Platelets &: Hemostasis

Introduction to Hemostasis

Hemostasis is the physiological process that stops bleeding at the site of vascular injury while maintaining normal blood flow elsewhere. It involves interactions between blood vessels, platelets, and coagulation factors. Dysregulation leads to hemorrhage (excessive bleeding) or thrombosis (inappropriate clotting).

Platelets (thrombocytes) are small, anucleated cell fragments that play a central role in primary hemostasis – the initial formation of a platelet plug at the site of injury.

I. Morphology of Platelets

Physical Traits

Size/Shape: Tiny (2-4 µm), discoid (lens-shaped) when inactive. Upon activation, they become spherical with pseudopods (finger-like projections) to enhance adhesion.
Anucleated: Lack a nucleus; cannot synthesize proteins. Limited lifespan (7-10 days).

Membrane

Rich in glycoproteins (e.g., GP Ib/IX/V, GP Ia/IIa, GP IIb/IIIa) acting as receptors for adhesion molecules (vWF, collagen, fibrinogen).

Cytoplasmic Granules

The cytoplasm contains critical granules and organelle systems.

Alpha-granules

Contain proteins for adhesion/coagulation:

Fibrinogen
von Willebrand factor (vWF)
Platelet factor 4 (PF4)
PDGF, P-selectin

Dense (delta) granules

Contain non-protein activators:

ADP, ATP
Serotonin
Calcium

Lysosomes

Contain hydrolytic enzymes for digesting material.

II. Formation of Platelets (Thrombopoiesis)

Occurs in bone marrow, regulated by Thrombopoietin (TPO).

1. Origin:

Hematopoietic Stem Cells (HSCs) → Common Myeloid Progenitor (CMP).

2. Megakaryoblast:

Progenitor undergoes endoreduplication (DNA replication without division), becoming polyploid.

3. Megakaryocyte:

Largest marrow cell (up to 100 µm). Highly lobulated nucleus.

4. Platelet Release:

Megakaryocytes extend proplatelets into sinusoidal capillaries. Blood shear flow fragments these into thousands of platelets (1,000-3,000 per megakaryocyte).

Regulation: TPO (from liver) stimulates megakaryocytes. Platelets bind and degrade TPO. High platelet mass = Low free TPO = Downregulated production (Negative Feedback).

III. Function of Platelets in Hemostasis (Primary Hemostasis)

1. Adhesion

Injury exposes subendothelial collagen.
Platelets adhere via GP Ib receptor binding to von Willebrand factor (vWF) (bridge between platelet and collagen).
Direct binding via GP Ia/IIa also occurs.
Result: Anchors platelets to injury site.

2. Activation

Triggered by adhesion, Thrombin, and ADP. Causes shape change (discoid → spherical + pseudopods) and granule release.

Key Molecules Released:

ADP: Potent activator, recruits more platelets.
Thromboxane A2 (TxA2): Synthesized via COX-1; powerful vasoconstrictor and aggregator.
Serotonin: Vasoconstriction.
vWF/Fibrinogen: Aid further adhesion/aggregation.

3. Aggregation

Activated platelets express GP IIb/IIIa receptor.
Fibrinogen acts as a bridge, binding to GP IIb/IIIa on adjacent platelets.
Links platelets together to form the primary hemostatic plug.

4. Procoagulant Activity

Activated platelets provide a negatively charged phospholipid surface (phosphatidylserine). This surface concentrates coagulation factors (Tenase/Prothrombinase complexes), accelerating Thrombin generation to convert fibrinogen to fibrin, stabilizing the plug.

Summary of Platelet Function

When a vessel is damaged, platelets:

Adhere to exposed matrix.
Activate (shape change + release substances).
Aggregate to form primary plug.
Provide surface for Secondary Hemostasis (Coagulation Cascade).

Steps and Components of the Coagulation Cascade

While primary hemostasis (platelet plug formation) provides an initial, temporary seal at the site of vascular injury, it is not strong enough to withstand arterial pressure or provide long-term protection. Secondary hemostasis reinforces the platelet plug with a meshwork of fibrin, a strong, insoluble protein. This process is known as blood coagulation or the coagulation cascade, and it involves a series of enzymatic reactions involving plasma proteins called coagulation factors.

