‐20  Intrinsic  and  Extrinsic  Tenase  Complexes  

 Figure  24-­‐21  

Prothrombinase  Complex  

14

Common  Pathway  

•  [FXa  +  FVa  +  PF3  +  Ca2+]  –  prothrombinase  complex    –    converts    prothrombin  (II)  to  thrombin  (IIa)    

•  FVa  –  a  cofactor  posi+ons  the  tenase  complex  (extrinsic  or  intrinsic)  to  interact  with  FII  on  the  platelet  phospholipid  surface  

•  Prothrombin  à  THROMBIN  (IIa)  

•  IIa  à  circula+ng  fibrinogen  (FI)  to  soluble  fibrin  monomers  

•  IIa    à  FXIII  to    FXIIIa    

•  FXIIIa  cross-­‐links  the  fibrin  monomers  into  an  insoluble  fibrin  polymer  (CLOT)    

15

Figure  24-­‐16  

Addi+onal  Fact  about  the  Common  Pathway  

•  Intrinsic  and  extrinsic  pathways  converge  on  the  common  pathway  •  Involves  the  intrinsic  and/or  extrinsic  tenase  complex  •  Prothrombinase  complex    à  Prothrombin  to  THROMBIN    

•  Thrombin    has  mul$ple  func$ons  1.  Converts  fibrinogen  to  fibrin  2.  Ac+vates  factor  XIII  3.  Ac+vates  platelets  4.  Ac+vates  FVIII  5.  Ac+vates  FV  6.  Ac+vates  FXI  

7.  Ac+vates  Protein  C  8.  Binds  to  Thrombomodulin  9.  Mitogen  for  fibroblasts  

Prothrombinase  Complex  

Xa  

Va  

Platelet  (PF3)  

Vitamin  K-­‐Dependent  Factors  

•  II—Common  pathway  •  VII—Extrinsic  pathway  •  IX—Intrinsic  pathway  •  X—Common  pathway  

•  C—Protein  C  pathway,  inhibitor  •  S—Protein  C  pathway,  inhibitor  •  Z—Probably  a  cofactor      

–  Protein  Z-­‐related  inhibitor  (ZPI)—inhibits  factor  Xa  and  XIa  

17

prothrombo+c  

an+thrombo+c  

Mechanism  of  Ac+on  of  VKDF’s  •  Contain  glutamic  acid  residue  

–  Terminal  GLA  located  at  the  COOH  terminal  end  

–  γ-­‐carboxyglutamic  acid  

•  Allows  calcium  to  alach  the  VKDF’s  to  the  phospholipid  surface  of  the  platelet  

•  Absence  of  vitamin  K  à  to  reduced  func+on  of  the  VKDF’s  

–  PIVKA’s—”proteins  induced  by  vitamin  K  antagonists”  

•  Func+on  of  the  GLA  domains  1.  Binds  the  complex  to  PF3  using  

Ca2+  as  a  bridge  2.  Folds  the  protein  into  the  proper  

configura+on  for  complex  forma+on  

18  

Ac+on  of  Thrombin  on  Fibrinogen  

•  Fibrinogen  molecule  consists  of  6  chains  –  Aα,  Bβ,  γγ  chains  

•  Thrombin  proteoly+cally  cleaves  a.  Fibrinopep+de    A  from  the  Aα-­‐

chain  b.  Fibrinopep+de  B  from  the  Bβ-­‐

chain  Ø  Leads  to  a  forma+on  of  a  

soluble  fibrin  monomer  (from  the  remaining  chains)  

•  Fibrin  monomers  polymerize  “end-­‐to-­‐end”  and  “side-­‐to-­‐side”    

•  Factor  XIIIa  catalyzes  the  cross-­‐linking  of  glutamine  and  lysine  bonds  à  polymeriza/on  of  soluble  fibrin  monomers  à  to  insoluble  fibrin  meshwork  

19

Forma+on  of  a  Fibrin  Clot  

1.  Thrombin  proteolyzes  fibrinopep+des  A  and  B  from  fibrinogen  to  yield    soluble  fibrin  monomer  

2.  Soluble  fibrin  monomer  then  associates  side-­‐to-­‐side  and  end-­‐to-­‐end  to  form  fibrin  polymers  

3.  Thrombin-­‐ac+vated  FXIIIa  covalently  cross-­‐links  the  fibrin  polymers  into  an  increasingly  complex  structure  and  an  ul+mately  insoluble  clot  

•  D  =  peripheral  domains  •  E  =  central  domain  

20  

Fibrinolysis  •  Presence  of  fibrin  triggers  the  ac+va+on  of  plasminogen  to  plasmin  

•  Plasminogen  is  the  target  substrate  of  the  ac+va+on  systems  and  ater  cleavage  becomes  the  ac+ve  enzyme  plasmin  

