Coagulation Cascade & Fibrinolysis

Quick Answer

Haemostasis is the physiological process that stops bleeding following vascular injury while maintaining blood fluidity. It involves three interconnected phases: primary haemostasis (platelet plug formation), secondary haemostasis (fibrin clot formation via the coagulation cascade), and fibrinolysis (clot dissolution). The modern cell-based model of coagulation recognises that coagulation occurs on cell surfaces (platelets, tissue factor-bearing cells) through initiation, amplification, and propagation phases, rather than as isolated intrinsic and extrinsic cascades.

Key Numbers:

• Platelet count: 150-400 × 10⁹/L
• PT: 11-13 seconds (INR 0.9-1.1)
• APTT: 25-35 seconds
• Fibrinogen: 2-4 g/L (critical threshold <1.5 g/L in bleeding)
• D-dimer: <0.5 mg/L FEU
• Thrombin burst generates ~100 nM thrombin in seconds

Critical Concepts:

• Tissue factor (TF) is the primary physiological initiator of coagulation
• Thrombin is the central enzyme with multiple amplification and feedback roles
• Factor XIII cross-links fibrin, creating stable clot resistant to fibrinolysis
• Antithrombin neutralises thrombin and factor Xa (enhanced 1000-fold by heparin)
• Protein C pathway provides negative feedback, activated by thrombin-thrombomodulin
• Plasmin degrades fibrin to D-dimers and fibrin degradation products

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CICM Exam Focus

First Part Written (SAQ)

Common Question Stems:

• "Describe the cell-based model of coagulation" (10 marks)
• "Outline the role of thrombin in haemostasis" (8 marks)
• "Describe the natural anticoagulant mechanisms" (10 marks)
• "Explain the mechanism of action of heparin and its reversal" (8 marks)
• "Describe the pathophysiology of DIC" (10 marks)
• "Outline the physiological regulation of fibrinolysis" (8 marks)

Expected Depth:

• Detailed understanding of cell-based model phases
• Factor half-lives and vitamin K dependence
• Specific inhibitor mechanisms (AT, Protein C/S, TFPI)
• Fibrinolytic pathway components and regulation
• Laboratory test interpretation and limitations

First Part Viva

Common Viva Topics:

• "Tell me about the coagulation cascade" (opening question)
• "How does thrombin amplify its own generation?"
• "What are the natural anticoagulants and how do they work?"
• "Explain how warfarin works and why INR rises"
• "What is the mechanism of heparin anticoagulation?"
• "How does tranexamic acid work?"
• "Describe the pathophysiology of DIC"

Examiner Expectations:

• Structured answer distinguishing primary and secondary haemostasis
• Cell-based model preferred over classical cascade (but know both)
• Quantitative data (half-lives, concentrations, test values)
• Clinical relevance to ICU practice
• Understanding of coagulation monitoring

High-Yield Topics

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Key Points

1. Primary haemostasis involves platelet adhesion (via vWF-GPIb), activation (shape change, granule release), and aggregation (GPIIb/IIIa-fibrinogen bridges) forming an unstable platelet plug.

2. The cell-based model describes coagulation as occurring on cell surfaces through three overlapping phases: initiation (TF-bearing cells), amplification (platelet surface, thrombin feedback), and propagation (activated platelet surface, thrombin burst).

3. Tissue factor (TF) is the primary physiological trigger of coagulation, forming a complex with factor VIIa that activates factors X and IX.

4. Thrombin is the central enzyme of coagulation with multiple functions: cleaves fibrinogen to fibrin, activates factors V, VIII, XI, XIII, activates platelets, and activates protein C (anticoagulant feedback).

5. Fibrin cross-linking by factor XIIIa (transglutaminase) creates covalent bonds between fibrin monomers, producing stable clot resistant to fibrinolysis.

6. Natural anticoagulants include antithrombin (inhibits thrombin, Xa), protein C/S system (degrades Va, VIIIa), and TFPI (inhibits TF-VIIa-Xa).

7. Fibrinolysis is mediated by plasmin (from plasminogen activation by tPA), regulated by PAI-1 and alpha-2-antiplasmin, producing D-dimers.

8. Coagulation tests: PT/INR reflects extrinsic pathway (factors VII, X, V, II, I), APTT reflects intrinsic pathway (XII, XI, IX, VIII + common), both insensitive to in vivo haemostasis.

9. Viscoelastic testing (TEG/ROTEM) provides global assessment of clot formation and lysis, enabling targeted blood product therapy.

10. DIC is a consumptive coagulopathy with simultaneous thrombosis and bleeding, diagnosed by ISTH scoring (platelets, PT, fibrinogen, D-dimer), treated by addressing the underlying cause.

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Primary Haemostasis

Primary haemostasis describes the formation of a platelet plug at sites of vascular injury. This process occurs within seconds of vessel damage and provides the initial haemostatic response, which is subsequently stabilised by fibrin formation during secondary haemostasis.

Vascular Response

Vasoconstriction: Immediate reflex vasoconstriction reduces blood flow to the injured vessel. This is mediated by local myogenic response, sympathetic nervous system activation, and release of vasoconstrictors including endothelin-1 and thromboxane A2 (TXA2) from activated platelets. Vasoconstriction is transient (minutes) and reduces blood loss while haemostatic processes are initiated.

Endothelial exposure: Vessel injury exposes the subendothelial matrix containing collagen (types I, III, VI), von Willebrand factor (vWF), fibronectin, and laminin. These components provide binding sites for platelet adhesion. Under high shear conditions (arteries, arterioles), vWF is essential for platelet adhesion; under low shear (veins), direct collagen-platelet binding is more important (PMID: 10892008).

Platelet Adhesion

Von Willebrand factor (vWF): A large multimeric glycoprotein synthesised by endothelial cells and megakaryocytes. vWF circulates bound to factor VIII (protecting it from degradation) and is released from Weibel-Palade bodies upon endothelial activation. Under high shear stress, vWF undergoes conformational change exposing the A1 domain, which binds to platelet GPIb-IX-V complex. vWF multimer size is regulated by ADAMTS13; deficiency causes thrombotic thrombocytopenic purpura (TTP) (PMID: 9731070).

GPIb-IX-V complex: The primary platelet receptor for vWF. This receptor complex contains GPIbα (vWF A1 domain binding), GPIbβ, GPIX, and GPV. Deficiency causes Bernard-Soulier syndrome (giant platelets, thrombocytopenia, bleeding). The GPIb-vWF interaction is a "catch bond" that strengthens under shear stress, enabling platelet capture from flowing blood (PMID: 17962512).

Collagen receptors: Direct platelet-collagen binding occurs via GPVI (immunoglobulin superfamily, activating receptor) and integrin α2β1 (GPIa/IIa, adhesion receptor). GPVI binding to collagen initiates intracellular signalling cascades leading to platelet activation. These receptors are more important under low shear conditions (PMID: 15528345).

Platelet Activation

Platelet activation involves shape change, granule secretion, and surface receptor activation, transforming platelets from quiescent discs to active haemostatic cells.

Agonists: Multiple agonists activate platelets:

• Collagen (via GPVI, α2β1)
• Thrombin (via PAR1, PAR4 in humans)
• ADP (via P2Y1, P2Y12)
• Thromboxane A2 (via TP receptor)
• Epinephrine (via α2-adrenergic receptor)

Signalling cascades: Platelet activation involves phospholipase C activation, calcium mobilisation from intracellular stores, protein kinase C activation, and cytoskeletal reorganisation. These pathways converge on "inside-out" signalling that activates integrin αIIbβ3 (GPIIb/IIIa) (PMID: 17170707).

Shape change: Activated platelets transform from discoid shape (3 μm diameter) to spherical with pseudopod extensions, increasing surface area for adhesion and aggregation. This shape change is driven by actin polymerisation and actomyosin contraction (PMID: 15659541).

Granule secretion: Platelets contain three types of granules:

Dense granule ADP release amplifies platelet activation through autocrine/paracrine signalling. Platelet polyphosphates accelerate factor XII activation and enhance factor V activation (PMID: 19965649).

Phosphatidylserine exposure: Activated platelets "flip" phosphatidylserine (PS) from inner to outer membrane leaflet. PS provides a negatively charged surface for assembly of coagulation factor complexes (tenase, prothrombinase), dramatically accelerating coagulation reactions. This links primary and secondary haemostasis (PMID: 11157530).

Platelet Aggregation

GPIIb/IIIa (integrin αIIbβ3): The most abundant platelet surface receptor (~80,000 copies/platelet). In resting platelets, GPIIb/IIIa is in a low-affinity conformation. Upon activation, "inside-out" signalling causes conformational change to high-affinity state, enabling fibrinogen and vWF binding. Deficiency causes Glanzmann thrombasthenia (PMID: 15914556).

Fibrinogen bridging: Each fibrinogen molecule has two RGD sequences that bind GPIIb/IIIa receptors on adjacent platelets, creating platelet-platelet bridges. This fibrinogen-dependent aggregation forms the basis of the platelet plug.

