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A. Overview
[Figure] "The Clotting Pathways" navigator

  1. The clotting pathway is presented in reverse order for ease of understanding
  2. Platelets are required in initial plugging of vessel hole and for clotting protein function
  3. A clot composed of fibrin is then built up on the platelets, which eventually dissolve
  4. The key step in fibrin clot formation is the conversion of fibrinogen (340K) to fibrin
  5. Thrombin is the enzyme which catalyzes this reaction
  6. Most of the coagulants circulate in inactive (precursor) forms
    1. This serves to control and limit coagulation
    2. In addition, anticoagulant proteins circulate to further regulate clot formation
  7. There are two known pathways for activating thrombin from prothrombin [2]
    1. These are the "intrinsic" and the "extrinsic" pathways
    2. Older view proposed that these pathways were separate
    3. Pathways are likely integrated: extrinsic pathway initiates the clotting cascade
    4. The physiological role of the intrinsic pathway is not entirely clear [1]
  8. Production of Clotting Proteins
    1. Most of the coagulation proteins are produced in the liver (in precursor or zymogen form)
    2. Endothelial cells also produce various clotting proteins
    3. Coagulation precursors are called Factors
    4. These factors require activation (usually proteolytic cleavage)
    5. Activated factors given suffix "a" (such as IIa for activated factor IIa)
  9. "Decision" to clot depends on balance between anti- and procoagulant proteins
    1. Each vascular bed appears to have a unique balance between specific anti- and pro- coagulant factors [2]
    2. Clotting disorders can usually be understood in terms of balance between these factors
  10. Genetic factors contribute to 40-70% of the variation in levels of plasma clotting factors [18]

B. Platelet Plug [4] navigator

  1. When blood vessels are traumatized, the endothelial lining is disrupted [5]
    1. Disruption of endothelial cells leads to exposure of subendothelial matrix
    2. Collagens, fibronectins, laminin and vWF is exposed
    3. Key "early" platelet adhesion molecules recognize these substances
    4. Early proteins include GPIb/IX/V and VP VI, integrins a2b1, a5b1, and a6b1
    5. Platelet activation ensues (with help from other activators such as ADP)
    6. Platelet activation culminates in conversion of GPIIb/IIIa from resting to active state
  2. Von Willebrand Factor (vWF) [6]
    1. vWF is required for initial platelet binding and aggregation
    2. Large protein (2050 amino acid monomer) produced by endothelial cells
    3. Forms polymers through disulfide bonds when released
    4. In circulation vWF is cleaved by plasma metalloproteinase in a shear-dependent maner
    5. Cleavage decreases size of polymers and forms dimers of 176K and 140K polypeptides
    6. Multimers are thrombogenic; they bind well to platelets and initiate plugs
    7. Shear stress in vessels increases binding of mutlimeric vWF complexes
    8. vWF binds to platelet protein GP IIa/IX/V and activated IIb/IIIa to stabilize interactions
    9. The vWF multimers are cleaved in serum by vWF cleaving protease (176K and 140K)
    10. Failure to cleave vWF multimers can produce thrombotic thrombocytopenic purpura [6,9]
  3. Platelet - Subendothelial Interactions [5]
    1. Von Willebrand Factors bound to endothelium are critical to good platelet binding
    2. Platelets also adhere to collagen types I, III (interstitial) and IV (basement membrane)
    3. Platelet glycoprotein Ia/IIa is a major receptor for collagen
    4. Platelets localize to the plug within seconds, narrowing the stream of leaking blood
    5. A series of integrins on the platelet membrane mediate adhesion and aggregation [2]
    6. Glycoprotein alpha2b/ß3a (GPIIb/IIIa) is last step in binding to a variety of proteins
    7. Platelet granule content deposition and shape changes strongly activated by thrombin
    8. Platelets also contain Factor Va, likely physiologically more significant than blood Va
  4. Enhancement of Platelet Aggregation
    1. Thromboxane A2 (also potent vasoconstrictor)
    2. Adenosine Diphosphate (ADP)
    3. Thrombin (and Thrombin bound to Thrombomodulin)
    4. Serotonin
    5. Subendothelial collagen, laminin, and fibronectins
    6. VWF multimers
    7. Epinephrine
  5. A lipid surface is required for rapid coagulation, and platelets provide this function
    1. In platelet poor plasma, coagulation times are on the order of 2-4 minutes
    2. In the presence of sufficient platelets, coagulation times are ~1 minute
  6. Within 1 minute of vessel damage, in the presence of platelets, fibrin strands form
    1. The platelet plug remains intact for a few hours
    2. It is eventually completely replaced by the fibrin clot
  7. Blocking platelet plug formation
    1. Antibodies to integrin 2b/3a are extremely potent inhibitors of platelet actions
    2. "Humanized" form of anti-GP 2b/3a is Abciximab (ReoPro®) very potent
    3. RGD (arginine-glycine-aspartate) peptides block platelet binding to fibronectin
    4. Aspirin and other irreversible cyclooxygenase inhibitors are potent blockers of platelet plug formation

