Coagulation

Hemostasis
 Balance between bleeding and clotting is ensured by a complex cascade
o Primary hemostasis: formation of PLT plug
o Secondary hemostasis: formation of fibrin clot
o Fibrinolysis: breakdown of fibrin clot
Component roles: blood cells
 RBCs: add bulk and structural integrity to final fibrin clot
 WBCs: help stimulate wound healing
o Monos and lymphs have TF on surface that can trigger coagulation
 PLTs: procoagulant only: Initiate and control hemostasis
o PLTs do not prevent clotting or breakdown the clot, instead they adhere, aggregate, and secrete
their granule contents to help form a clot
 Plasma components:
o Zymogens (procoagulants), cofactors, control proteins (ACs), fibrinogen (substrate)
Primary Hemostasis
 Triggered by small injury to vessel walls exposing subendothelial collagen.
 Vasoconstriction: helps to seal wound or reduce blood flow
 PLTs become activated
o Adhere to site of injury, secrete granule contents, aggregate with other PLTs, all to form a PLT
plug
 (A), Normal blood flow in intact vessels. RBCs and platelets flow near the center, and WBCs marginate
and roll evenly along the smooth endothelium. Endothelial cells and the subendothelial matrix, which
contains collagen, smooth muscle cells (in arteries) and collagen-producing fibroblasts, have several
mechanisms by which they can limit blood clotting (TM, TFPI, HS) and platelet activation (NO,
PGI2). (B), Trauma to the blood vessel wall exposes subendothelial collagen and tissue factor, triggering
platelet adhesion. (C), VWF serves as a bridge between subendothelial collagen and the platelet GP
Ib/IX/V receptor. Platelet interaction with collagen via α2β1 and GP VI receptors triggers release of
TXA2 and ADP and subsequent activation of the αIIbβ3 receptor. (D), Activation of αIIbβ3 supports
platelet-platelet aggregation through binding of arginine-glycine-aspartate (RGD)–containing ligands
such as fibrinogen and VWF. ADP, Adenosine diphosphate, FG, fibrinogen; HS, heparan
sulfate; NO, nitric oxide; PGI2, prostacyclin; RBC, red blood cells; TF, tissue factor; TFPI, tissue factor
pathway inhibitor; TM, soluble thrombomodulin; TXA2, thromboxane A2; VWF, von Willebrand
 factor; WBC, white blood cells

.
 Vasocontriction and PLT plug formation= rapid, short-lived response vessel damage
 To control major bleeding long term the plug must be reinforced by fibrin
 Defects in primary hemostasis can result debilitating chronic hemorrhage (sometimes fatal)
o Seen with collagen abnormalities, thrombocytopenia, qualitative PLT disorders, or in VWD
Secondary hemostasis: activation of coagulation and fibrin clot formation
 Series or cascade of plasma coag protein activation that results in fibrin clot formation
 Activated by large injuries to BVs and surrounding tissues
 Triggered by activator tissue factor (TF)
o TF membrane
o Found in subendothelial SMCs, fibroblasts (therefore, released by disruption of endothelial cells
in vasculature), monocytes and lymphs.
 Fibrin Clot
o Fibrillar protein that gives semisolid character to blood clot or thrombus
o Breakdown product of fibrinogen
 Fibrinogen: plasma GP that is converted to fibrin by thrombin (serine protease)
digestion
 Fibrinogen has no binding sites, while fibrin monomers can polymerize locally to site of
trauma/injury
Coagulation Cascade
 Series of plasma protein interactions
o Coag factors are mostly serine proteases that circulate at zymogens (inactive enzymes)
o During process of coag, they become activated and through a domino effect of activation (one
activates another and so on), produce thrombin
 After injury
o PLTs activated and ashere + aggregate to damaged areas until new endothelial cells can grow.
TF in subendothelial cells exposed by injury
o Coag system initiation occurs on TF-bearing cells
o Coag reactions then propagated on PLT surface
 o Thrombin generated- fibrin clot stabilized- fibrinolysis