Clot retraction: After aggregation, platelet-myosin interactions contract the clot, reducing clot volume by 50-80%. This consolidates the plug and brings wound edges together. Clot retraction requires functional GPIIb/IIIa and fibrinogen (PMID: 16020511).

Antiplatelet Pharmacology

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Cell-Based Model of Coagulation

The cell-based model, developed by Hoffman and Monroe, represents the modern understanding of coagulation as occurring on specific cell surfaces rather than as isolated plasma cascades. This model explains clinical observations that classical cascade models cannot, including why haemophilia causes bleeding despite normal extrinsic pathway and why factor XII deficiency does not cause bleeding (PMID: 11520473).

Key Principles

1. Coagulation occurs on cell surfaces: Coagulation factor complexes assemble on phospholipid membranes (phosphatidylserine-exposing surfaces), particularly tissue factor-bearing cells and activated platelets.

2. Different cell surfaces have different roles: TF-bearing cells (fibroblasts, smooth muscle cells, monocytes) initiate coagulation; platelets amplify and propagate the thrombin burst.

3. Small amounts of thrombin activate platelets: Trace thrombin generated during initiation activates platelets, providing the surface for the massive thrombin burst during propagation.

4. Factor complexes dramatically accelerate reactions: Factor Va-Xa (prothrombinase) is 300,000-fold more active than Xa alone; factor VIIIa-IXa (tenase) is 200,000-fold more active than IXa alone.

Initiation Phase

The initiation phase occurs on tissue factor-bearing cells when vascular injury exposes TF to blood.

Tissue factor (TF, factor III, CD142): A transmembrane glycoprotein constitutively expressed on adventitial fibroblasts, vascular smooth muscle cells, and organ capsules. TF forms a "haemostatic envelope" around blood vessels. Under pathological conditions, TF expression is induced on monocytes and endothelial cells by inflammatory cytokines (TNF-α, IL-1β, endotoxin), contributing to thrombosis in sepsis and DIC (PMID: 10700172).

TF-VIIa complex formation: When TF is exposed to blood, it binds factor VIIa (the only coagulation factor that circulates in active form at ~1% of total factor VII). The TF-VIIa complex activates both factor X and factor IX. Factor Xa generated on TF-bearing cells combines with factor Va to form the prothrombinase complex, generating small amounts of thrombin. This initial thrombin generation is limited because tissue factor pathway inhibitor (TFPI) rapidly inhibits the TF-VIIa-Xa complex (PMID: 9792242).

Products of initiation:

• Factor Xa (small amounts, on TF cell surface)
• Factor IXa (diffuses to nearby platelets)
• Thrombin (small amounts, ~10 nM, insufficient for stable clot)

The initiation phase produces enough thrombin to activate platelets but not enough to form a stable fibrin clot.

Amplification Phase

The amplification phase occurs on the platelet surface and amplifies the coagulation signal.

Platelet activation by thrombin: The small amounts of thrombin generated during initiation activate platelets through protease-activated receptors (PAR1 and PAR4 in humans). Activated platelets undergo shape change, expose phosphatidylserine, and release granule contents including partially activated factor V. Thrombin also cleaves vWF from factor VIII, releasing factor VIII for activation (PMID: 16917131).

Cofactor activation: Thrombin activates factors V and VIII on the platelet surface:

• Factor Va is the cofactor for factor Xa in the prothrombinase complex
• Factor VIIIa is the cofactor for factor IXa in the tenase complex

Factor XI activation: Thrombin activates factor XI on the platelet surface. This "feedback" activation of factor XI bypasses the need for factor XII (contact activation), explaining why factor XII deficiency does not cause bleeding. Platelet-bound factor XIa activates factor IX, generating more IXa for the propagation phase (PMID: 15746011).

Products of amplification:

• Activated platelets with exposed phosphatidylserine
• Factor Va (platelet-bound)
• Factor VIIIa (platelet-bound)
• Factor XIa (platelet-bound)

Propagation Phase

The propagation phase produces the "thrombin burst" necessary for stable fibrin clot formation.

Intrinsic tenase complex (VIIIa-IXa): Factor IXa (generated during initiation and by factor XIa during amplification) binds to factor VIIIa on the activated platelet surface. This complex activates factor X 200,000-fold faster than IXa alone. The IXa-VIIIa complex is the primary generator of factor Xa during propagation, explaining why haemophilia A (factor VIII deficiency) or B (factor IX deficiency) causes severe bleeding (PMID: 9144644).

Prothrombinase complex (Va-Xa): Factor Xa binds to factor Va on the platelet surface, forming the prothrombinase complex. This complex converts prothrombin (factor II) to thrombin 300,000-fold faster than Xa alone. The prothrombinase complex generates the "thrombin burst" (~100 nM thrombin in seconds) necessary for stable fibrin formation (PMID: 16476888).

Thrombin burst: Approximately 96% of total thrombin is generated during the propagation phase. This massive thrombin generation:

• Cleaves fibrinogen to fibrin monomers
• Activates factor XIII for fibrin cross-linking
• Further activates factors V, VIII, XI (positive feedback)
• Activates thrombin-activatable fibrinolysis inhibitor (TAFI)
• Activates protein C (negative feedback via thrombomodulin)

Cell-Based Model vs Classical Cascade

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Classical Cascade Model

While the cell-based model better represents in vivo haemostasis, the classical cascade model remains important for understanding laboratory tests (PT, APTT) and is still examined. The cascade was described independently by Macfarlane and Davie in 1964 (PMID: 14175596).

Intrinsic Pathway

The intrinsic pathway is initiated by contact activation on negatively charged surfaces. All factors required are present (intrinsic) in plasma.

Contact activation: Factor XII (Hageman factor) binds to negatively charged surfaces (glass, kaolin, collagen, polyphosphates) and is activated to XIIa. Factor XIIa activates factor XI to XIa. This pathway requires high molecular weight kininogen (HMWK) and prekallikrein as cofactors. Contact activation does not contribute significantly to physiological haemostasis (factor XII deficiency does not cause bleeding) but may contribute to pathological thrombosis (PMID: 18096831).

Factor XI activation: Factor XIa activates factor IX to IXa in a calcium-dependent reaction. Factor IX is a vitamin K-dependent serine protease with a half-life of 18-24 hours. Factor IX deficiency causes haemophilia B (Christmas disease).

Tenase complex: Factor IXa combines with factor VIIIa on a phospholipid surface in the presence of calcium to form the intrinsic tenase complex. This complex activates factor X. Factor VIII is the rate-limiting cofactor; its deficiency causes haemophilia A.

APTT measurement: The activated partial thromboplastin time measures the intrinsic and common pathways. Phospholipid (substitute for platelet membrane) and activator (kaolin, ellagic acid, or silica) are added to citrated plasma, then calcium is added to start the reaction. APTT is prolonged by deficiency of factors XII, XI, IX, VIII, X, V, II, or I, or by inhibitors (heparin, lupus anticoagulant, factor-specific antibodies).

Extrinsic Pathway

The extrinsic pathway is initiated by tissue factor, which is "extrinsic" to blood.

Tissue factor pathway: TF-VIIa complex directly activates factor X (and factor IX). This is the primary physiological initiator of coagulation. The TF pathway is rapidly inhibited by TFPI, limiting its role to initiation.

Factor VII: A vitamin K-dependent serine protease with the shortest half-life (4-6 hours) of all coagulation factors. Approximately 1% circulates as factor VIIa. Factor VII levels fall most rapidly during vitamin K deficiency or warfarin therapy, explaining the early prolongation of PT.

PT measurement: The prothrombin time measures the extrinsic and common pathways. Tissue factor (thromboplastin) and calcium are added to citrated plasma. PT is prolonged by deficiency of factors VII, X, V, II, or I. PT is reported as INR (International Normalised Ratio) to standardise for thromboplastin reagent variability.

Common Pathway

The common pathway comprises factors X, V, II (prothrombin), and I (fibrinogen), converging from both intrinsic and extrinsic pathways.

Prothrombinase complex: Factor Xa binds factor Va on phospholipid surface (activated platelet membrane) with calcium. This complex cleaves prothrombin to thrombin. Factor V is a cofactor, not an enzyme; it is activated by thrombin (positive feedback) and inactivated by activated protein C (negative feedback).

Prothrombin to thrombin: Prothrombin (factor II) is a vitamin K-dependent zymogen. The prothrombinase complex cleaves prothrombin at two sites, releasing the active serine protease thrombin. Thrombin has multiple substrates and regulatory functions.

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Coagulation Factors

Factor Nomenclature and Properties

Vitamin K-Dependent Factors

Factors II, VII, IX, and X (plus proteins C and S) require vitamin K for gamma-carboxylation of glutamic acid residues. Gamma-carboxyglutamic acid (Gla) residues bind calcium and enable membrane binding. Warfarin inhibits vitamin K epoxide reductase, preventing gamma-carboxylation and producing non-functional factors (PMID: 18565894).

