Coag - Claude

Define hemostasis.

The process by which the body ceases bleeding at the site of vessel injury while maintaining blood flow within the vascular system.

Define coagulation.

The specific process that results in the formation of a blood clot. It is a sub-process of hemostasis.

What causes pathological bleeding?

Deficiency or dysfunction of coagulation proteins, platelets, or vascular integrity.

What happens when anticoagulant (inhibitor) mechanisms are dysfunctional or deficient?

Excessive or inappropriate clotting (thrombosis).

What are the 3 stages of hemostasis in order?

1) Primary Hemostasis (platelet plug formation), 2) Secondary Hemostasis (fibrin-platelet plug), 3) Fibrinolysis (clot breakdown).

What is the goal of Primary Hemostasis?

Formation of a platelet plug.

What is the goal of Secondary Hemostasis?

Stabilization of the platelet plug by forming a fibrin-platelet plug.

What is the goal of Fibrinolysis?

Removal/breakdown of the fibrin clot once healing is complete.

Name the 3 layers of the vascular wall from outermost to innermost.

1) Tunica externa (adventitia), 2) Tunica media, 3) Tunica intima.

What is the Tunica externa composed of?

Collagen fibers.

What is the Tunica media composed of?

Smooth muscle cells and sheets of elastin.

What is the Tunica intima composed of?

A layer of endothelial cells and a subendothelial layer.

What is the net surface charge of a normal intact endothelium?

Negative.

What 3 substances does the NORMAL endothelium secrete to inhibit platelet activation/aggregation?

Prostacyclin (PGI2), Nitric oxide (NO), and ADPase.

What 2 substances does the NORMAL endothelium secrete to inhibit blood coagulation?

Heparan sulfate and Tissue Factor Pathway Inhibitor (TFPI).

How does the NORMAL endothelium activate fibrinolysis?

By releasing Tissue Plasminogen Activator (tPA).

How does the endothelium functionally change upon injury?

It shifts from non-thrombogenic/antifibrinolytic to thrombogenic and antifibrinolytic.

What substance does INJURED endothelium secrete to facilitate initial platelet adhesion?

von Willebrand Factor (vWF), released from Weibel-Palade bodies.

What substance does INJURED endothelium produce to initiate fibrin formation?

Tissue Factor (TF).

What does endothelin do?

Induces vasoconstriction.

Platelets are cytoplasmic fragments derived from which precursor cell?

Megakaryocytes, in the bone marrow. They are ultimately derived from hematopoietic stem cells.

Which hormone is the primary regulator of megakaryocyte and platelet development?

Thrombopoietin.

Describe the Megakaryoblast (Stage I) of platelet maturation.

15–20 µm diameter, high N:C ratio (4:1 to 3:1), oval/round nucleus with 2+ nucleoli, coarse non-clumped chromatin, scant deep basophilic agranular cytoplasm. Endomitosis begins.

Describe the Promegakaryocyte (Stage II) of platelet maturation.

25–30 µm, high N:C ratio (4:1 to 2:1), indented or bilobed nucleus without nucleoli, coarse chromatin with some clumping. Endomitosis ENDS. Demarcation Membrane System (DMS) begins.

What is Endomitosis?

Nuclear replication without cytoplasmic division, resulting in a polyploid nucleus. Begins in Stage I (Megakaryoblast) and ends in Stage II (Promegakaryocyte).

What is the Demarcation Membrane System (DMS)?

An internal membrane system that forms during the Promegakaryocyte stage (visible only by electron microscopy) and ultimately creates the platelet cell membrane.

Describe the Megakaryocyte (Stage III & IV) of platelet maturation.

40–80 µm, multilobed nucleus (4, 8, or 16 lobes), no nucleoli, coarse & clumped chromatin, abundant light blue-pink cytoplasm with pink & azurophilic granules. Demarcation zones present.

What are Proplatelets?

Long (2–10 µm), uniform cytoplasmic extensions from megakaryocytes that are released between vascular sinus endothelial cells and break apart into mature platelets in peripheral blood.

What are the key characteristics of a NORMAL mature platelet?

2–3 µm diameter, small lavender-blue/colorless with reddish-purple granules, 7–10 day lifespan, normal count 150–450 x 10³/µl.

How is a Giant Platelet defined?

Larger than a normal RBC; 8–20 µm in diameter.

What is the Glycocalyx?

The outer membrane surface (peripheral zone) of the platelet, rich in glycoproteins (membrane receptors).

What does the Structural Zone (Sol-Gel Zone) contain and what is its function?

Microtubules (maintain discoid shape) and microfilaments made of actin & myosin (form pseudopods during platelet activation).

What are the 4 main types of granules/organelles in the Platelet Organelle Zone?

Mitochondria (energy), Glycogen granules (metabolism), Lysosomal granules (hydrolytic enzymes), and Dense/Alpha granules.

How many dense granules are typically in a platelet?