Coagulation Cascade
 Cellular base of negatively charged phospholipid (subendothelial cells or PLT surface)
 Ca2+ to bind factors/co-factors to a negatively charged phospholipids (coag limited to site of injury)
 Plasma coag proteins
o Serine proteases for enzyme activity
o Co-factors for stabilizing and enhancing enzyme activity
 Phospholipid (-ve) + Ca2+ + SP + CF
Plasma-Based Coagulation Cascade
 The coagulation cascade consists of the contact system (simplified here) and the intrinsic, extrinsic, and
common pathways. In the intrinsic pathway (red), the contact factors XII, prekallikrein (pre-K), and
high-molecular-weight kininogen (HMWK) are activated and proceed to activate factors XI, IX, VIII, X,
and V and prothrombin, which converts fibrinogen to fibrin. In the extrinsic pathway (green), tissue
factor (TF) activates factor VII, which activates factors X, V, and prothrombin, cleaving fibrinogen to
fibrin. Both the intrinsic and extrinsic pathways converge with the activation of factor X, so factors X, V,
prothrombin, and fibrinogen are called the common pathway (blue). Dashed boxes indicate the
coagulation factor complexes that assemble on phospholipid (yellow symbol). These pathways are the
basis of clinical coagulation laboratory tests. Thr, Thrombin
 3 in-vivo complexes formed + contact factors
o VIIa and TF (extrinsic tenase)
o IXa and VIIIa (intrinsic tenase)
o Xa and Va (prothrombinase)
o Contact factor pathway (XII, pre-K and HMWK)
Extrinsic Pathway

 TF (FIII), VII + common

 TF + VII + Ca2+ + PL= extrinsic tenase complex. Extrinsic because TF not found in blood.
 Initiates coagulation after tissue injury and TF exposure. Initiates the common pathway and also
intrinsic pathway
 Extrinsic: TF exposed in subendothelium, activates FVII and forms extrinsic tenase complex TF: VIIa
 Small ampunt of FX is activated by the TF: VIIa complex and initiates the common pathway- a small
amount of thrombin is produced
 Extrinsic pathway also activates a small smount of FIX to IXa
o Initiates activation of the intrinsic pathway
Intrinsic pathway
  All factors found in plasma. True in vivo intrinsic pathway starts at FIXa and Factor FXIa
o FIX activated by extrinsic pathway
o FXI activated by thrombin. Contact factor complex can also activate FXI
 Extrinsic pathway activates small amount of FIX. FXIa further activates FIX to FIXa
 FIXa combines with cofactor FVIIIa forming intrinsic tenase complex
o Occurs on PLT surface. Complex also activates FX (much more effectively), initiating the
common pathway
 Contact factor complex
o Can also activate FXI. Contact factors= HMWK, pre-K, XIIa
o Activated by contact with negatively charged foreign surfaces.
Common Pathway

 FX is activated by both extrinsic and intrinsic pathways

 FXa activates FV- Va and forms the prothrombinase complex (Xa and Va on PLT membrane in presence
of Ca2+)
 Prothrombinase complex activates prothrombin (FII) into thrombin (FIIa)
 Thrombin then converts fibrinogen (FI) to fibrin (red clot formation)
 Thrombin activates FXIII to cross-link fibrin or stabilize clot
 Thrombin positive feedback loop creates a cascade of activation
o Thrombin loops back into cascade to further activate V, VIII, XI, and PLTs until solid clot is
formed
o Once intrinsic pathway is active, extrinsic gets shut down
  TF inhibitors stop the extrinsic system (no more activation of X and IX)
o Antithrombin comes in to prevent further initiation of thrombin once clot is solid.
Cell-Based Coagulation

 Can be described via interdependent phases: initiation, amplification, propagation