Clinical implications:

• Factor VII has shortest half-life (4-6 hours) → PT prolonged first with warfarin
• Factor II has longest half-life (60-72 hours) → antithrombotic effect delayed 5-7 days
• Factor X half-life (40-45 hours) important for actual anticoagulant effect
• Vitamin K reversal: oral 1-3 days, IV 6-12 hours (faster synthesis)

Factor Complexes

Tenase complex (intrinsic): IXa-VIIIa-Ca²⁺-phospholipid

• Activates factor X
• 200,000-fold rate enhancement over IXa alone
• Factor VIII is the rate-limiting component (deficiency = haemophilia A)

Prothrombinase complex: Xa-Va-Ca²⁺-phospholipid

• Activates prothrombin to thrombin
• 300,000-fold rate enhancement over Xa alone
• Factor V activated by thrombin (positive feedback)
• Factor V inactivated by APC (negative feedback)

Extrinsic tenase: TF-VIIa-Ca²⁺

• Activates factors X and IX
• Primary physiological initiator
• Rapidly inhibited by TFPI

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Tissue Factor Pathway

Tissue Factor Biology

Structure: Tissue factor (CD142) is a 47 kDa transmembrane glycoprotein, the only coagulation factor that is a true receptor. TF has an extracellular domain (factor VII/VIIa binding), transmembrane domain, and short cytoplasmic tail (signalling).

Distribution: TF is constitutively expressed on:

• Adventitial fibroblasts (perivascular)
• Vascular smooth muscle cells
• Organ capsules (brain, lung, heart, kidney, placenta)
• Epithelial cells (skin, mucosa)

This distribution creates a "haemostatic envelope" around blood vessels. TF is not normally expressed on cells in contact with blood (endothelium, leukocytes), but expression can be induced (PMID: 17510281).

Inducible TF expression: Pathological TF expression occurs on:

• Monocytes (LPS, cytokines, immune complexes)
• Endothelial cells (TNF-α, IL-1β, thrombin, hypoxia)
• Cancer cells (constitutive in many malignancies)
• Activated platelets (controversial, may be microparticle-derived)

Inducible TF contributes to thrombosis in sepsis, cancer, and inflammatory states.

TF-VIIa Complex

Formation: When TF is exposed to blood, it binds factor VII/VIIa. Factor VIIa circulates at ~1% of total factor VII (~10 pM). TF binding increases factor VIIa activity by 10⁷-fold. Factor VII can be activated to VIIa by the TF-VIIa-Xa complex (autoactivation), factor IXa, factor Xa, or thrombin.

Substrates:

• Factor X → Xa (primary)
• Factor IX → IXa (important for sustained coagulation)

Inhibition: TFPI binds to factor Xa, then this complex binds to TF-VIIa, forming an inactive quaternary complex. This limits TF-dependent coagulation to the initiation phase.

TFPI (Tissue Factor Pathway Inhibitor)

Structure: TFPI is a 40 kDa Kunitz-type serine protease inhibitor with three Kunitz domains (K1 binds TF-VIIa-Xa complex, K2 binds Xa, K3 binds heparin).

Mechanism: TFPI inhibits factor Xa first, then the TFPI-Xa complex inhibits TF-VIIa. This "product inhibition" means that the TF pathway inhibits itself once factor Xa is generated.

Distribution: TFPI is produced by endothelial cells. ~80% is bound to endothelium (released by heparin), ~20% in plasma (mostly bound to lipoproteins), ~2.5% in platelets. Heparin releases endothelial TFPI, contributing to its anticoagulant effect (PMID: 10699111).

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Thrombin Generation

Thrombin (factor IIa) is the central enzyme of the coagulation cascade with multiple procoagulant, anticoagulant, and cellular effects.

Thrombin Structure and Activation

Prothrombin (factor II): A vitamin K-dependent glycoprotein (72 kDa) synthesised in the liver. Contains Gla domain (membrane binding), two kringle domains, and serine protease domain. Plasma concentration ~100 μg/mL (1.4 μM).

Activation: The prothrombinase complex (Xa-Va-Ca²⁺-phospholipid) cleaves prothrombin at two sites:

1. Arg271-Thr272: Releases fragment 1.2 + prethrombin 2
2. Arg320-Ile321: Generates active α-thrombin

Meizothrombin is an intermediate form with partial activity.

Thrombin Functions

Procoagulant functions:

1. Cleaves fibrinogen to fibrin monomers
2. Activates factor XIII → XIIIa (fibrin cross-linking)
3. Activates factor XI → XIa (intrinsic pathway amplification)
4. Activates factor V → Va (prothrombinase cofactor)
5. Activates factor VIII → VIIIa (tenase cofactor)
6. Activates platelets via PAR1/PAR4 (amplification)
7. Activates TAFI → TAFIa (fibrinolysis inhibition)

Anticoagulant functions (when bound to thrombomodulin):

1. Activates protein C → APC (inactivates Va, VIIIa)
2. Reduced fibrinogen cleavage
3. Reduced platelet activation

Cellular functions:

1. Platelet activation (shape change, aggregation, secretion)
2. Endothelial activation (vWF release, P-selectin expression)
3. Smooth muscle proliferation
4. Leukocyte activation

Thrombin Kinetics

Thrombin generation curve: Measured by calibrated automated thrombography (CAT) or thrombin generation assay. Parameters include:

• Lag time: Time to initiation of thrombin generation
• Peak thrombin: Maximum thrombin concentration (~100-400 nM)
• Time to peak: Time to reach maximum thrombin
• Endogenous thrombin potential (ETP): Area under curve, total thrombin generated

Thrombin generation testing better reflects global coagulation than PT/APTT and predicts bleeding/thrombosis risk (PMID: 16934139).

Thrombin Inhibition

Direct thrombin inhibitors: Bind to active site and/or exosite 1

• Hirudin (bivalirudin): Exosite 1 + active site, irreversible
• Argatroban: Active site only, reversible
• Dabigatran: Active site, reversible, oral

Indirect thrombin inhibitors: Require antithrombin

• Unfractionated heparin: Enhances AT inhibition of thrombin and Xa
• Requires chain length ≥18 saccharides for thrombin inhibition

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Fibrin Formation

Fibrinogen Structure

Structure: Fibrinogen is a 340 kDa glycoprotein consisting of two sets of three polypeptide chains (Aα, Bβ, γ)₂ held together by disulfide bonds. The molecule has a trinodular structure with central E domain and two lateral D domains connected by coiled-coil regions. Plasma concentration is 2-4 g/L (PMID: 15842653).

Binding sites: Fibrinogen contains:

• Thrombin cleavage sites (releases fibrinopeptides A and B)
• GPIIb/IIIa binding sites (RGD sequences for platelet aggregation)
• Factor XIIIa cross-linking sites (lysine and glutamine residues)
• Plasmin cleavage sites (fibrinolysis)

Fibrin Polymerisation

Fibrinopeptide release: Thrombin cleaves fibrinopeptides A and B from the N-termini of Aα and Bβ chains:

• Fibrinopeptide A (FPA, 16 aa): Released first, exposes "A" polymerisation site
• Fibrinopeptide B (FPB, 14 aa): Released second, exposes "B" polymerisation site

Fibrin monomer polymerisation: Fibrin monomers spontaneously polymerise through knob-hole interactions:

• "A" knob (exposed by FPA release) binds to "a" hole in D domain
• "B" knob (exposed by FPB release) binds to "b" hole in D domain

Initial polymerisation forms two-stranded protofibrils, which then laterally aggregate into thicker fibrin fibres. This produces a mesh-like clot structure (PMID: 11500106).

Clot structure: Fibrin clot structure affects mechanical properties and susceptibility to fibrinolysis:

• Thin fibres (high thrombin): Dense clot, resistant to lysis
• Thick fibres (low thrombin): Loose clot, susceptible to lysis
• Clot architecture influenced by fibrinogen concentration, thrombin concentration, calcium, factor XIII, and plasma proteins

Factor XIII Cross-Linking

Factor XIII structure: Factor XIII is a transglutaminase that circulates as an A₂B₂ heterotetramer (A subunits contain active site, B subunits are carrier/regulatory). Platelet factor XIII lacks B subunits.

Activation: Thrombin cleaves the activation peptide from A subunits. Calcium induces dissociation of B subunits, exposing the active site. Factor XIIIa is also activated by fibrin (fibrin accelerates thrombin cleavage 80-fold).

Cross-linking reactions: Factor XIIIa catalyses transamidation reactions between glutamine and lysine residues, forming isopeptide bonds:

1. γ-chain cross-linking: First and fastest, links adjacent D domains (γ-γ dimers)
2. α-chain cross-linking: Slower, forms α-polymer networks
3. α-γ heteropolymers: Further cross-linking

Cross-linked fibrin is resistant to mechanical disruption and plasmin degradation. Factor XIII deficiency causes delayed bleeding (initial plug forms but fails) and poor wound healing (PMID: 23992447).

D-Dimer Formation

Plasmin degradation: Plasmin cleaves fibrin at specific sites, producing fibrin degradation products (FDPs). Because cross-linked fibrin contains covalent bonds between D domains, plasmin degradation produces D-dimer (two cross-linked D domains). D-dimer is a specific marker of cross-linked fibrin degradation (not fibrinogen degradation) (PMID: 18556770).