•  Plasmin  then  splits  fibrin  and  fibrinogen  into  fragments  –  Interfere  with  thrombin  ac+vity,  platelet  func+on,  and  fibrin  polymeriza+on,  leading  to  clot  

dissolu+on    

21  

Func+on  of  Fibrinolysis  •  Fibrinolysis  and  Repair  

1.  Fibrinolysis  1.  Physiologic  process  of  removing  unwanted  fibrin  deposits  2.  Gradual  progressive  enzyma+c  cleavage  of  fibrin  to  soluble  

fragments  3.  Fragments  are  removed  by  RES  4.  Re-­‐establishes  blood  flow  in  vessels—maintains  patency  5.  Involves  proteoly+c  diges+on  of  fibrinogen  and  fibrin  by  plasmin  6.  In+mately  related  to  fibrin  forma+on  

2.  Repair  •  Involves  release  of  PDGF    

Key  Player  in  Fibrinolysis  •  Plasminogen  

–  Becomes  bound  to  fibrin  at  the  +me  of  fibrin  polymeriza+on  –  Bound  plasminogen  is  converted  to  a  two-­‐chain  ac+ve  plasmin  molecule  

•  Plasmin  –  Serine  protease  that  systema+cally  digests  fibrin  

•  Also  digests  FV,  FVIII,  Fibrinogen  –  Binding  to  fibrin  clot  via  lysine  bonds  prevents  systemic  ac+vity  –  Plasmin  is  the  main  enzyme  in  fibrinoly+c  system  

23  

Ac+vators  and  Inhibitors  of  Fibrinolysis  

Ac/vators  

1.  Tissue  plasminogen  ac+vator  (t-­‐PA)  –  Plasmin  acts  primarily  on  fibrin,  but  is  a  

nonspecific  destroyer  •  Hydrolyzes  plasminogen  bound  to  

the  fibrin  clot  à  ini+ates  fibrinolysis  

•  2  forms  of  tPA  –  Synthe+c  recombinant  TPA  

used  in  thromboly+c  therapy  –  Secreted  from  endothelial  

cells  

2.  Urokinase  –  Secreted  from  urinary  tract  epithelial  

cells—intrinsic  plasminogen  ac+vator  –  Does  not  bind  firmly  to  fibrin,  so  it  is  not  

extremely  effec+ve  

3.  Streptokinase  –  Extrinsic,  therapeu+c  ac+vator  

Ac+vators  and  Inhibitors  of  Fibrinolysis  

Inhibitors  

1. Plasminogen  Ac+vator  Inhibitor  (PAI-­‐1)  – Prevents  t-­‐PA  and  urokinase  from  ac+va+ng  free  fluid-­‐phase  plasminogen  

 2. α2-­‐An+plasmin  

– α2AP  rapidly  inhibits  free  plasma    

– Produced  in  the  liver  

PAI-­‐1  

Naturally  Occurring  Inhibitors  (Regulators)  of  the  Coagula+on  Cascade    

1.  An+thrombin  (AT)—major  regulator  of  coagula+on  cascade  a.  Inhibits  IIa,  Xa,  and  the  other  serine  

proteases  b.  Serves  as  a  cofactor  for  heparin  and  other  

heparinoids  

2.  Ac+vate  Protein  C  (aPC)—inhibits  Va  and  VIIIa  a.  PC—when  ac+vated  becomes  APC  b.  PS—serves  as  a  cofactor  to  PC  

3.  Heparin  Cofactor  II  (HCII)—Similar  to  AT,  but  only  inhibits  IIa  

4.  Tissue  Factor  Pathway  Inhibitor  (TFPI)—inhibits  extrinsic  tenase  

5.  Thrombin  Ac+vatable  Fibrinolysis  Inhibitor  (TAFI)—thrombin  ac+vatable  fibrinlysis  inhibitor  

6.  Thrombomodulin  (Tm)—thrombomodulin—regulates  thrombin—alenuates  thrombin’s  procoagulant  ac+vity  conver+ng  it  into  an  an+coagulant  

26

Regulators  of  Coagula+on  Cascade  

TFPI   Protein  C/S  An+coagulant  Pathway  

27  

Kallikrein-­‐Kinin  System  

•  PK  and  HMWK  needed  to  enhance  or  amplify  the  contact  factors  –  XIIa  +  HMWK  converts  PK  to  

kallikrein    –  Kallikrein  feeds  back  to  

accelerate    XII  à  XIIa  –  Speeds  up  the  intrinsic  ac+va+on  

•  Ac+va+on  of  FXIIa  acts  as  the  common  link  between  A.  Fibrinoly+c  system  B.  Kinin  system  C.  Complement  system  D.  Coagula+on  system  

28

Kallikrein-­‐Kinin  System  

•  System  of  proteins  that  when  ac+vated  à  to  the  release  of  vasoac+ve  kinins  

•  Kinins  are  ac+vated  by  Kallikrein  à  release  both  HMWK  and  LMWK    •  Kinins  are  involved  in  physiological  and  pathological  processes  

–  Regula+on  of  blood  pressure  and  flow  (via  modula+on  of  the  renin-­‐angiotensin  pathway  –  Blood  coagula+on  –  Cellular  prolifera+on  and  growth,  Angiogenesis,  Apoptosis,  and  Inflamma+on  