 Initiation: triggered with formation of the extrinsic tenase complex. Complex activates low levels of
factors IX and X to generate a small amount of thrombin
 Amplification: thrombin feedback loop creates a cascade of activation
o Activates small amounts of factors FV, VIII, XI and activates more PLTs (COAT PLT formation also
seen)
o Serves to amplify more thrombin generation
o Begins fibrin formation
 Propagation:
o cofactors Va and VIIIa activated by thrombin during amplification phase bind to activated PLT
membranes
 Become receptors for Xa and IXa, respectively
o IXa generated from initiation phase binds to VIIIa on PLT membrane forming intrinsic temase
complex
 Thrombin activation of FXI in amplification phase also activates more FIX. This process
initiates intrinsic pathway
 Platelet factor 3
 A phospholipid from the platelet membrane that contributes to the blood
clotting cascade by forming a phospholipid-protein complex (THROMBOPLASTIN)
which serves as a cofactor with FVIIa to activate FX in the extrinsic pathway
o Intrinsic tenase complex activates FX at 50-100 fold higher rate than extrinsic complex,
producing more activated FXa
o Activated Xa binds to Va forming the prothrombinase complex
 This complex activates prothrombin and generate a bust of thrombin
o Thrombin cleaves fibrinogen to fibrin forming fibrin clot
o Thrombin activates FXIII to cross-link or stabilize clot (RBCs incorporated in clot as well- forms
red clot)
Secondary Hemostasis
 Cofactors: bind serine proteases ton stabilize and enhance their reactivity
 FV
o Procoagulant cofactor- soluble GP circulating in plasma and stored in platelet alpha-granules
 During PLT activation, PLTs release/secrete partially activated FV (by thrombin) at the
site of injury
o Primarily synthesized in BM megakaryocytes. Unstable in plasma. Aka labile factor
o Cofactor to FXa in the prothrombinase complex
 FVIII
o Procoagulant factor- soluble plasma protein circulating in plasma
 Extremely unstable in plasma except during coagulation. Circulates bound to VWF,
which increases its stability while circulating (from minutes to 12h)
o Produced primarily by hepatocytes and by microvascular endothelial cells (i.e in the lung)
o Aka Anti-hemophilic factor
 X-linked recessive factor
 Deficiency in VIIII results in hemophilia A (severe bleeding)
o Cofactor to FIX, which form the intrinsic tenase complex

 Serine Proteases
o IIa, VIIa, IXa, Xia, XIIa, prekallikrein (pre-K)
o Start as zymogens (inactive enzymes) that circulate in plasma
 o Become proteolytic enzymes
 Activated by cleavage (at one or more sites)
 One zymogens gets activated and becomes a serine protease- which activates the next
zymogen- which becomes protease
 Local activation on cell or PLT surface at site of injury or disruption of BV membrane
o Most coag factors are produced in the liver- when liver fails patient has increased tendency to
bleed
o Some factors require vitamin K for normal production: Factors II, VII, IX and X are vitamin-K
dependent

 Vitamin K
o In liver, vitamin K carboxylates certain factors so they can bind Ca2+ and PL and participate in
coag reactions
o Without the second carboxyl group, factors cannot bind Ca2+ and PL- ineffective factors
o Absence of VitK= non functional factors
o Vitamin K dependent factors: FII, VII, IX, X and regulatory control proteins, protein C, S and Z
o Blood thinner or oral AC coumadin/Warfarin works by blocking vitamin K
 Factors cant bind Ca2+ and PL, essential to complex formation
 No clotting
Serine Proteases
 FIX: Xmas factor
o Vitamin K dependent factor- cofactor is FVIIa and together form the intrinsic tenase complex
that will activate FX
o Deficiency leads to hemophilia B or Xmas disease
 X-linked recessive factor, severe bleeding results
 FXI
o Serine protease: not part of any complex
o Activated by contact factor complex or thrombin-in turn activates FIX
o Deficiency leads to hemophilia C or Rosenthal syndrome (mild and variable bleeding results
 FXII
o Serine protease that forms part of the contact factor complex
 Activated by contact with negatively charged foreign surfaces
 FXIIa transforms pre-K into active Kallikrein
 Kallikrein cleaves or activates HWMK to Bradykinin
 Activates FXI
o Deficiencies in contact factors do not cause clinical bleeding disorders but will prolong in vitro
coag testing and from this, require further investigation
 Thrombin (FIIa)
o Main enzyme of coag cascade with multiple key activities (prothrombin to thrombin)
o Triggers positive feedback loop to generate more of itself
  Activates cofactors FV and FVIII=> Va and VIIIa
 Activates FXI=> FXIa
 Induces PLT aggregation and activation
 Cleaves fibrinogen to fibrin (FI=> FIa) (primary function)
 Activates FXIII=> FXIIIa (factor stabilizing factor)
 Thrombin also plays a role in regulation of coag and in fibrinolysis
 Thrombin: final serine protease
 Fibrinogen (FI)
o Large plasma GP produced by liver
o Absorbed, carried and released by PLTs (alpha granules)
o Highest mean plasma conc or procoagulants
o Involved in PLT aggregation
 Linking activated PLTs together via their GP Ib/IIIa receptors
o Primary substrate of thrombin
 Cleaved or digested by thrombin into fibrin monomers which spontaneously polymerize
to form fibrin polymer (fibrillar lattice network of fibrin)
 FXIII
o A transglutaminase
o Fibrin stabilizing factor
 Crosslinks adjacent D-domains of fibrin polymers
 Final product is a meshwork of crosslinked or stabilized fibrin polymers resistant to
fibrinolysis (insoluble)
 Von Willebrand Factor (VWF)
o Plasma procoagulant
o Large multimeric GP
o Produced by megakaryocytes and endothelial cells
 Stored in PLTs (alpha granules) and endothelial cells (Weibel-Palade bodies)
 Released when injury or disruption occurs via variety of henmostatic stimuli
 Blood group O individuals have lower levels of VWF
o Primary function
 Bridges PLTs and subendothelial collagen during PLT adhesion- helps to initiate primary
hemostasis through this function
 Receptor site to support PLT aggregation
o Carrier protein for FVIII
o Abnormalities in VWF molecular structure and concentration can lead to severe bleeding
Regulatory Mechanisms (Inhibitors and Fibrinolysis)
 Inhibitors (natural AC) and their cofactors regulate serine proteases/cofactors in the coag cascade
through AC feedback loops
 Ensures coag is localized (not systemic) and maintain balance between abnormal thrombosis and
bleeding via
o Slowing procoagulant activation, suppressing thrombin production
 Main control proteins/regulators
o TFPI, APC and protein S, AT and heparin cofactor II, protein Z and protein Z dependent protease
inhibitor (ZPI)
Tissue Factor Pathway Inhibitor
 TFPI is a serine protease inhibitor (SERPIN)
 Synthesized primarily by endothelial cells and also expressed on PLT membrane
 Principal regulator of the extrinsic pathway
 o Inhibits extrinsic complex and FXa from this pathway
 Cofactor-Protein S
o Enhances enzyme reaction tenfold