Clinical significance: D-dimer is elevated in:

• Venous thromboembolism (PE, DVT)
• DIC
• Recent surgery or trauma
• Malignancy
• Pregnancy
• Inflammation, infection
• Advanced age

D-dimer has high negative predictive value for VTE (normal D-dimer with low clinical probability effectively excludes VTE) but low specificity.

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Natural Anticoagulants

The natural anticoagulant systems prevent excessive clot formation and limit coagulation to sites of injury. Deficiency of these proteins causes thrombophilia.

Antithrombin (AT)

Structure: Antithrombin is a 58 kDa serine protease inhibitor (serpin) synthesised in the liver. Plasma concentration ~150 μg/mL (2.5 μM), half-life 2.5-3 days. AT contains a reactive centre loop that acts as "bait" for target proteases.

Mechanism: AT forms irreversible 1:1 complexes with target serine proteases:

• Factor IIa (thrombin): Primary target
• Factor Xa: Major target
• Factors IXa, XIa, XIIa: Minor targets

The inhibition reaction is slow (second-order rate constant ~10⁴ M⁻¹s⁻¹) but is accelerated ~1000-fold by heparin (rate constant ~10⁷ M⁻¹s⁻¹). Heparin binding causes conformational change in AT, improving accessibility of the reactive centre loop (PMID: 15105442).

Heparin cofactor activity: Unfractionated heparin binds to AT via a specific pentasaccharide sequence. For thrombin inhibition, heparin must also bind to thrombin (requires chain length ≥18 saccharides). For factor Xa inhibition, heparin binding to AT alone is sufficient (pentasaccharide adequate). Low molecular weight heparins (average 15 saccharides) preferentially inhibit Xa over thrombin.

Clinical significance: AT deficiency (50% of normal) increases VTE risk 5-50 fold. Acquired deficiency occurs in:

• DIC (consumption)
• Liver disease (decreased synthesis)
• Nephrotic syndrome (urinary loss)
• Heparin therapy (consumption)
• L-asparaginase therapy

Protein C System

The protein C system provides negative feedback on coagulation by inactivating factors Va and VIIIa (PMID: 15125018).

Protein C: A vitamin K-dependent serine protease (62 kDa) synthesised in the liver. Circulates as zymogen, activated by the thrombin-thrombomodulin complex. Half-life 6-8 hours.

Thrombomodulin (TM): A transmembrane glycoprotein on endothelial cells. When thrombin binds to TM, its substrate specificity changes:

• Decreased: Fibrinogen cleavage, factor activation, platelet activation
• Increased: Protein C activation (~1000-fold enhancement)

This converts thrombin from a procoagulant to an anticoagulant enzyme.

Protein S: A vitamin K-dependent cofactor (69 kDa) for APC. ~40% circulates free (active), ~60% bound to C4b-binding protein (inactive). Protein S enhances APC activity ~10-fold by increasing affinity for phospholipid surfaces.

APC mechanism: Activated protein C (APC) proteolytically inactivates factors Va and VIIIa:

• Factor Va → Factor Vi (cleavage at Arg506, Arg306, Arg679)
• Factor VIIIa → Factor VIIIi (cleavage at Arg336, Arg562)

This reduces tenase and prothrombinase activity, limiting thrombin generation.

Factor V Leiden: A point mutation (Arg506Gln) in factor V that eliminates the primary APC cleavage site. Factor V Leiden is resistant to APC inactivation, causing a hypercoagulable state. Present in 5% of Caucasian population, increases VTE risk 3-7 fold (heterozygous) or 80-fold (homozygous) (PMID: 7989937).

TFPI (Tissue Factor Pathway Inhibitor)

As described above, TFPI inhibits the TF-VIIa-Xa complex, limiting TF-dependent coagulation to the initiation phase. TFPI deficiency is embryonically lethal in mice, indicating its essential role.

Other Anticoagulant Mechanisms

Heparan sulfate: Endothelial surface glycosaminoglycans that enhance AT activity, providing anticoagulant properties to intact endothelium.

Thrombin-activatable fibrinolysis inhibitor (TAFI): While primarily antifibrinolytic, TAFI also removes C-terminal lysine residues from partially degraded fibrin, reducing plasminogen binding and protecting the clot from lysis.

Prostacyclin (PGI₂) and nitric oxide (NO): Endothelial-derived vasodilators that inhibit platelet activation.

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Fibrinolysis

Fibrinolysis is the enzymatic degradation of fibrin clots, essential for wound healing and maintaining vascular patency.

Plasminogen and Plasmin

Plasminogen: A 92 kDa glycoprotein synthesised in the liver. Circulates as single-chain zymogen at ~200 μg/mL. Contains five kringle domains (K1-K5) with lysine-binding sites (LBS) that mediate fibrin binding. Two forms exist:

• Glu-plasminogen (native, closed conformation)
• Lys-plasminogen (partially degraded, open conformation, more readily activated)

Plasmin: Active serine protease formed by cleavage at Arg561-Val562. Plasmin degrades fibrin, fibrinogen, and other proteins including factors V and VIII. Plasmin also activates matrix metalloproteinases and releases growth factors from extracellular matrix (PMID: 10910927).

Fibrin-bound plasminogen: Plasminogen binds to fibrin via lysine-binding sites, colocalising with tPA for efficient activation. Fibrin acts as a cofactor, increasing tPA activation of plasminogen 500-fold.

Plasminogen Activators

Tissue-type plasminogen activator (tPA):

• 68 kDa serine protease, primarily from endothelial cells
• Fibrin-dependent activation (low activity without fibrin)
• Cleaves plasminogen at Arg561-Val562
• Half-life: 4-6 minutes (rapidly cleared by liver)
• Therapeutic forms: alteplase, reteplase, tenecteplase

Urokinase-type plasminogen activator (uPA):

• 54 kDa serine protease, from kidney, monocytes
• Fibrin-independent activation
• Important in cell migration, wound healing, cancer metastasis
• Therapeutic: urokinase

Streptokinase: Bacterial protein (streptococci) that forms complex with plasminogen, activating other plasminogen molecules. Not fibrin-specific, causes systemic fibrinolytic state. Antigenic, single use only.

Fibrinolysis Inhibitors

Plasminogen activator inhibitor-1 (PAI-1):

• 50 kDa serpin, primary inhibitor of tPA and uPA
• Secreted by endothelium, platelets, adipocytes
• Forms irreversible 1:1 complexes with tPA/uPA
• Elevated in obesity, metabolic syndrome, sepsis
• Elevated PAI-1 impairs fibrinolysis, promoting thrombosis (PMID: 11023213)

Alpha-2-antiplasmin (α₂AP):

• 70 kDa serpin, primary inhibitor of plasmin
• Rapidly inhibits free plasmin (half-life <0.1 second)
• Slowly inhibits fibrin-bound plasmin (protected by fibrin)
• Cross-linked to fibrin by factor XIIIa (further protects clot)

TAFI (Thrombin-activatable fibrinolysis inhibitor):

• Carboxypeptidase activated by thrombin-TM complex
• Removes C-terminal lysine residues from partially degraded fibrin
• Reduces plasminogen binding to fibrin
• Links coagulation and fibrinolysis (high thrombin → TAFI activation → clot protection)

Regulation of Fibrinolysis

The balance between plasminogen activators (tPA, uPA) and inhibitors (PAI-1, α₂AP, TAFI) determines net fibrinolytic activity.

Profibrinolytic: Enhanced by:

• Endothelial activation (tPA release)
• Stasis (venous occlusion test)
• Exercise
• DDAVP (releases vWF and tPA)

Antifibrinolytic: Enhanced by:

• PAI-1 elevation (inflammation, sepsis, obesity)
• TAFI activation (high thrombin generation)
• Factor XIII cross-linking (α₂AP incorporation into clot)

Antifibrinolytic Drugs

Tranexamic acid (TXA):

• Synthetic lysine analogue
• Binds to lysine-binding sites on plasminogen
• Prevents plasminogen binding to fibrin
• Inhibits plasmin(ogen)-mediated fibrinolysis
• Does not inhibit free plasmin directly

CRASH-2 trial (PMID: 20554319): TXA (1g loading + 1g over 8 hours) within 3 hours of injury reduced all-cause mortality (14.5% vs 16.0%) and death from bleeding (4.9% vs 5.7%) in trauma patients. TXA given after 3 hours increased death from bleeding. This established TXA as standard of care in trauma and has been extended to PPH (WOMAN trial) and surgery.

Aminocaproic acid: Similar mechanism to TXA but less potent and shorter half-life. Rarely used.

Aprotinin: Serine protease inhibitor (bovine), directly inhibits plasmin. Withdrawn due to increased mortality (BART trial), limited availability for compassionate use.

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Endothelial Function

The endothelium plays a central role in haemostatic regulation, providing both anticoagulant and procoagulant functions depending on the physiological state.

Anticoagulant Endothelium (Resting State)

Thrombomodulin (TM): Constitutively expressed on endothelium. Binds thrombin and converts it from procoagulant to anticoagulant (protein C activation). TM expression is reduced by inflammatory cytokines.

