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Coagulation
Jason Ryan, MD, MPH
Thrombus Formation
ENDOTHELIAL DAMAGE

FIBRIN ACTIVATED PLATELETS

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THROMBUS
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Vasoconstriction
• 1st line of defense against bleeding
• Occurs in response to endothelial damage
• Key mediator: endothelins
• Proteins
• Potent vasoconstrictors
• Released by endothelial cells near site of damage
• Endothelin receptor blockers used in pulmonary hypertension
Coagulation Factors
• Proteins synthesized in liver
• Soluble in plasma
• Activate when triggered by endothelial damage
• Form an insoluble protein: Fibrin
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• Fibrin mesh prevents blood loss
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Coagulation Factors
• Named by Roman numerals: I, V, X
• Circulate as inactive enzymes (zymogens)
• Activated forms: Ia, Va, Xa
• Many activate to become serine proteases
• Serine: amino acid
• Protease: cleaves proteins
• Serine protease: protein cleavage enzyme, contains serine

Serine
Coagulation Cascade
• Sequential activation of clotting factor zymogens
• Constant low level of activation in serum
• Amplification occurs with endothelial damage
• Leads to fibrin generation
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Coagulation Cascade

Joe D/Wikipedia
Coagulation Cascade
• Center of cascade is activation of X → Xa
• Xa converts prothrombin (II) → thrombin (IIa)
• Thrombin (IIa): Fibrinogen (I) → fibrin (Ia)
• Fibrin forms plug to stop bleeding
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• Activation X → Xa makes fibrin

X Xa
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Coagulation Cascade

X Xa

Prothrombin (II) Thrombin (IIa)

Fibrinogen (I) Fibrin (Ia)

Coagulation Cascade
Direct Thrombin
Inhibitors (DTIs)
Xa inhibitors Hirudin
Rivaroxaban Lepirudin
Apixaban Bivalirudin
↓Thrombin Desirudin
AfraTafreeh.com Argatroban
Dabigatran (PO)
X Xa ↓Fibrin

Prothrombin (II) Thrombin (IIa)

Fibrinogen (I) Fibrin (Ia)

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Tissue Factor
Thromboplastin

• Glycoprotein
• Constitutively expressed in sub-endothelial cells
• Not expressed by endothelial cells
• No significant contact of with circulating blood
• Exposed by endothelial damage
• Major activator of coagulation system
• Basis for Prothrombin Time and INR
• Tissue factor added to blood sample
• Time to form clot = PT
Coagulation Cascade
• Primary event: Exposure of tissue factor
• Interacts with factor VII → VIIa
• TF:VIIa activates Xa

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TF:VIIa
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Coagulation Cascade

TF:VIIa
X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)

Fibrinogen (I) Fibrin (Ia)

Thrombin
• Thrombin (IIa) makes more thrombin
• Can activate cascade (positive feedback)
• Factor V → Va
• Factor XI → XIa IXa:VIIIa
• Factor VIII → VIIIa AfraTafreeh.com
• Factor XIa activates IX → IXa Xa
• IX uses VIIIa as a co-factor
Va
• IXa can also activate Xa VIIIa
• More amplification Thrombin XIa
IIa

Fibrin
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Coagulation Cascade
IXa:VIIIa

Hemophilia
Va
VIIIa
TF:VIIa XIa

X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)

Fibrinogen (I) Fibrin (Ia)

Factor VIII
• Produced in endothelial cells (not the liver)
• Circulates bound to von Willebrand Factor
• vWF critical for platelet aggregation
• vWF produced by endothelial cells and megakaryocytes
• Binding to vWF increases VIII plasma half life
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• Released from vWF in response to vascular injury
• Vascular injury → ↑ thrombin → becomes VIIIa

VIII—vWF
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Multicomponent Complexes
• Two complexes for conversion X → Xa
• Three components bound together:
• Active clotting factor functioning as enzyme
• Co-factor
• Substrate
• Require phospholipids and calcium
• Phospholipid: Occur on surfaces of cells
• TF-bearing cells or platelets
• Calcium: Co-factor
Multicomponent Complexes
X
• Extrinsic Xase
Ca+
• Phospholipid: TF-bearing cells VIIa
TF X
• Enzyme: Factor VIIa
Phospholipid
• Co-factor: Tissue factor
• Substrate: Factor X AfraTafreeh.com