 Inhibits coagulation via 2 step process

o TFPI bind and inactivates FXa
o TFPI: Xa complex binds and inactivates TF:VIIa on subendothelial cell membrane
 Once intrinsic pathway is active, TFPI shuts down extrinsic tenase complex and Xa
 Xa + IXa production shifts to intrinsic pathway (as propagation phase occurs)
Protein C regulatory System
 The protein C system changes thrombin’s function from a procoagulant to an AC
 Thrombin binds to endothelial cell membrane protein thrombomodulin
o Thrombin becomes inactive and can no longer activate procoagulant factors or PLTs
 Together, thrombin-thrombomodulin/ T-T complex activates protein C system
o Thrombin activates protein C (activated protein C/ APC)- serpin
o APC disassociates from EPCR and binds cofactor protein S (stabilizes APC)
o APC-protein S complex inactivates (by further cleaving or digesting) FVa and FVIIIa
o Slows or blocks thrombin generation and coagulation

Protein S Regulatory System

 Cofactor that binds and stabilizes APC and TFPI
 Enhances factor Xa inhibition by TFPI tenfold
 Augments action T-T complex fivefold. Synthesized in liver and circulates in free (cofactor form) and
bound (unavailable) forms
Antithrombin
 Serine protease inhibitor (SERPIN)
  Binds and neutralizes serine proteases
o Thrombin (FIIa) and FIXa, FXIa, FXIUIa, pre-K) and plasmin
 Heparin is required for effective AC activity
 AT activity is accelerated 2000 fold by binding to heparin
 Basis for the AC activity of pharmaceutical heparin. Also, circulating in plasma is heparin cofactor II=
serpin that exclusively inactivates thrombin
 Acts to prevent, amongst others further activation of thrombin
Protein Z + Protein Z Dependent Protease Inhibitor (ZPI)
 ZPI: serine protease inhibitor (SERPIN).
 Protein Z: nonproteolytic factor
o Vitamin K -dep plasma GP synthesized in liver
o Increases the ability of ZPI to inhibit factor Xa 2000-fold
 Binds to ZPI
o ZPI + Protein Z/ PL + Ca2+= serpin- potent inhibitor of FXa
 ZPI also inhibits FXIa
o Separate reaction that does not require protein Z, PL, and Ca2+
o The inhibition of FXIa is accelerated twofold by the presence of heparin
Fibrinolysis Control Mechanisms
Fibrinolysis
 Systemic hydrolysis of fibrin- digesting clot (final stage of coag)
 Restores normal blood flow: occurs as vasculature heals or disruption repairs
 Begins a few hours after the insoluble and stable fibrin clot produced (fibrin polymerization and
crosslinking)
 Initiates with fibrinolytic proteins assembly on fibrin during clotting (localizes fibrinolysis to site of
injury/disruption)
o Plasminogen, tissue plasminogen activator (TPA), Urokinase Plasminogen Activator (UPA)
 Digestion of clot leaves Fibrin degradation products