I. Overview

The coagulation cascade is traditionally described as having two main pathways that converge on a common pathway. However, a more modern and physiologically relevant view is the cell-based model. We will present both models for a comprehensive understanding.

II. Traditional Model: Intrinsic, Extrinsic, and Common Pathways

This model helps to understand individual factors and their interactions, especially in laboratory testing.

1. Extrinsic Pathway (Initiation)

Initiated when blood is exposed to Tissue Factor (TF), expressed by subendothelial cells (fibroblasts, smooth muscle) upon injury.

Step 1: TF binds to circulating Factor VII (VIIa) → Forms TF-VIIa complex.

Step 2: TF-VIIa complex activates Factor X to Xa and Factor IX to IXa.

Rapid pathway; primarily responsible for initiation.

2. Intrinsic Pathway (Amplification)

Activated by contact of Factor XII with negatively charged surfaces (collagen, platelets) or by XIIa itself.

Step 1: Factor XII → XIIa.

Step 2: XIIa activates Factor XI → XIa.

Step 3: XIa activates Factor IX → IXa.

Step 4: IXa + Factor VIIIa (activated by thrombin) → Tenase Complex (IXa/VIIIa).

The Tenase complex activates Factor X → Xa.

Slower pathway; significant contribution to amplification.

3. Common Pathway

Both pathways converge at the activation of Factor X.

Step 1: Activated Factor X (Xa) + Factor Va (cofactor activated by thrombin) → Prothrombinase Complex (Xa/Va).
Assembles on activated platelet surfaces.

Step 2: Prothrombinase converts Prothrombin (Factor II) → Thrombin (Factor IIa).
Central event of the cascade.

Step 3: Roles of Thrombin:

Converts Fibrinogen (I) → Fibrin monomers.
Activates Factor XIII → XIIIa.
Activates cofactors V and VIII.
Activates Factor XI (feedback amplification).
Activates platelets (positive feedback).

Step 4: Fibrin monomers spontaneously polymerize → Unstable clot.

Step 5: Factor XIIIa (transglutaminase) cross-links fibrin monomers → Stable, insoluble fibrin mesh.

III. Cell-Based Model of Coagulation

This model emphasizes the role of cellular surfaces (TF-bearing cells and activated platelets) and occurs in three overlapping phases.

1. Initiation Phase (on TF-bearing cells)

Vascular injury exposes TF on subendothelial cells.
TF binds VIIa → TF-VIIa.
TF-VIIa activates small amounts of X to Xa and IX to IXa.
Xa + Va generates a small "Thrombin Spurt". Crucial for activating platelets/cofactors.

2. Amplification Phase (on Activated Platelets)

The initial thrombin activates platelets (shape change, exposure of phosphatidylserine).
Thrombin activates cofactors V, VIII, and Factor XI.
Activated platelets provide negatively charged phospholipid surface.
Factor IXa binds VIIIa on platelet surface → Tenase Complex.
Tenase efficiently converts large amounts of X → Xa.

3. Propagation Phase (on Activated Platelets)

Large amounts of Xa + Va assemble on platelet surface → Prothrombinase Complex.
Converts massive amounts of prothrombin → thrombin. Result: "Thrombin Burst".
Thrombin burst rapidly converts fibrinogen to fibrin, activates XIIIa (cross-linking), and further activates platelets → Robust, stable clot.

IV. Key Coagulation Factors & Components

Plasma proteins, mostly synthesized in the liver.

Vitamin K-Dependent Factors:

Factors II, VII, IX, X, Protein C, Protein S. Require Vitamin K for synthesis in liver.

Fibrinogen Group:

Factors I (Fibrinogen), V, VIII, XIII. Consumed during coagulation.

Contact Group:

Factors XII, XI, PK, HMWK. Intrinsic pathway initiation.

V. Role of Calcium (Ca²⁺):

Essential cofactors for activation/function of several factors, particularly for assembly of Tenase/Prothrombinase complexes on phospholipid surfaces.

VI. Goal of Secondary Hemostasis:

Generate a stable, cross-linked fibrin mesh that traps RBCs/cellular elements, providing mechanical strength to the platelet plug and forming a definitive blood clot.