C. Fibrin Clotting Pathwaynavigator

  1. The final clot is formed on the platelet surface by conversion of fibrinogen (340K) to fibrin
    1. Initially the fibrin is layed down in weak strands
    2. Strength of the clot is provided by cross-linking the fibrin strands
    3. Cross-linking is carried out by Factor XIII
  2. The converting enzyme is a protease called thrombin (Factor IIa)
    1. Thrombin (t1/2 seconds) is a vitamin K dependent protease
    2. Thrombin is generated from prothrombin (t1/2 days) by "Prothrombinase"
    3. Thrombomodulin binds to thrombin and reduces its function
    4. Thrombomodulin limits clotting; high levels are protective against clots
  3. Prothrombinase is calcium dependent and consists of Factors Xa and Va
    1. Factor Xa is produced by proteolysis of Factor X
    2. Extrinsic Pathway Factor Xa Generation: Factors VIIa and Tissue Factor (Factor III or TF)
    3. Intrinsic Pathway Factor Xa Generation: Factors IXa and VIIIa with PL and calcium
    4. Factor Va is from V, by action of low levels of circulating thrombin (always present)
    5. Maximum thrombin production occurs after clot formed
  4. The critical and rate limiting point is conversion of Factor X to Xa
  5. Thrombin Effects
    1. Direct activation of platelets (procoagulant)
    2. Feedback amplification of Factors V, VIII, and XI to Va, VIIIa, and XIa
    3. Therefore has effects on both extrinsic and intrinsic pathways
    4. Critical role in conversion of Factor XIII to XIIIa
    5. Factor XIIIa is a transglutaminase that stabilizes clot by crosslinking the fibrin
  6. A variety of coagulation inhibitors play key roles in regulating these processes (below)

D. Extrinsic (Initiation) Pathwaynavigator

  1. Clotting is initiated by exposure of whole blood to tissue factor (TF or Factor III)
    1. TF is a membrane protein found abundantly in cells surrounding the vascular bed
    2. This is the mechanism for trauma induced clotting
    3. In vitro, clotting occurs in plasma to which phospholipid, TF and Ca have been added
  2. Factor VII (50K) is proconvertin: binds Ca and TF
    1. Factor VII circulates in a partially activated form (Factor VIIa)
    2. Feedback amplification for conversion of VII to VIIa occurs with IXa and Xa
    3. Note that Factor VIIa has a t1/2 ~2.5 hours, much longer than most factors
  3. Factor VIIa complexed with TF converts factors IX and X to IXa and Xa
    1. The conversion of X to Xa with Factor VII and TF is much more rapid than that by IXa
    2. Factor IXa (with VIIIa, extrinsic pathway) can also convert Factor X to Xa
  4. Factor V is the other critical component of the Xa-Va prothrombinase complex
    1. Factor Xa on phospholipid surface activates V to Va
    2. Thrombin (in solution and on surface) also activates V to Va
  5. Factors Xa and Va with phospholipid and calcium convert prothrombin to thrombin
  6. Tissue factor pathway inhibitor (TFPI) plays a role in regulating pathway (see below)
  7. Factor VII Genotype [8,19]
    1. Factor VII (FVII) plays key role in regulation of coagulation pathway
    2. Activated FVII levels have been associated with age and other risk factors
    3. FVII is a highly polymorphic gene; these mutations can affect serum FVII levels
    4. H7H7 (QQ) genotype has lowest risk of MI (8-40% of RR genotype)