Endothelial protein C receptor (EPCR): Binds protein C and presents it to the thrombin-TM complex, enhancing activation 20-fold. EPCR-bound APC also has cytoprotective effects via PAR1 signalling.

Heparan sulfate proteoglycans: Glycosaminoglycans on endothelial surface that enhance AT activity, providing anticoagulant glycocalyx.

TFPI: Endothelial cells synthesise and express TFPI on their surface, inhibiting TF-VIIa-Xa.

Prostacyclin (PGI₂): Synthesised from arachidonic acid by cyclooxygenase-2 and prostacyclin synthase. PGI₂ causes vasodilation (smooth muscle relaxation) and inhibits platelet activation (increases cAMP). Half-life 2-3 minutes (PMID: 8632973).

Nitric oxide (NO): Synthesised by endothelial nitric oxide synthase (eNOS) from L-arginine. NO causes vasodilation and inhibits platelet adhesion, activation, and aggregation (increases cGMP). NO also inhibits leukocyte adhesion and smooth muscle proliferation (PMID: 23011416).

Ecto-ADPase (CD39): Degrades ADP released by activated platelets, limiting platelet recruitment.

Procoagulant Endothelium (Activated State)

Endothelial activation by inflammatory cytokines (TNF-α, IL-1β), thrombin, histamine, or hypoxia shifts the balance to a procoagulant state.

TF expression: Normally absent on endothelium, induced by cytokines and LPS. Contributes to thrombosis in sepsis and DIC.

Reduced TM expression: Inflammatory cytokines reduce thrombomodulin expression, decreasing protein C activation.

vWF release: Endothelial activation releases ultra-large vWF multimers from Weibel-Palade bodies. These are highly thrombogenic before ADAMTS13 cleavage.

PAI-1 release: Endothelial cells release PAI-1, inhibiting fibrinolysis.

P-selectin expression: Weibel-Palade body fusion expresses P-selectin, mediating platelet and leukocyte adhesion.

Loss of glycocalyx: Inflammatory conditions degrade the endothelial glycocalyx, exposing procoagulant subendothelium.

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Coagulation Tests

Prothrombin Time (PT) and INR

Principle: Measures time for clot formation after adding tissue factor (thromboplastin) and calcium to citrated plasma. Reflects extrinsic and common pathways (factors VII, X, V, II, I).

Normal values: PT 11-13 seconds (varies with reagent); INR 0.9-1.1

INR calculation: INR = (Patient PT / Mean Normal PT)^ISI

• ISI (International Sensitivity Index) corrects for thromboplastin variability
• Standardises monitoring for warfarin therapy

Prolonged by:

• Warfarin/vitamin K deficiency
• Liver disease
• DIC
• Factor VII, X, V, II, I deficiency
• Lupus anticoagulant (usually, may shorten)
• Direct oral anticoagulants (variable effect)

Limitations:

• Does not reflect in vivo haemostasis
• Insensitive to hypercoagulable states
• Affected by sample handling (under-filling, delayed processing)

Activated Partial Thromboplastin Time (APTT)

Principle: Measures time for clot formation after adding phospholipid, contact activator (kaolin, ellagic acid, silica), and calcium to citrated plasma. Reflects intrinsic and common pathways (factors XII, XI, IX, VIII, X, V, II, I).

Normal values: 25-35 seconds (varies with reagent)

Prolonged by:

• Heparin (UFH, not LMWH)
• Factor XII, XI, IX, VIII, X, V, II, I deficiency
• Lupus anticoagulant
• Factor inhibitors (antibodies)
• DIC

Uses:

• UFH monitoring (target ratio 1.5-2.5)
• Haemophilia screening
• Lupus anticoagulant testing
• Mixing studies (corrects with deficiency, not with inhibitor)

Limitations:

• Does not reflect in vivo haemostasis
• Factor XII deficiency prolongs APTT but does not cause bleeding
• Variable sensitivity to different heparin preparations

Fibrinogen

Clauss method: Measures time for clot formation after adding excess thrombin to diluted plasma. Clotting time inversely proportional to fibrinogen concentration.

Normal values: 2-4 g/L

Critical threshold: <1.5 g/L associated with increased bleeding risk; <1.0 g/L significantly impairs clot formation (PMID: 31022311).

Elevated in:

• Acute phase response (infection, inflammation, surgery)
• Pregnancy
• Malignancy

Decreased in:

• DIC (consumption)
• Massive haemorrhage (dilution, consumption)
• Liver failure (decreased synthesis)
• Thrombolytic therapy
• Congenital afibrinogenaemia/hypofibrinogenaemia

D-Dimer

Principle: Immunoassay detection of D-dimer (cross-linked fibrin degradation product).

Normal values: <0.5 mg/L FEU (varies with assay)

Age-adjusted cut-off: Age × 10 μg/L for patients >50 years (improves specificity without losing sensitivity)

Clinical use:

• VTE exclusion (high NPV with low clinical probability)
• DIC diagnosis (ISTH scoring)
• Not useful for VTE diagnosis (low specificity)

Elevated in: VTE, DIC, surgery, trauma, pregnancy, malignancy, sepsis, inflammation, advanced age

Thrombin Time (TT)

Principle: Time for clot formation after adding thrombin to plasma. Measures fibrinogen-to-fibrin conversion.

Normal values: 14-21 seconds

Prolonged by:

• Heparin (very sensitive)
• Hypofibrinogenaemia
• Dysfibrinogenaemia
• Fibrin degradation products
• Direct thrombin inhibitors

Use: Heparin detection, dysfibrinogenaemia screening

Reptilase Time

Principle: Snake venom enzyme cleaves fibrinogen (not inhibited by heparin or hirudin).

Use: Distinguishes heparin effect from fibrinogen abnormality

• Prolonged TT + normal reptilase time = heparin present
• Prolonged TT + prolonged reptilase time = fibrinogen abnormality

Anti-Xa Assay

Principle: Measures inhibition of factor Xa by heparin-AT complex. Patient plasma (with heparin) added to known amount of Xa. Residual Xa activity measured by chromogenic substrate.

Uses:

• LMWH monitoring (target 0.5-1.0 U/mL peak for treatment)
• UFH monitoring (alternative to APTT, especially with lupus anticoagulant)
• Fondaparinux monitoring
• Direct Xa inhibitor detection (rivaroxaban, apixaban, edoxaban)

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Viscoelastic Testing

Viscoelastic haemostatic assays (VHAs) provide real-time, global assessment of clot formation, strength, and lysis. The two main platforms are thromboelastography (TEG) and rotational thromboelastometry (ROTEM) (PMID: 25891637).

TEG (Thromboelastography)

Principle: Blood sample in oscillating cup; pin suspended in sample attached to torsion wire. As clot forms, fibrin strands connect cup to pin, transmitting rotation. Output is graphical tracing reflecting clot kinetics.

Parameters:

Modifications:

• Kaolin TEG: Standard activator
• Rapid TEG: Tissue factor and kaolin (faster result)
• TEG with heparinase: Removes heparin effect
• Functional fibrinogen TEG: Platelet inhibition (isolates fibrinogen contribution)
• TEG-PM (platelet mapping): Assesses antiplatelet effect

ROTEM (Rotational Thromboelastometry)

Principle: Similar to TEG but pin oscillates rather than cup. Different terminology and slightly different reference ranges.

Parameters:

ROTEM assays:

• EXTEM: Tissue factor activation (extrinsic pathway)
• INTEM: Contact activation (intrinsic pathway)
• FIBTEM: EXTEM + platelet inhibitor (isolates fibrinogen)
• APTEM: EXTEM + antifibrinolytic (detects hyperfibrinolysis)
• HEPTEM: INTEM + heparinase (detects heparin effect)

Clinical Applications

Massive transfusion protocols: VHA-guided transfusion reduces blood product use compared to standard coagulation tests (PMID: 26010738).

Algorithm example (ROTEM-guided):

• Low EXTEM CT → FFP
• Low FIBTEM MCF (<10 mm) → Fibrinogen concentrate or cryoprecipitate
• Low EXTEM MCF with normal FIBTEM → Platelets
• APTEM MCF > EXTEM MCF → Hyperfibrinolysis → TXA

Cardiac surgery: VHA predicts bleeding and guides haemostatic intervention. Reduces transfusion requirements.

Trauma: PROPPR trial showed balanced transfusion improved outcomes; VHA enables targeted therapy.

Liver transplantation: Guides management in complex coagulopathy.

Obstetrics: Detects hyperfibrinolysis in postpartum haemorrhage.

Limitations of VHAs

• Point-of-care, but requires trained personnel
• Whole blood test (includes cellular components)
• Does not detect platelet dysfunction (aspirin, clopidogrel) without special assays
• Does not reflect vWF function
• Not standardised across platforms
• Affected by hypothermia, acidosis

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DIC Pathophysiology

Disseminated intravascular coagulation (DIC) is a syndrome of dysregulated coagulation characterised by widespread microvascular thrombosis and consumption of platelets and coagulation factors, leading to both organ dysfunction from thrombosis and bleeding from consumptive coagulopathy (PMID: 19714458).