Regulation of Clotting and Fibrinolysis

Hemostasis is a delicate balance. While rapid clot formation is vital to stop bleeding, uncontrolled or excessive clotting can lead to thrombosis, blocking blood vessels and causing severe damage (e.g., heart attack, stroke). Therefore, the body has sophisticated mechanisms to regulate the coagulation cascade and dissolve clots once they are no longer needed.

I. Regulation of Coagulation (Anticoagulation Systems)

These systems work to limit the size and propagation of the clot to the site of injury, preventing it from spreading unnecessarily.

1. Antithrombin (AT)

Mechanism: Major plasma protein that inactivates several factors, particularly Thrombin (IIa), Xa, and lesser amounts of IXa, XIa, and XIIa.

Action: Forms a stable, irreversible complex with these serine proteases, rendering them inactive.

Enhancement by Heparan Sulfate/Heparin: Activity is accelerated (1,000-fold+) when binding to negatively charged polysaccharides like heparan sulfate (natural on endothelium) or heparin (drug). This induces a conformational change exposing the active site.

2. Protein C System

Components: Thrombomodulin, Protein C, and Protein S.

Activation:

Thrombin binds to Thrombomodulin (receptor on healthy endothelium).
This complex activates Protein C into Activated Protein C (APC).

Action of APC:

APC + cofactor Protein S inactivates cofactors Va and VIIIa (via cleavage).
Shuts down prothrombinase and tenase complexes, stopping thrombin generation.

Clinical Relevance: Deficiency in Protein C or S increases thrombosis risk.

3. Tissue Factor Pathway Inhibitor (TFPI)

Mechanism: Directly inhibits the initial step of the extrinsic pathway.

Action: Binds and inactivates Factor Xa. The TFPI-Xa complex then binds and inactivates the TF-VIIa complex.

Result: "Turns off" the tissue factor pathway, limiting the initial thrombin burst.

4. Dilution of Factors

Flowing blood dilutes activated factors, washing them away from the injury site preventing expansion.

5. Hepatic Clearance

Liver clears activated factors and inhibitors from circulation to maintain balance.

II. Clot Dissolution (Fibrinolysis)

Once repair occurs, the stable fibrin clot must be removed (fibrinolysis) to restore flow.

Key Enzyme: Plasmin

Mechanism: Serine protease that cleaves fibrin and fibrinogen, breaking the meshwork.
Formation: Circulates as inactive Plasminogen.
Activation: Converted to Plasmin by Plasminogen Activators.

Plasminogen Activators

Tissue Plasminogen Activator (t-PA)

Released from damaged endothelium.
High affinity for fibrin.
Binds fibrin within clot → activates plasminogen locally.

Urokinase Plasminogen Activator (u-PA)

Found in tissues/fluids (urine).
Converts plasminogen to plasmin.
Role in local fibrinolysis and tissue remodeling.

Inhibitors of Fibrinolysis

Ensures clot doesn't dissolve prematurely.

Plasminogen Activator Inhibitor-1 (PAI-1): Inhibits t-PA and u-PA (reduces plasmin generation).
Alpha-2-antiplasmin (α2AP): Primary inhibitor of free plasmin in circulation. Prevents systemic fibrinogen breakdown.
TAFI (Thrombin Activatable Fibrinolysis Inhibitor): Activated by thrombin. Removes lysine residues from fibrin, making it resistant to plasmin. Links coagulation to fibrinolysis regulation.

Products of Fibrinolysis

Breakdown produces soluble Fibrin Degradation Products (FDPs).

D-Dimer: Specific FDP formed when cross-linked fibrin (by XIIIa) is degraded.
Clinical Significance: Elevated levels indicate recent/ongoing clot formation and breakdown.

Summary of Regulation and Fibrinolysis

Anticoagulation systems (AT, Protein C, TFPI) prevent expansion beyond injury.
Fibrinolysis (Plasminogen/Plasmin via t-PA/u-PA) ensures timely removal.
The balance is crucial for vascular patency and preventing bleeding/thrombosis.

Laboratory Tests and Disorders of Hemostasis

Laboratory tests distinguish between bleeding and clotting disorders, identify specific deficiencies, and guide therapy. They are generally categorized by the phase of hemostasis they assess.

I. Tests of Primary Hemostasis (Platelet Function)

Evaluate platelet number, adhesion, and aggregation.