E. Intrinsic Pathway Coagulation navigator

  1. Unclear physiological role in the initiation of coagulation
    1. Likely plays a role in maintaining and amplifying clotting
    2. Deficiencies in intrinsic pathway proteins are asymptomatic except F XI
    3. F XI deficiency leads to a moderately severe bleeding disorder
  2. Initiation of intrinsic pathway by conversion of Factor XII (Hageman factor) to XIIa
  3. Factor XIIa then converts factor Factor XI to XIa
    1. Factor XI can also be activated by thrombin (Factor IIa)
    2. Prekallikrein and high-molecular weight kininogen are required for contact XI --> XIa
    3. Activated Hageman factor (XIIa) converts prekallikrein to kallikrein, a serine protease
  4. Factor XIa then converts factor IX (K dependent) to IXa (see above)
    1. Calcium is required for this reaction
    2. Tissue Factor (F-III) also plays a role here and links intrinsic and extrinsic pathways
  5. Together, F-IXa and F-VIIIa convert F-X to F-Xa
    1. Complex of Factor IXa and VIIIa is called the tenase complex
    2. This occurs on a membrane surface, probably platelets in vivo
    3. Factor VIII to VIIIa conversion is by thrombin (positive feedback)
    4. Activated protein C, in the presence of protein S, inactivates F-VIIIa
    5. Elevated Factor VIII levels also confer ~7X risk for recurrent DVT after initial DVT [10]
  6. Integrated newer view of clotting
    1. Factors VIII, IX and XI required for maintaining hemostasis initiated via extrinsic route
    2. Factor IX is activated by Tissue Factor (TF), which also activates F VII (extrinsic path)
    3. Factor IX (and VII) activation are inhibited by tissue factor pathway inhibitor (TFPI)
    4. TFPI main role is inhibition of extrinsic pathway (see below)
    5. Thrombomodulin reduces thrombin activity

F. Cofactorsnavigator

  1. Both pathways require Vitamin K and calcium ("Factor IV")
  2. Vitamin K dependent factors are II (prothrombin), VII (extrinsic), IX (intrinsic), and X
  3. Vitamin K is necessary for the gamma carboxylation of glutamic acid residues
    1. Gamma carboxylation of Glu forms dicarboxates or Gla residues
    2. Gla residues are found only in the N terminal portions of the above molecules
    3. They are also found on the Vitamin K dependent anti-coagulants Proteins S and C
    4. Vitamin K is oxidized during the carboxylation steps and must be recycled
  4. The warfarin anticoagulants (warfarin, coumarin) block the recycling of vitamin K
    1. They prevent reduction of Vitamin K
    2. Therefore, active vitamin K is depleted and coagulation inhibited
  5. Calcium is required for the dicarboxylate - anion bridges
  6. These form between X/IXa and phospholipid surfaces