Aetiology

Pathophysiology

Initiating event: Tissue factor exposure (trauma, surgery, cancer) or TF expression on monocytes/endothelium (sepsis, inflammation) triggers massive thrombin generation.

Uncontrolled thrombin generation:

1. TF-VIIa activates coagulation cascade
2. Impaired natural anticoagulants (AT, protein C consumption; reduced TM expression)
3. Inflammatory cytokines amplify TF expression and impair fibrinolysis (PAI-1 elevation)

Microvascular thrombosis: Widespread fibrin deposition in small vessels causes:

• Organ ischaemia and dysfunction (kidney, liver, lung, brain)
• Microangiopathic haemolytic anaemia (schistocytes)
• Tissue necrosis (purpura fulminans in severe cases)

Consumptive coagulopathy: Continuous coagulation and fibrinolysis consume:

• Platelets → thrombocytopenia
• Fibrinogen → hypofibrinogenaemia
• Factors V, VIII, antithrombin, protein C → factor depletion

Secondary fibrinolysis: Plasminogen activation attempts to lyse microthrombi, producing:

• D-dimer elevation (marker of cross-linked fibrin breakdown)
• FDPs (impair platelet function, contribute to bleeding)

Clinical Presentation

Acute DIC: Rapid consumption with severe bleeding

• Oozing from wounds, IV sites, lines
• Mucosal bleeding
• Haematuria
• GI bleeding
• Intracranial haemorrhage

Chronic DIC: Compensated, more thrombotic than bleeding

• Recurrent VTE
• Arterial thrombosis
• Trousseau syndrome (malignancy-associated)

ISTH DIC Scoring System

The International Society on Thrombosis and Haemostasis (ISTH) scoring system provides a standardised diagnostic approach (PMID: 11816725).

Prerequisites: Underlying disorder known to be associated with DIC

Interpretation:

• Score ≥5: Compatible with overt DIC, repeat daily
• Score <5: Suggestive, not affirmative; repeat in 1-2 days

DIC Management

Treat underlying cause: Most important intervention

• Source control in sepsis
• Delivery in obstetric causes
• Chemotherapy in APL (ATRA)

Supportive care:

• Platelet transfusion if <50 × 10⁹/L and bleeding (or <20 × 10⁹/L prophylactically)
• FFP if PT/APTT prolonged and bleeding (15-30 mL/kg)
• Fibrinogen replacement if <1.5 g/L (cryoprecipitate or fibrinogen concentrate)
• Avoid antifibrinolytics (contraindicated in DIC due to risk of widespread thrombosis)

Anticoagulation: Consider in chronic DIC with thrombotic predominance (e.g., Trousseau syndrome). Not routinely recommended in acute DIC.

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Clinical Applications

Massive Transfusion

Definition: Transfusion of ≥10 units PRBCs in 24 hours, or ≥4 units in 1 hour with ongoing bleeding, or replacement of entire blood volume in 24 hours.

Pathophysiology of trauma coagulopathy:

1. Tissue injury → TF release → thrombin generation
2. Shock → endothelial activation → thrombomodulin expression → APC generation → hyperfibrinolysis
3. Hypoperfusion → acidosis, hypothermia → enzyme dysfunction
4. Dilution from crystalloid resuscitation

PROPPR Trial (PMID: 25647203): Compared 1:1:1 (plasma:platelets:PRBCs) vs 1:1:2 ratio in severe trauma. 1:1:1 ratio achieved haemostasis more frequently (86% vs 78%) and reduced 24-hour mortality from exsanguination (9.2% vs 14.6%), though 24-hour and 30-day all-cause mortality were similar.

Key principles:

• Activate massive transfusion protocol early
• Balanced blood product resuscitation (1:1:1 or VHA-guided)
• Tranexamic acid within 3 hours (CRASH-2)
• Treat hypothermia, acidosis, hypocalcaemia
• Consider fibrinogen replacement (target >1.5-2 g/L)
• Point-of-care VHA testing enables targeted therapy

Anticoagulation in ICU

Anticoagulation Reversal

Warfarin reversal (for serious bleeding):

• Vitamin K 5-10 mg IV (onset 6-12 hours)
• 4-factor PCC 25-50 U/kg (immediate)
• FFP if PCC unavailable (15-30 mL/kg)

Target INR reversal doses (4-factor PCC):

• INR 2-4: 25 U/kg
• INR 4-6: 35 U/kg
• INR >6: 50 U/kg

Heparin reversal:

• Protamine 1 mg per 100 U UFH (max 50 mg)
• Only ~60% effective for LMWH
• Slow administration to avoid hypotension, anaphylaxis

DOAC reversal:

• Dabigatran: Idarucizumab 5g IV (complete reversal in minutes)
• Rivaroxaban/Apixaban: Andexanet alfa 400-800 mg bolus + infusion, or 4-factor PCC 50 U/kg if unavailable

Heparin-Induced Thrombocytopenia (HIT)

Pathophysiology: Antibodies to PF4-heparin complexes activate platelets, causing:

• Thrombocytopenia (platelet consumption)
• Paradoxical thrombosis (venous > arterial)

4T Score for clinical probability:

• Thrombocytopenia
• Timing (day 5-10 or immediate if prior exposure)
• Thrombosis or other sequelae
• Other causes for thrombocytopenia

Management:

• Stop all heparin (including line flushes, coated catheters)
• Start alternative anticoagulation (argatroban, bivalirudin, fondaparinux)
• Do not give warfarin until platelet recovery (causes skin necrosis)
• No platelet transfusion (fuels thrombosis)

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Australian/NZ Context

Australian Haemophilia Centre Guidelines

The Australian Haemophilia Centre provides comprehensive guidance for factor replacement therapy, perioperative management of patients with bleeding disorders, and management of inhibitors.

Key considerations:

• Access to recombinant factor concentrates
• Extended half-life products available
• Gene therapy emerging
• Emicizumab for haemophilia A with inhibitors

ANZICS Massive Transfusion Protocols

ANZICS-CORE recommends implementation of institutional massive transfusion protocols with:

• Early activation criteria (clinical + laboratory triggers)
• Balanced product administration (1:1:1 ratio)
• Fibrinogen replacement to maintain >1.5 g/L
• Tranexamic acid for trauma within 3 hours
• Point-of-care VHA testing where available
• Calcium replacement to maintain ionised Ca >1.0 mmol/L

Indigenous Health Considerations

Higher VTE rates: Aboriginal and Torres Strait Islander peoples have elevated rates of venous thromboembolism, particularly in context of chronic disease burden, higher rates of immobility from hospitalisations, and obesity (PMID: 32144954).

Rheumatic heart disease: High prevalence in Indigenous Australians with need for anticoagulation for mechanical valves, atrial fibrillation. Challenges with warfarin monitoring in remote areas.

Access issues in remote areas:

• Blood product availability (limited storage, transport)
• Laboratory testing delays
• Specialist haematology access
• Cultural considerations for blood transfusion
• Role of Royal Flying Doctor Service (RFDS) in retrieval

Māori health (New Zealand): Similar considerations with higher cardiovascular disease burden, VTE risk, and access challenges in rural areas. Whānau involvement in transfusion decisions.

Blood Product Availability

Australian Red Cross Lifeblood: Supplies blood products nationally

• Universal donor blood for emergencies (O negative PRBCs, AB plasma)
• Fractionated products (FFP, cryoprecipitate, platelets)
• Specialist products (PCC, fibrinogen concentrate, factor concentrates)

Remote/rural considerations:

• Limited cold storage capacity
• Product expiry (platelets 5 days, FFP 1 year frozen)
• Emergency product requests via blood bank
• Walking blood bank protocols for remote settings
• Group-specific vs emergency universal products

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SAQ Practice

Question 1: Cell-Based Model of Coagulation (15 marks)

A 45-year-old man with haemophilia A (severe, factor VIII <1%) presents with acute haemarthrosis following minor trauma.

(a) Describe the cell-based model of coagulation, explaining why this patient bleeds despite having an intact extrinsic pathway (10 marks)

(b) Outline the principles of factor replacement therapy for this patient (5 marks)

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Model Answer:

(a) Cell-Based Model of Coagulation (10 marks)

The cell-based model describes coagulation as occurring on specific cell surfaces through three overlapping phases: initiation, amplification, and propagation.