1. Platelet Count

Normal: 150k - 450k/µL

Thrombocytopenia (<150k)

Bleeding (petechiae, purpura). Causes: Marrow failure, ITP/TTP, splenomegaly.

Thrombocytosis (>450k)

Risk of thrombosis or paradoxical bleeding (dysfunction).

Platelet Function Analyzer (PFA-100)

Screening test simulating vessel injury. Detects vWD, aspirin use, intrinsic defects.

Platelet Aggregometry

Definitive test. Measures response to agonists (ADP, collagen, ristocetin). Diagnoses vWD, Bernard-Soulier.

Note: Bleeding Time is largely historical and replaced by PFA-100.

II. Tests of Secondary Hemostasis (Coagulation Cascade)

Prothrombin Time (PT) & INR

Extrinsic/Common

Normal PT: 10-14 sec. | Normal INR: 0.8-1.2

Prolonged in: Deficiency of VII, X, V, II, Fibrinogen. Liver disease, Vit K deficiency, Warfarin therapy.

INR Use: Standardizes PT for monitoring Warfarin (Target usually 2.0-3.0).

Activated Partial Thromboplastin Time (aPTT)

Intrinsic/Common

Normal Range: 25-35 sec.

Prolonged in: Deficiency of XII, XI, IX, VIII, X, V, II. Heparin therapy, Hemophilia A/B, Lupus anticoagulant.

Use: Monitoring Heparin therapy.

Thrombin Time (TT)

Measures Fibrinogen → Fibrin conversion. Prolonged by low fibrinogen, heparin, FDPs.

Fibrinogen Level

Normal: 200-400 mg/dL. Low in DIC/Liver disease. High in inflammation.

III. Tests of Fibrinolysis (D-Dimer)

D-Dimer

Specific degradation product of cross-linked fibrin.

Clinical Significance:

Elevated: Indicates clot formation/breakdown. Screening for DIC, PE, DVT.
Negative: High negative predictive value to rule out DVT/PE in low-risk patients.

Common Disorders of Hemostasis

I. Primary Hemostasis Disorders (Platelet/Vessel)

Symptoms: Mucocutaneous bleeding (petechiae, epistaxis).

1. Thrombocytopenia

Decreased Production: Marrow suppression, leukemia, B12/folate deficiency.
Increased Destruction: ITP (Autoimmune), TTP/HUS (Microangiopathic).
Sequestration: Splenomegaly.

2. Platelet Function Disorders

Inherited: Glanzmann's (GP IIb/IIIa), Bernard-Soulier (GP Ib).
Acquired: Aspirin/NSAIDs, Uremia.

3. Von Willebrand Disease (vWD)

Most common inherited bleeding disorder. Deficiency/defect in vWF (platelet adhesion + Factor VIII carrier).

Lab Findings: Normal Platelet Count, Prolonged Bleeding Time, Prolonged aPTT (low FVIII), Abnormal Ristocetin Aggregation.

II. Secondary Hemostasis Disorders (Coagulation Factors)

Symptoms: Deep tissue bleeding (hemarthroses, hematomas).

Hemophilia A (VIII) & B (IX)

X-linked recessive. Deep bleeding.

Labs: Prolonged aPTT, Normal PT.

Vitamin K Deficiency

Affects II, VII, IX, X. Diet/Malabsorption/Warfarin.

Labs: Prolonged PT (sensitive) & aPTT.

Liver Disease

Reduced synthesis of factors. Bleeding + Thrombosis risk.

Labs: Prolonged PT/aPTT, Low Platelets.

DIC (Disseminated Intravascular Coagulation)

Widespread activation (sepsis/trauma) → Consumption of factors → Bleeding + Clotting.

Labs: ↓ Platelets, ↑ PT/aPTT, ↓ Fibrinogen, ↑ D-Dimer.

III. Thrombotic Disorders (Thrombophilia)

Inherited Thrombophilias

Factor V Leiden: Resistance to APC (Most common).
Prothrombin Gene Mutation: High prothrombin.
Deficiencies: Antithrombin, Protein C, Protein S.

Acquired Thrombophilias

Antiphospholipid Syndrome (APS): Autoimmune. Paradoxical prolonged aPTT.
Others: Malignancy, Pregnancy, Immobilization, HIT (Heparin-Induced).

Biochemistry: Platelets and Hemostasis Quiz

Platelets and Hemostasis

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