Xa
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Multicomponent Complexes
X
• Intrinsic Xase
• Phospholipid: Platelets
• Enzyme: Factor IXa
• Co-factor: Factor VIII (VIIIa)
• Substrate: Factor X

Ca+
VIIIa IX X
Phospholipid

Xa
Coagulation Cascade
IXa:VIIIa

Va
VIIIa
TF:VIIa AfraTafreeh.com XIa

X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)

Fibrinogen (I) Fibrin (Ia)

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Coagulation Cascade
IXa:VIIIa
Phospholipids
Calcium

Va
VIIIa
TF:VIIa XIa

X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)

Fibrinogen (I) Fibrin (Ia)

Calcium
• Factor IV
• Required for clot formation
• Activated platelets release calcium
• EDTA binds calcium in blood samples
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• Prevents clotting

Tannim101/Wikipedia-
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Factor XIII
• Crosslinks fibrin
• Stabilizes fibrin plug
• Absence of XIII → inadequate clot formation
• Requires calcium as co-factor
• Activated by thrombin (IIa) formation
Coagulation Cascade
IXa:VIIIa

Va
VIIIa
TF:VIIa AfraTafreeh.com XIa

X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)

Fibrinogen (I) Fibrin (Ia)

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Coagulation Cascade
IXa:VIIIa

Va
VIIIa
TF:VIIa XIa

X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)
XIIIa
Ca++
Fibrinogen (I) Fibrin (Ia)
Factor XII
Hageman factor

• Can activate factor XI (XIa)

• Physiologic significance unclear
• Important for testing of coagulation system
• Activated by contact with negatively charges
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• Factor XII → XIIa via contact with silica
• Basis for partial thromboplastin time (PTT)

XII XIIa

XI XIa
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Coagulation Cascade
IXa:VIIIa

Va
VIIIa
TF:VIIa XIa

X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)
XIIIa

Fibrinogen (I) Fibrin (Ia)

Coagulation Cascade
XII XIIa

XI T XIa

IX IXa:VIIIa VIII
T

TF:VIIa Va
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X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)
XIIIa

Fibrinogen (I) Fibrin (Ia)

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Coagulation Cascade
XII XIIa Intrinsic Pathway

Extrinsic
Pathway
XI T XIa

IX IXa:VIIIa VIII
T

TF:VIIa Va
X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)
XIIIa

Fibrinogen (I) Fibrin (Ia)

Coagulation Cascade Activated Partial
Thromboplastin Time
(PTT)
XII XIIa Add Plasma to (-) charge
substance (silica)
XI T XIa Time to form clot

IX IXa:VIIIa VIII
T

TF:VIIa Va
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X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)
XIIIa

Fibrinogen (I) Fibrin (Ia)

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Coagulation Cascade
Prothrombin Time (PT)
Add Plasma to TF
Time to form clot

Va
TF:VIIa
X Xa
Endothelial
Damage
Prothrombin (II) Thrombin (IIa)
XIIIa

Fibrinogen (I) Fibrin (Ia)

Intrinsic Pathway
Contact Pathway

• Requires kinins for normal function

• Kinins = peptide hormones/signaling molecules
• Short half lives
• Circulate as inactive precursors: kininogens
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• Activated by kallikreins
• Kinins link coagulation with inflammation
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Intrinsic Pathway
Kinin System

• Bradykinin
• Vasodilator
• Increases vascular permeability
• Pain
• Degraded by angiotensin converting enzyme (ACE)
• ACE inhibitors can raise bradykinin levels
• Dangerous side effect: angioedema
• Also degraded by C1 inhibitor (complement system)
• C1 inhibitor deficiency → hereditary angioedema
Intrinsic Pathway
Factor XII

• Activates clotting and produces bradykinin

• Requires PK, HMWK for normal function

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XII XIIa

Prekallikrein (PK) Kallikrein

High molecular weight kininogen

Bradykinin
HMWK
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Prekallikrein Deficiency
• Rare condition
• Results in markedly prolonged PTT
• XII cannot activate normally
• No bleeding problems