Initiation Phase (3 marks)

• Occurs on tissue factor (TF)-bearing cells (fibroblasts, smooth muscle cells)
• TF binds circulating factor VIIa (1% of total VII is active)
• TF-VIIa complex activates factor X to Xa and factor IX to IXa
• Small amounts of thrombin generated (~10 nM)
• Rapidly inhibited by TFPI (tissue factor pathway inhibitor)
• Insufficient thrombin for stable clot formation

Amplification Phase (3 marks)

• Occurs on activated platelet surface
• Small amounts of thrombin from initiation activate platelets via PAR1/PAR4
• Thrombin activates cofactors: factor V → Va, factor VIII → VIIIa
• Thrombin activates factor XI → XIa on platelet surface
• Thrombin releases factor VIII from vWF
• Platelets expose phosphatidylserine (provides coagulation surface)

Propagation Phase (3 marks)

• Massive thrombin generation ("thrombin burst") on activated platelet surface
• Factor IXa + VIIIa form intrinsic tenase complex (200,000-fold rate enhancement)
• Tenase generates large amounts of factor Xa
• Factor Xa + Va form prothrombinase complex (300,000-fold rate enhancement)
• ~96% of total thrombin generated in this phase
• Thrombin cleaves fibrinogen to fibrin, activates factor XIII

Why Haemophilia A Causes Bleeding (1 mark)

• Factor VIII deficiency prevents formation of functional tenase complex (VIIIa-IXa)
• Initiation phase intact (normal PT)
• Propagation phase severely impaired—cannot generate adequate thrombin burst
• Explains why extrinsic pathway (TF-VIIa) cannot compensate
• Explains why factor XII deficiency does not cause bleeding (not required for propagation)

(b) Factor Replacement Principles (5 marks)

Initial Treatment (3 marks)

• Recombinant factor VIII concentrate preferred (reduced pathogen transmission risk)
• Target factor VIII level 50-100% for acute haemarthrosis
• Dosing: Factor VIII dose (IU) = Body weight (kg) × Desired rise (%) × 0.5
• For severe haemarthrosis: 25-40 IU/kg (~50 kg patient = 1250-2000 IU)
• Repeat dosing every 12 hours (VIII half-life 8-12 hours) until bleeding controlled

Ongoing Management (2 marks)

• Extended half-life products available (reduced dosing frequency)
• Consider prophylaxis for recurrent bleeding
• Emicizumab for patients with inhibitors
• Joint protection, physiotherapy
• Multidisciplinary haemophilia centre care

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Question 2: DIC and Fibrinolysis (15 marks)

A 32-year-old woman is admitted to ICU with septic shock from urosepsis. Her laboratory results show: platelets 45 × 10⁹/L, PT 22 seconds (control 12 s), fibrinogen 0.8 g/L, D-dimer >10 mg/L FEU. She is oozing from IV sites.

(a) Calculate the ISTH DIC score and interpret the result (4 marks)

(b) Describe the pathophysiology of DIC in sepsis (6 marks)

(c) Outline the management of this patient's coagulopathy (5 marks)

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Model Answer:

(a) ISTH DIC Score (4 marks)

Interpretation (1 mark): Score ≥5 is compatible with overt DIC. Score of 8 confirms overt DIC in context of sepsis (prerequisite met: underlying disorder known to cause DIC).

(b) Pathophysiology of DIC in Sepsis (6 marks)

Initiating Event (1.5 marks)

• Gram-negative bacteria release endotoxin (LPS); Gram-positive release exotoxins
• LPS activates monocytes and endothelial cells via TLR4
• Inflammatory cytokines (TNF-α, IL-1β, IL-6) are released
• Tissue factor expression induced on monocytes and endothelium

Uncontrolled Thrombin Generation (1.5 marks)

• TF-VIIa complex triggers coagulation cascade
• Massive thrombin generation throughout circulation
• Natural anticoagulants overwhelmed:
  • Antithrombin consumed
  • Protein C/S consumed; thrombomodulin expression reduced
  • TFPI inadequate

Microvascular Thrombosis (1.5 marks)

• Widespread fibrin deposition in small vessels
• Organ ischaemia → multiorgan dysfunction (kidney, liver, lung)
• Microangiopathic haemolytic anaemia (red cell fragmentation)
• Tissue necrosis (purpura fulminans in severe cases)

Consumptive Coagulopathy (1.5 marks)

• Continuous coagulation consumes:
  • Platelets → thrombocytopenia
  • Fibrinogen → hypofibrinogenaemia
  • Factors V, VIII → factor depletion
• Secondary fibrinolysis attempts to lyse microthrombi
• However, PAI-1 elevated in sepsis (impaired fibrinolysis)
• D-dimer markedly elevated from fibrin breakdown
• Fibrin degradation products impair platelet function → bleeding

(c) Management of DIC (5 marks)

Treat Underlying Cause (Essential, 2 marks)

• Source control for urosepsis (IV antibiotics, consider drainage)
• This is the most important intervention
• DIC will not resolve without treating the underlying cause

Supportive Blood Product Therapy (2 marks)

• Platelet transfusion: Target >50 × 10⁹/L in actively bleeding patient
  • This patient requires platelets (45 × 10⁹/L with bleeding)
• Fibrinogen replacement: Target >1.5 g/L
  • Cryoprecipitate (10 units) or fibrinogen concentrate (3-4 g)
  • This patient requires fibrinogen (0.8 g/L)
• Fresh frozen plasma: 15-30 mL/kg if PT prolonged >1.5× normal and bleeding
  • Consider if bleeding continues despite fibrinogen replacement
• Avoid aggressive FFP if no bleeding (fluid overload risk)

Additional Considerations (1 mark)

• Do NOT give antifibrinolytics (TXA) in DIC—risk of widespread thrombosis
• Anticoagulation generally NOT indicated in acute bleeding DIC
• Maintain core temperature, correct acidosis
• Repeat coagulation tests regularly (ISTH score daily)
• Consider VHA (TEG/ROTEM) to guide targeted therapy

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Viva Scenarios

Viva Scenario 1: Coagulation Cascade and Anticoagulation (20 marks)

Opening Stem: "Tell me about the coagulation cascade."

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Candidate: The coagulation cascade is the enzymatic process by which blood clots form. The modern understanding is the cell-based model, which describes coagulation occurring on cell surfaces through three phases: initiation, amplification, and propagation.

In the initiation phase, tissue factor—exposed by vascular injury—binds factor VIIa, forming the TF-VIIa complex. This activates factors X and IX, generating small amounts of thrombin. However, this is rapidly inhibited by TFPI.

In the amplification phase, the small amount of thrombin activates platelets via PAR1 and PAR4 receptors. Thrombin also activates cofactors V and VIII, and factor XI on the platelet surface.

In the propagation phase, factor IXa combines with VIIIa on the activated platelet surface, forming the intrinsic tenase complex. This generates large amounts of factor Xa, which combines with Va to form the prothrombinase complex. This produces the "thrombin burst"—approximately 96% of total thrombin—which cleaves fibrinogen to fibrin.

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Examiner: "How does the classical cascade model differ from the cell-based model?"

Candidate: The classical cascade, described in the 1960s by Macfarlane and Davie, depicts two parallel pathways—intrinsic and extrinsic—converging on a common pathway. The intrinsic pathway is initiated by factor XII on negatively charged surfaces, while the extrinsic pathway is initiated by tissue factor.

The key differences are:

1. Factor XII role: The classical model suggests factor XII initiates coagulation, but factor XII deficiency does not cause bleeding. The cell-based model correctly predicts this—factor XII is not required for normal haemostasis.

2. Factor XI activation: In the classical model, XI is activated by XIIa. In the cell-based model, XI is activated by thrombin on the platelet surface—a feedback loop.

3. Haemophilia explanation: The classical model cannot explain why haemophilia (VIII or IX deficiency) causes severe bleeding despite an intact extrinsic pathway. The cell-based model explains this—VIII and IX are essential for the tenase complex on platelets, which generates the thrombin burst.

The classical model remains useful for interpreting laboratory tests—PT reflects the extrinsic pathway, APTT reflects the intrinsic pathway.

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Examiner: "You mentioned thrombin has multiple functions. What are these?"

Candidate: Thrombin is the central enzyme of coagulation with both procoagulant and anticoagulant functions.

Procoagulant functions:

• Cleaves fibrinogen to fibrin monomers
• Activates factor XIII to XIIIa for fibrin cross-linking
• Activates factor XI (positive feedback)
• Activates factor V to Va (positive feedback)
• Activates factor VIII to VIIIa (positive feedback)
• Activates platelets via PAR1 and PAR4
• Activates TAFI, inhibiting fibrinolysis

Anticoagulant function:

• When thrombin binds to thrombomodulin on endothelial cells, it activates protein C. Activated protein C, with protein S as cofactor, inactivates factors Va and VIIIa, providing negative feedback.

This dual role means thrombin initially promotes clot formation at the injury site but subsequently limits clot propagation through the protein C pathway.

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Examiner: "How does heparin work?"

Candidate: Heparin is an indirect anticoagulant that works by enhancing antithrombin activity.

Antithrombin is a serine protease inhibitor that slowly inhibits thrombin, factor Xa, and other coagulation factors. Heparin binding causes a conformational change in antithrombin, accelerating its activity approximately 1000-fold.

Unfractionated heparin has two anticoagulant mechanisms:

1. Binding to antithrombin alone is sufficient to inhibit factor Xa
2. For thrombin inhibition, heparin must simultaneously bind to both antithrombin and thrombin—this requires a chain length of at least 18 saccharides

Low molecular weight heparins (average 15 saccharides) preferentially inhibit factor Xa over thrombin because they are too short to bridge antithrombin to thrombin.