•  Kinin  ac+on  on  endothelial  cells  à  1.  Vasodila+on  2.  Increased  vascular  permeability  3.  Release  of  tPA  4.  Produc+on  of  nitric  oxide  (NO)  5.  Mobiliza+on  of  arachidonic  acid  à  PGI2  produc+on  6.  Chemotaxis  

•  In  hemostasis  –bradykinin  is  released  from  HMWK  

29  

Hageman  Factor    Kinin—CloEng—Kinin—Fibrinoly+c—Complement  Systems  

30  

Cell-­‐based  Model  of  Hemostasis    

•  Proposed  to  replace  the  tradi+onal  cascade  

•  Specific  cellular  surface  receptors  for  coagula+on  proteins  promote  hemostasis  

•  Occurs  in  3  phases  1.   Ini/a/on  2.   Amplifica/on  3.   Propaga/on    

31

The Role of Platelets in Hemostasis

Collagen Other factors TF

Thrombin

Platelet

Platelet Platelet

Platelet

Platelet

Activated platelet

Activated platelet

Activated platelet

Platelet Platelet

Platelet Platelet

Adhesion

Aggregation

Contraction

Secretion

Primary Haemostasis

=

Activated platelet

Activated platelet

Activated platelet

Activated platelet

This plug of activated platelets, localised to the site of injury, provides the phospholipid surface upon which Secondary Haemostasis takes place

Initiation, Amplification, Propagation

VIIIa IXa

Hoffman M & Munroe DM. A cell-based model of hemostasis. Thromb Haemost 2001; 85: 958-965

+ activates various factors

Initiation

Amplification

Propagation

Control Mechanisms in Coagulation

VIIIa IXa

+ activates various factors

APC/PS

TFPI

Antithrombin

CELL  BASED  MODEL  OF  HEMOSTASIS  

Hemostasis

Ø Vasoconstric+on  Ø Platelet  plug  forma+on  

l Ac+va+on  l Adherence  l Aggrega+on  

Ø Stabiliza+on  of  platelet  plug  l Coagula+on  cascade  l Fibrin    

XII  

XIIa  

XI  XIa  

IX  

X   X  

VII  

IIa  II  

Fibrin  Fibrinogen  

VIIa  TF  

TF  

IXa  VIIIa  

Xa  Va  

Cascade  Model  

XII  

XIIa  

XI  XIa  

IX  

X   X  

VII  

IIa  II  

Fibrin  Fibrinogen  

VIIa  TF  

TF  

IXa  VIIIa  

Xa  Va  

Intrinsic

Common

Extrinsic

XII  

XIIa  

XI  XIa  

IX  

X   X  

VII  

IIa  II  

Fibrin  Fibrinogen  

VIIa  TF  

TF  

IXa  VIIIa  

Xa  Va  

Tests  

aPTT

PT

TT

Cell Based Theory

Ø Platelets  and  IIa  are  central  

Ø Sequence  l Ini+a+on  l Amplifica+on  l Propaga+on  

XII  

XIIa  

XI  XIa  

IX  

X   X  

VII  

IIa  II  

Fibrin  Fibrinogen  

VIIa  TF  

TF  

IXa  VIIIa  

Xa  Va  

Xa  Va  

XIa  

IIa  

Fibrin  Fibrinogen  

VIIa  TF  

IXa  VIIIa  

Xa  

IIa  

Fibrin  Fibrinogen  

XIa  

VIIa  TF  

IXa  VIIIa  

Plt  

Xa   IIa  VIIa  TF  

Xa  Va  

IIa  Plt  

Xa  

IXa  VIIIa  

Xa  Va  

IIa  

Plt  

Plt  

Fg  

Fb  

vWF  

Fb  

Plt  

Plt   Fb  

Fb  

Fb  

vWF  

Tissue factor bearing cell

Subendothelial collagen

Plt  

IIa  

Ini+a+on  

Amplifica+on  

Propaga+on  

Platelet activation

Ø Triggered  by  l Sub-­‐endothelial  collagen  l IIa  l ADP  l TXA2  l 5HT  l Epinephrine  

Platelet activation

•  Results in; – Greatly increased surface area

– Dense granules release Ca2+, 5HT, TXA2, ADP

– Alpha granules release factor V, fibrin, VWF

Platelet activation

•  Platelet phospholipid bilayer is actively controlled

•  Resting internal surface is procoagulant •  Bilayer everts during activation, exposing

procoagulant surface

Plt  

Xa   IIa  VIIa  TF  

Xa  Va  

Plt  

XIa  

IIa  

IXa  

VIIIa  

Va  

Fg  vWF  

IIa  Plt  

Xa  

IXa  VIIIa  

Xa  Va  

Plt  

Plt  

Plt  IIa  

TXA2

5HT

Plt  

Plt  

Fg  

Fb  

vWF  

Fb  

Plt  

Plt   Fb  

Fb  

Fb  

vWF  

Tissue factor bearing cell

Subendothelial collagen

IIa  

IIa  

IIa  

Plt  

IIa  

IXa  VIIIa  

ADP

Propaga+on