XII XIIa

Prekallikrein (PK) Kallikrein

High molecular weight kininogen

Bradykinin
HMWK
Kinin System
Key Points

• Activated by factor XII

• Link between coagulation and inflammation
• Bradykinin
• ACE inhibitors
• Hereditary angioedemaAfraTafreeh.com
• Prekallikrein Deficiency: ↑PTT
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Coagulation Inhibitors
• Important deactivators of coagulation
• Antithrombin III
• Proteins C and S
• Tissue factor pathway inhibitor
Antithrombin III
• Serpin (inhibitor of serine proteases)
• Inhibits serine proteases: factors II, VII, IX, X, XI, XII
• Produced by liver
• Activated by endothelium
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• Endothelium makes heparan sulfate molecules
• Activate antithrombin
• Basis for role of heparin drug therapy
• Deficiency: Hypercoagulable state
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Proteins C and S
• Glycoproteins synthesized in liver
• Protein C: zymogen
• Active form: activated protein C (APC)
• APC primarily inactivates factors Va and VIIIa
Proteins C and S
• Protein C activated by thrombomodulin
• Cell membrane protein
• Found on endothelial cells
• Thrombomodulin binds thrombin
• Complex activates protein C to APC
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Proteins C and S
• APC requires protein S as co-factor
• Protein S circulates in active form (not a zymogen)

Thrombomodulin: Thrombin
Protein C APC
Inactivation
Va, VIIIa

Protein
S
TFPI
Tissue factor pathway inhibitor

• Inactivates Xa via two mechanisms

• Directly binds Xa
• Binds TF/FVIIa complex → prevents X activation
• Plasma levels increased with heparin administration
• May contribute to antithrombotic effect
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Plasminogen and Plasmin

• Plasminogen synthesized by liver (zymogen)
• Converted to active enzyme: plasmin
• Main role of plasmin is breakdown of fibrin
• Broad substrate specificity
• Also degrades clotting factors, fibrinogen
Plasminogen Activators
• Tissue plasminogen activator (tPA) and urokinase
• Synthesized by endothelial and other cells
• Used as drug therapy for acute MI and stroke
• Streptokinase: Streptococcal protein; activates plasminogen
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FDPs and D-dimer

Fibrin/Clot

Plasminogen Plasmin

tPA
Fibrin
Urokinase Degradation D-dimers
Products
FDPs and D-dimer
• Fibrinogen has two domains: E (central) and D (side)
• Crosslinking of fibrin (XIII) creates E linked two Ds

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Mpt-matthew/Wikipedia
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FDPs and D-dimer

• D-dimer is a special type of FDP
• Presence of D-dimers indicates clot breakdown
• Breakdown of crosslinked fibrin from XIII
• Elevated D-dimer used for diagnosis of DVT/PE

Fibrin/Clot

Plasmin

D-dimers FDPs
FDPs and D-dimer
• ↑ FDPs seen in breakdown of clot
• Also seen in absence of clot from fibrinogen breakdown
• Plasmin can convert fibrinogen → FDPs
• FDPs indicate plasmin activity only
• Not necessarily clot breakdown
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Fibrin/Clot Fibrinogen

Plasmin

FDPs
D-dimers FDPs
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Primary Fibrinolysis
• Rarely phenomena: Plasmin overactive
• Causes ↑ FDP with normal D-dimer
• “Hyperfibrinolysis”
• Plasmin breakdown of fibrinogen (not fibrin) → FDPs
• No clot or crosslinked fibrin → No d-dimers
• Plasmin can deplete clotting factors
• Increased PT/PTT with bleeding (like DIC)
• Prostate cancer: release of urokinase
• Cirrhosis: Loss of alpha2 antiplasmin from liver
FDPs and D-dimer
Key Points

• Clot breakdown: FDPs and D-dimers

• Hyperfibrinolysis: FDPs with normal D-dimer levels
• ↑ D-dimer used to diagnosis thrombotic disorders
• Elevated levels seen in DVT/PE
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• Sensitive but not specific
• Elevated in many other disorders
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Vitamin K
• Required for synthesis of many clotting factors
• “Vitamin K dependent clotting factors”
• Vitamin K dependent factors: II, VII, IX, X, C, S
• Vitamin K deficiency: bleeding
• Warfarin: Vitamin K antagonist

Gonegonegone /Wikipedia
ESR
Erythrocyte Sedimentation Rate

• Rate of RBC sedimentation in test tube

• Normal 0-22 mm/hr for men; 0-29 mm/hr for women
• Increased in inflammatory conditions

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MechESR/Wikipedia
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ESR
Erythrocyte Sedimentation Rate

• ESR increased by “acute phase reactants” in plasma

• Serum proteins that rise in inflammation or tissue injury
• Driven by cytokines
• Most come from liver
• Key acute phase reactants
• Fibrinogen
• Ferritin
• C-reactive protein (binds bacteria; activates complement)