G. Natural Coagulation Inhibitors [2] navigator

  1. Endothelium
    1. Intact, healthy endothelium is a natural coagulation inhibitor
    2. Expresses adenosine diphosphatase (ADPase), which degrades ADP
    3. Also expresses thrombomodulin, heparin-like molecules, TPA, prostacyclin, annexin V
    4. All of these prevent platelet binding, aggregation, and activation
    5. Endothelial injury leads to adhesion molecule expression and decreases in anti- platelet and other anti-clotting molecules, as well as appearance of TF
    6. Adhesion molecules permit leukocyte migration
  2. Antithrombin (AT)
    1. AT (Formerly antithrombin III) is a 58K serine protease inhibitor
    2. Inhibits factors IXa and Xa activity and also blocks XIa and IIa action
    3. Therefore it increases the partial thromboplastin time (PTT or APTT)
    4. AT's Arg active site can block Serine protease triad of all clotting proteases
    5. Platelet bound Xa is highly resistant to proteolysis by AT
    6. Heparin (like substance) required for efficient function of AT
    7. Heparin interacts with AT in the AT-Lysine rich region
    8. Heparin binding to AT increases the rate at which AT inhibits F-Xa and thrombin ~1000X
    9. Vascular bed heparin-like substance is possibly heparan sulfate glycosaminoglycan
  3. Protein C
    1. Inactive zymogen activated by proteolysis by thrombin-thrombomodulin complex
    2. Protein C needs to bind to endothelial protein C receptor and thrombomodulin for activation
    3. Activated Pro C attacks factors VIIIa and Va (enhanced by activated Protein S)
    4. Factors IXa and Xa protect VIIIa and Va against Protein C attack
    5. Protein C is also vitamin K dependent and fibrinolytic
    6. Endothelial protein C receptor and thrombomodulin are both downregulated in severe meningococcal sepsis [11]
  4. Protein S
    1. Contains Gla (that is, Vitamin K dependent dicarboxylated amino acid)
    2. Acts as nonproteolytic cofactor of Protein C
    3. May actually prevent Xa from inhibiting Pro C
    4. Normally some Protein S is complexed with complement C4b
    5. In inflammation, most of factor S is complexed in inactive form
    6. This may explain the hypercoagulable states often found in inflammatory diseases
  5. Thrombomodulin (TM)
    1. TM is an endothelial cell membrane glycoprotein receptor for thrombin
    2. Thrombin binding to TM leads to protein C activation
    3. However, TM does not block thrombin induced platelet activation or fibrin formation
    4. TM is excreted in kidney and in liver
    5. Soluble thrombomodulin may be a marker for endothelial damage
    6. Soluble (serum) thrombomodulin levels are inversely related to risk of first coronary artery disease [12]
  6. Tissue Factor Pathway Inhibitor (TFPI)
    1. Endogenous inhibitor mainly of tissue factor (F III) activated clotting
    2. Also blocks intrinsic pathway IX--> IXa conversion
    3. TFPI levels are usually limiting allowing a titration of response to vessel injury
    4. In presence of sufficient TF (vessel injury), TFPI is overwhelmed by complex of TF/VIIa
  7. alpha-2-macroglobulin
    1. 726K homotetramer of 180K
    2. Forms irreversible complex with thrombin (Factor IIa), rapidly cleared from circulation
  8. Annexin V [7]
    1. Annexin V also called placental anticoagulant protein 1, vascular anticoagulant alpha
    2. Appears to be important endothelial and placental anticoagulant protein
    3. Anti-phospholipid Ab reduce levels of annexin V and accelerate plasma coagulation

H. Extracellular Signals Influencing Endothelial Cell Clotting Functions [2]navigator

  1. Inflammatory Signals
    1. Tumor necrosis factor alpha (TNFa)
    2. Interleukin 1
    3. Interleukin 6
  2. Factors Involved in Angiogenesis and Tissue Repair
    1. Tranforming growth factor ß (TGFß)
    2. Vascular endothelial growth factor (VEGF)
    3. Platelet-Derived growth factor (PGDF)
  3. Shear Stress
    1. Increased thrombomodulin (anticoagulant)
    2. Increased tissue-type plasminogen activator (anticoagulant)
    3. Increased nitric oxide synthetase (anticoagulant)
    4. Increased tissue factor (procoagulant)
  4. Hypoxia
    1. Increased plasminogen-activator inhibitor type 1 (PAI-1; procoagulant)
    2. Decreased tissue-type plasminogen activator (TPA; anticoagulant)
  5. Airplane travel may influence endothelium and/or pathways to increase coagulation [17]