3–8 dense granules per platelet.

What are stored in DENSE granules (δ-granules)?

ADP (promotes activation), ATP (activates Ca²⁺ channels), Calcium (primary/secondary messenger), and Serotonin (vasoconstriction).

How many Alpha granules (α-granules) are in a platelet?

~50–80 per platelet — most numerous granule type.

What HEMOSTATIC proteins are in Alpha granules?

Fibrinogen, Factor V, vWF, Plasminogen, PAI-1, and α₂-antiplasmin.

What NON-HEMOSTATIC proteins are in Alpha granules?

β-thromboglobulin, Platelet-derived growth factor (PDGF — promotes smooth muscle growth), and Thrombospondin (promotes platelet-to-platelet interaction).

What is the Open Canalicular System (OCS)?

A membrane system that releases the contents of dense and alpha granules during platelet activation.

What is the Dense Tubular System (DTS)?

A membrane system that serves as the storage site for calcium within the platelet.

What are the 3 sub-steps of platelet plug formation in order?

1) Platelet Adhesion, 2) Shape Change & Secretion, 3) Platelet Aggregation.

What event triggers the start of platelet adhesion?

Vascular injury exposes collagen in the subendothelial layer.

What happens immediately after vessel injury to initiate platelet adhesion?

Circulating vWF (and vWF released from Weibel-Palade bodies) binds to exposed collagen.

Which platelet receptor complex binds to vWF during adhesion?

GPIb/IX/V complex — allows the platelet to "roll" on the vessel wall.

Which 2 platelet receptors bind DIRECTLY to collagen to secure platelet adhesion?

GPVI and GPIa/IIa.

Describe vWF's structure and where it is stored.

Large multimeric glycoprotein circulating in plasma. Ultra-large vWF (ULVWF) is stored in Weibel-Palade bodies (endothelial cells) and alpha granules (platelets). Cleaved by ADAMTS-13.

What are the dual roles of vWF in hemostasis?

Primary Hemostasis: mediates platelet adhesion to vessel wall. Secondary Hemostasis: carrier protein for Factor VIII.

What shape change occurs during platelet activation?

Platelets change from discoid to a sphere with pseudopods.

What receptor does ADP activate during shape change, and what does this cause?

ADP activates GPIIb/IIIa receptor, enabling it to bind fibrinogen for aggregation.

What are STRONG platelet agonists?

Thrombin (most potent) and Collagen.

What are WEAK platelet agonists?

ADP and Epinephrine.

What is Thromboxane A₂ (TxA₂) and what is its function?

A platelet agonist synthesized from Arachidonic Acid (AA) via the cyclooxygenase pathway. Stimulates platelet activation, secretion, and enhances vasoconstriction.

How does Aspirin affect platelets?

Aspirin irreversibly inhibits cyclooxygenase, blocking TxA₂ synthesis for the platelet's entire life (~7–10 days). Prevents platelet activation. Used to reduce MI and stroke risk.

What are the platelet receptors for each of these ligands: vWF, Collagen, Fibrinogen, ADP, Thrombin?

vWF → GPIb/IX/V

Collagen → GPIa/IIa & GPVI

Fibrinogen → GPIIb/IIIa

ADP → P2Y₁ and P2Y₁₂

Thrombin → PAR1 and PAR4.

How does platelet aggregation work? What molecule links platelets?

ADP activates GPIIb/IIIa receptors. Fibrinogen bridges adjacent platelets (Ca²⁺ is required) to form the platelet plug.

Describe the structure of Fibrinogen.

Trinodular glycoprotein composed of 2α, 2β, and 2γ chains. Has a center E nodule and D nodules on each side. Terminal ends of α and γ chains on D domain bind GPIIb/IIIa. Reference Range: 200–400 mg/dL.

What are Zymogens?

Inactive forms of enzymes (coagulation factors circulate as zymogens). An "a" after the Roman numeral indicates an activated factor (e.g., VIIa).

List all coagulation factors and their common names.

I=Fibrinogen

II=Prothrombin

III=Tissue Factor/Thromboplastin

IV=Calcium

V=Labile factor/Proaccelerin

VII=Stable factor/Proconvertin

VIII=Antihemophilic factor

IX=Christmas factor

X=Stuart-Prower factor

XI=Plasma thromboplastin antecedent

XII=Hageman factor

XIII=Fibrin-stabilizing factor

HK/HMWK= High-Molecular-Weight Kininogen, Fitzgerald factor

PK= Prekallinkrein, Fletcher factor.

What are the 3 functional classifications of coagulation factors?

Substrate (Fibrinogen)

Cofactor (Factors V, VIII, and HK)

Enzyme (Serine protease + Transaminase)

What are the 3 structural groups of coagulation factors?

Contact Group (XII, XI, HKWK, PK)

Prothrombin Group (II, VII, IX, X — Vitamin K dependent)

Fibrinogen Group (I, V, VIII, XIII).