Fibrinolytic Proteins
 Fibrinolytic proteins Plasminogen, TPA, UPA are incorporated into fibrin as clot forms
 Several hours after clot formation and in response to inflammation and coag, TPA, UPA are activated
(or released) and act to convert plasminogen to plasmin (the primary serine protease of fibrinolytic
system)
 This process begins fibrin degradation and helps to restore normal blood flow
Tissue Plasminogen Activator (TPA)
 Serine protease. Secreted by endothelial cells
 Released in response to IF and coag and covalently binds fibrin clot
o Hydrolyzes fibrin bound plasminogen
  Converts plasminogen=> plasmin
 Initiates fibrin degradation
 Helps to restore normal blood flow
 Circulating TPA is bound to fibrinolysis inhibitor, plasminogen activator inhibitor-1 (PAI-1) is neutralized
and taken out of circulation
Urokinase Plasminogen Activator (UPA)
 Becomes incorporated into the mix of fibrin-bound plasminogen and TPA while clot is being formed
o Does not bind fibrin firmly and has a minor physiologic effect
 UPA converts plasminogen to plasmin
o Initiates fibrin degradation and helps to restore normal blood flow
Plasminogen and Plasmin
 Plasma zymogen (serine protease) produced by the liver. Abundant in plasma
 Plasminogen becomes incorporated into clot as fibrin polymerizes
 Plasminogen becomes converted into plasmin when cleaved by fibrin bound TPA and/or UPA
 Bound plasmin digests clots (fibrin), restores blood flow-localized and prevents systemic clotting (slow
digestion
 Free plasmin is capable of digesting plasma fibrinogen (FI), FV and FVIII
o Free plasmin levels controlled by a2-antiplasmin
Fibrinolysis Inhibitors
 A2-antiplasmin (AP): SERPIN
o Synthesized in liver and primary inhibitor of plasmin
o Slow fibrinolysis
 Competes for plasminogen binding with fibrin
 Binds directly (rapidly and irreversibly) to plasmin. Inactivates plasmin
 Plasminogen Activator Inhibitor-1 (PAI-1)
o Serine protease inhibitor (SERPIN).
o Produced by endothelial cells, megakaryocytes, smooth muscle cells, fibroblasts, monocytes,
adipocytes, hepatocytes, and other cell types
 PLTs store a pool of PAI-1, accounting for more than half of its availability and fir its
availability and for its delivery to the fibrin clot
o Principal inhibitor of plasminogen activation
o Inactivates TPA and UPA
 Prevents conversion of plasminogen to active plasmin
 Thrombin Activatable Fibrinolysis Inhibitor (TAFI)
o Plasma procarboxypeptidase. Synthesized in the liver
o Activated by thrombin-thrombomodulin complex (same complex that activates Protein C
pathway)
o Functions as an antifibrinolytic enzyme
 Inhibits fibrinolysis
 Prevents binding of TPA and plasminogen to fibrin
 Blocks the formation of plasmin. Result is suppression of fibrinolysis
 Thrombin Activatable Fibrinolysis Inhibitor (TAFI)
o In coagulopathies with factor deficiencies, such as hemophilia
 Decreased thrombin production may reduce activation of TAFI
 Resulting in increased fibrinolysis that contributes to more bleeding
o In thrombotic disorders
 Increased thrombin generation may increase activation of TAFI
 Resulting in decreased fibrinolysis and can further contribute to thrombosis
 o TAFI also may play a role in regulating inflammation and wound healing
Fibrin Degradation Products
 Plasmin cleaves fibrin and fibrinogen and produces a series of identifiable fibrin fragments
o X,Y, D, E and D-Dimer
 Several of these fragments inhibit hemostasis
o Contribute to hemorrhage by preventing PLT activation
o Prevent fibrin polymerization
 Fragments X, Y, D, and E
o Produced by systematic digestion of either fibrin or fibrinogen by plasmin
 D-Dimer: specific product of digestion of crosslinked fibrin only
o Therefore, a marker of thrombosis and fibrinolysis
o Separately detectable by monoclonal ab for d-dimer antigen
o D-Dimer immunoassay
 Identify chronic and acute DIC: uncontrolled activation of thrombin and consumption of
coag factors PLTs and fibrinolytic proteins
o Rule out venous thromboembolism
o Suspected deep venous thrombosis (DVT) or pulmonary embolism (PE)