I. Fibrinolysis
[Figure] "Clot Degradation" navigator

  1. Eventually all clots are broken down with healing or fibrous tissue deposition
  2. The enzyme plasmin is responsible for breakdown of cross-linked fibrin
    1. Plasmin is produced from the inactive precursor plasminogen
    2. Plasminogen activators (enzymes) are either intrinsic or extrinsic
    3. Extrinsic fibrinolytic agents are used for therapy
  3. Extrinsic Fibrinolytic Pathway
    1. Urokinase like plasminogen activator: uPA
    2. Tissue type plasminogen activator: TPA
    3. Streptokinase is produced from bacteria and also increases plasmin production
  4. Plasmin Inhibitors
    1. Intrinsic Inhibitor: plasminogen activator inhibitor (PAI-1)
    2. alpha2-antiplasmin (circulates in serum)
    3. Medication: Epsilon (e)-aminocaproic acid (EACA, Amikar®)
  5. PAI-1
    1. PAI-1 is a pro-coagulant protein which inhibits the activity of plasmin
    2. PAI-1 is an anti-protease belonging to serine protease inhibitor superfamily
    3. PAI-1 is produced in response to thrombin as well as various inflammatory stimuli
    4. These inflammatory stimuli include cytokines, TNF, lipopolysaccharide
    5. In addition, PAI-1 is stimulated by apolipoprotein-B containing lipid particles
    6. In addition, PAI-1 (and TPA) is stimulated by insulin [16]
  6. Regulation of PAI-1
    1. The PAI-1 gene is transcriptionally regulated
    2. The PAI-1 promoter contains a polymorphic site which leads to variable PAI-1 mRNAs
    3. The 4G genotype is associated with increased levels of serum PAI-1 (versus 5G) [13]
    4. Increased levels of PAI-1 and the 4G genotype are associated with increased death due to meningococcal infection [13]
    5. The 4G genotype is also associated with increased risk of sepsis rather than meningitis in patients who contract meningococcus [14]
    6. The 4G genotype is associated with increased risk of death from sepsis of any cause [15]
  7. Fibrin split products (Fibrin Degradation Products, FDP) and D-Dimers
    1. Increase when marked fibrinolysis occurs, for example in HUS/TTP and DIC
    2. D-Dimers are derived from cross-linked fibrin and is part of a genuine clot
    3. FSP and D-Dimers produced during surgery / healing as well
    4. Elevated D-Dimer levels in healthy elderly associated with 1.5X increased risk of functional decline [20]
    5. High IL-6 with elevated D-Dimer levels associated with 2.0X risk of functional decline in elderly [20]

References navigator

  1. Dahlback B. 2000. Lancet. 355(9215):1627 abstract
  2. Lefkovits J, Plow EF, Topol EJ. 1995. NEJM. 332(23):1553 abstract
  3. Rosenberg RD and Aird WC. 1999. NEJM. 340(20):1555 abstract
  4. Schafer AI. 1996. Am J Med. 101(2):199 abstract
  5. Topol EJ, Byzova TV, Plow EF. 1999. Lancet. 353(9148):227 abstract
  6. Moake JL. 1998. NEJM. 339(22):1629 abstract
  7. Rand JH, Wu XX, Andree HAM, et al. 1997. NEJM. 337(3):154 abstract
  8. Iacoviello L, Di Castelnuovo A, de Knijff P, et al. 1998. NEJM. 338(2):79 abstract
  9. Tsai HM, Rice L, Sarode R, et al. 2000. Ann Intern Med. 132(10):794 abstract
  10. Kyrle PA, Minar E, Hirschl M, et al. 2000. NEJM. 343(7):457 abstract
  11. Faust SN, Levin M, Harrison OB, et al. 2001. NEJM. 345(6):408 abstract
  12. Salomaa V, Matei C, Aleksic N, et al. 1999. Lancet. 353(9166):1729 abstract
  13. Hermans PWM, Hibberd ML, Booy R, et al. 1999. Lancet. 354(9178):556 abstract
  14. Westendorp RGJ, Hottenga JJ, Slagboom PE. 1999. Lancet. 354(9178):561 abstract
  15. Mesters RM, Florke N, Ostermann H, Kienast J. 1996. Thromb Haemost. 75:902 abstract
  16. Carmassi F, Morale M, Ferrini L, et al. 1999. Am J Med. 107(4):344 abstract
  17. Schreijer AJ, Cannegieter SC, Meijers JC, et al. 2006. Lancet. 367(9513):832 abstract
  18. de Lange M, Snieder H, Ariens RAS, et al. 2001. Lancet. 357(9250):101 abstract
  19. Girelli D, Russo C, Ferraresi P, et al. 2000. NEJM. 343(11):774 abstract
  20. Cohen HJ, Harris T, Pieper CF. 2003. Am J Med. 114(3):180 abstract