Fondaparinux is a synthetic pentasaccharide that only inhibits factor Xa—it has no direct effect on thrombin.

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Examiner: "How would you reverse heparin anticoagulation?"

Candidate: Protamine sulfate reverses heparin by binding to the heparin molecule and neutralising its activity.

For unfractionated heparin:

• Dose: 1 mg protamine per 100 units of heparin
• Maximum dose 50 mg
• Administer slowly IV to avoid hypotension, bradycardia, anaphylaxis
• Protamine effect is immediate

For LMWH:

• Protamine only partially effective (approximately 60%)
• Neutralises anti-IIa but not anti-Xa activity
• For recent LMWH: 1 mg protamine per 1 mg enoxaparin (or per 100 anti-Xa units)
• Consider second dose if bleeding continues
• rFVIIa or andexanet alfa may be considered in life-threatening bleeding

For fondaparinux:

• Protamine is NOT effective
• Andexanet alfa may be used
• rFVIIa off-label in life-threatening bleeding
• Consider dialysis (fondaparinux partially dialysable)

Important caveats: Protamine can cause hypotension, bradycardia, and allergic reactions—particularly in patients with fish allergies, prior protamine exposure, or NPH insulin use.

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Examiner: "What are the indications for TEG or ROTEM in the ICU?"

Candidate: Viscoelastic haemostatic assays like TEG and ROTEM provide real-time, global assessment of clot formation and lysis.

Main indications:

1. Massive transfusion/major bleeding: Guides targeted blood product therapy. Algorithms based on ROTEM parameters can identify whether the patient needs plasma (low EXTEM CT), fibrinogen (low FIBTEM MCF), platelets (low EXTEM MCF with normal FIBTEM), or antifibrinolytic (improved APTEM versus EXTEM).

2. Cardiac surgery: Detects residual heparin effect, guides protamine dosing, identifies cause of post-bypass bleeding.

3. Liver transplantation: Complex coagulopathy with both bleeding and thrombotic risk; VHA guides individualised therapy.

4. Trauma: Detects acute traumatic coagulopathy and hyperfibrinolysis.

5. Obstetric haemorrhage: Identifies hyperfibrinolysis in PPH.

Advantages over standard tests:

• Faster results (15-30 minutes vs 45-60 minutes for lab tests)
• Whole blood (includes cellular components)
• Assesses clot strength and fibrinolysis (not just initiation)
• Enables targeted therapy (reduces unnecessary transfusion)

Limitations:

• Does not detect antiplatelet drug effects without special assays
• Does not reflect vWF function
• Requires trained personnel and quality assurance
• Not standardised across platforms

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Viva Scenario 2: Fibrinolysis and Antifibrinolytics (20 marks)

Opening Stem: "A trauma patient has been given tranexamic acid. Tell me how this drug works."

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Candidate: Tranexamic acid is a synthetic lysine analogue that inhibits fibrinolysis.

Mechanism of action: Plasminogen contains five kringle domains with lysine-binding sites. These sites normally bind to C-terminal lysine residues on fibrin, allowing plasminogen to localise to the clot for activation by tissue plasminogen activator (tPA).

Tranexamic acid competitively binds to these lysine-binding sites, preventing plasminogen from binding to fibrin. This prevents localisation of plasminogen to the clot surface and prevents its activation to plasmin.

Importantly, TXA does not directly inhibit plasmin—it prevents the plasminogen-fibrin interaction required for efficient fibrinolysis.

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Examiner: "What is the evidence for TXA in trauma?"

Candidate: The key trial is CRASH-2, published in Lancet in 2010.

CRASH-2 was a multinational randomised controlled trial of over 20,000 trauma patients at risk of significant haemorrhage.

Intervention: TXA 1g loading over 10 minutes, then 1g over 8 hours, versus placebo.

Primary outcome: All-cause mortality at 4 weeks was reduced from 16.0% to 14.5% (NNT 67).

Key findings:

• Death due to bleeding was reduced (5.7% to 4.9%)
• TXA given within 3 hours of injury reduced bleeding deaths
• TXA given after 3 hours increased bleeding deaths
• No increase in thrombotic events (PE, DVT, MI, stroke)

This established TXA as standard of care in trauma, with the critical caveat that it must be given early—within 3 hours of injury.

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Examiner: "Why might TXA given after 3 hours increase mortality?"

Candidate: This is not completely understood, but several hypotheses exist:

1. Established DIC: After 3 hours, trauma-induced coagulopathy may have progressed to DIC with microvascular thrombosis. Inhibiting fibrinolysis at this stage may worsen organ ischaemia by preventing clot dissolution.

2. Hyperfibrinolysis shutdown: Initial hyperfibrinolysis may be followed by fibrinolytic shutdown—at this stage, antifibrinolytics may promote thrombosis.

3. Different bleeding phenotype: Early bleeding is more likely to be due to surgical sources amenable to haemostatic intervention. Later bleeding may reflect different pathophysiology.

4. Selection bias: Patients surviving to 3+ hours may have different injury patterns or be in different physiological states.

This finding reinforces the importance of pre-hospital TXA administration in major trauma systems.

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Examiner: "Describe the normal fibrinolytic pathway."

Candidate: Fibrinolysis is the enzymatic process by which fibrin clots are degraded.

Plasminogen activation:

• Plasminogen is synthesised in the liver and circulates in plasma
• It binds to fibrin via lysine-binding sites on its kringle domains
• Tissue plasminogen activator (tPA), released from endothelial cells, binds to fibrin
• tPA activates plasminogen to plasmin by cleaving at Arg561-Val562
• Fibrin acts as a cofactor, increasing tPA activity 500-fold

Plasmin action:

• Plasmin is a serine protease that cleaves fibrin at specific sites
• Degradation of cross-linked fibrin produces D-dimer
• Plasmin also degrades fibrinogen (produces FDPs without D-dimer) and factors V/VIII

Regulation:

• PAI-1 (plasminogen activator inhibitor-1) inhibits tPA and uPA
• Alpha-2-antiplasmin rapidly inhibits free plasmin
• TAFI (thrombin-activatable fibrinolysis inhibitor) removes C-terminal lysines from fibrin, reducing plasminogen binding
• Factor XIIIa cross-links alpha-2-antiplasmin into the clot, protecting it from lysis

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Examiner: "What is hyperfibrinolysis and how would you diagnose it?"

Candidate: Hyperfibrinolysis is a pathological state of excessive fibrinolytic activity where clots are degraded faster than they can form.

Causes:

• Trauma (particularly severe, with tissue injury and shock)
• Liver disease (decreased clearance of tPA, decreased production of inhibitors)
• Acute promyelocytic leukaemia (APL)
• Thrombolytic therapy
• Cardiac surgery (particularly with cardiopulmonary bypass)
• Obstetric emergencies (amniotic fluid embolism, placental abruption)

Clinical features:

• Diffuse microvascular bleeding
• Clots that form then dissolve
• Bleeding from previously haemostatic sites

Diagnosis:

Laboratory:

• Markedly elevated D-dimer and FDPs
• Reduced fibrinogen
• Elevated plasmin-antiplasmin complexes (research test)

Viscoelastic testing (preferred):

• TEG: LY30 >3% (or LY60 >15%) indicates hyperfibrinolysis
• ROTEM: ML (maximum lysis) >15%
• ROTEM APTEM vs EXTEM: If MCF improves with APTEM (contains antifibrinolytic), hyperfibrinolysis is confirmed

Management:

• Tranexamic acid 1g IV (repeat if needed)
• Treat underlying cause
• Fibrinogen replacement may be needed
• In extreme cases (liver transplant), aminocaproic acid infusion considered

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Examiner: "What other clinical applications does TXA have?"

Candidate: Beyond trauma, TXA has established or emerging applications in:

1. Postpartum haemorrhage: The WOMAN trial showed TXA reduced death from bleeding when given within 3 hours of delivery. Now part of PPH guidelines.

2. Perioperative bleeding: Reduces blood loss and transfusion requirements in cardiac surgery, orthopaedic surgery (hip/knee arthroplasty), and spinal surgery.

3. Upper GI bleeding: HALT-IT trial (2020) showed no mortality benefit in GI haemorrhage—TXA is NOT recommended for upper GI bleeding.

4. Intracerebral haemorrhage: TICH-2 trial showed reduced haematoma expansion but no functional benefit. Currently not standard of care.

5. Menorrhagia: Oral TXA reduces menstrual blood loss by 30-50%.

6. Dental procedures in anticoagulated patients: Topical TXA mouthwash.

7. Hereditary angioedema: Reduces attack frequency.

The key principle is that TXA is most effective when hyperfibrinolysis contributes to bleeding—it does not help if fibrinolysis is not the primary problem.

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References

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Natural Anticoagulants

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Australian/NZ Context

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Related Topics

• Haematology in ICU
• Massive Transfusion
• Anticoagulation Management
• Heparin-Induced Thrombocytopenia
• Thromboembolic Disease
• Liver Failure Coagulopathy
• Trauma Coagulopathy
• Obstetric Haemorrhage