Which factors are Vitamin K dependent and why?

Factors II, VII, IX, X, Protein C, and Protein S.

• Vitamin K carboxylates glutamic acid into γ-carboxyglutamic acid, essential for calcium binding and activation.

• Essential for prothrombin Group factor

Describe the Extrinsic Pathway.

Vascular injury → Tissue Factor (TF) + FVII + Ca²⁺ → forms TF/VIIa Extrinsic Tenase Complex → activates Factor X (and Factor IX) → leads to thrombin generation.

Describe the Intrinsic Pathway.

Contact with collagen → activates FXII → activates XI (with HMWK) → activates IX (with Ca²⁺) → IXa + VIIIa forms Intrinsic Tenase Complex (on activated platelet surface + Ca²⁺) → activates Factor X.

Describe the Common Pathway.

Xa + Va + Ca²⁺ + platelet phospholipids → Prothrombinase Complex → Prothrombin → Thrombin → Fibrinogen → Fibrin monomers → polymers → XIIIa + Ca²⁺ → Cross-linked Fibrin Mesh.

How does fibrin formation occur step by step?

Thrombin cleaves fibrinopeptides on E domain → fibrin monomers form.

Cleaved binding sites on E domain bind γ-chains on D domain → fibrin polymer.

Thrombin activates Factor XIII, and XIIIa cross-links adjacent D-domains.

List the PRO-coagulant activities of Thrombin.

• Converts fibrinogen to fibrin

• Activates Factors V, VII, VIII, XI, XIII

• stimulates endothelial cells to release vWF, PAI, and tissue factor

• activates platelets.

List the ANTI-coagulant activities of Thrombin.

• Suppresses fibrinolysis by activating TAFI

• binds Thrombomodulin to activate Protein C

• stimulates endothelial cells to release tPA, prostacyclin, and nitric oxide.

What is the function of Protein C and how is it activated?

Activated by Thrombin/Thrombomodulin complex + Calcium + Protein S (cofactor).

Function: inhibits Factor Va and Factor VIIIa.

What is the function and mechanism of TFPI?

Synthesized in endothelium, megakaryocytes, and smooth muscle.

Released from alpha granules during activation.

Forms complex with Factor Xa to inhibit the TF/VIIa complex (made by the extrinsic pathway to blocks the extrinsic pathway).

What is the function of Antithrombin (AT)?

Synthesized in the liver.

Inhibits Thrombin, Xa, XIa, IXa, and VIIa. Its activity is greatly enhanced when bound to heparin.

What does the PT (Prothrombin Time) test screen for?

Deficiencies in the EXTRINSIC and COMMON pathways; factor inhibitors; monitoring Warfarin/Vitamin K antagonist therapy; Vitamin K deficiency.

Describe the PT test principle.

Thromboplastin reagent (tissue factor, phospholipids, calcium) is added to plasma. Time for clot formation is recorded in seconds. Manual testing is done in duplicate.

What is the reference range for PT?

11.9–15.1 seconds.

What is INR and why is it used?

International Normalized Ratio. Reported with every PT result to standardize PT across different reagents/instruments. Formula: INR = (Patient PT / Mean Normal PT)^ISI.

What are the INR therapeutic ranges?

Standard anticoagulant therapy: 2.0–3.0; Mechanical heart valve: 2.5–3.5; Recurrent VTE within therapeutic range: 3.0–4.0; Critical: >5.0; Normal: 0.9–1.1.

What does the APTT (Activated Partial Thromboplastin Time) test screen for?

Deficiencies in the INTRINSIC and COMMON pathways; factor inhibitors; lupus anticoagulants; monitoring Heparin therapy.

Describe the APTT test principle.

APTT reagent (contact activators + phospholipids) is added to plasma. After incubation, calcium chloride is added and clotting time is measured.

What is the reference range for APTT?

25–35 seconds.

What is fibrinolysis and what is its purpose?

Activated in response to coagulation/fibrin formation. Removes insoluble fibrin deposits by enzymatic digestion. Helps re-establish vessel integrity and blood flow.

What is Plasminogen and what does Plasmin do?

Plasminogen: zymogen that circulates in blood (synthesized in liver).

Plasmin: non-specific proteolytic enzyme that degrades fibrin to FDP/FSP; can also degrade fibrinogen, Factors V, VIII, and XII if uncontrolled.

What physiologic activators convert Plasminogen to Plasmin?

• Tissue Plasminogen Activator — tPA (synthesized by injured endothelial cells; binds plasminogen-fibrin complex)

• Urokinase Plasminogen Activator — uPA (produced by monocytes, macrophages, renal tubular epithelium; found in urine; important in wound healing/inflammation).

What contact activators can activate plasminogen?

XIIa, XI, and Kallikrein (accounts for ~15% of plasmin generating activity).

What exogenous fibrinolytic agents exist?

Streptokinase, Staphylokinase, tPA (Tenecteplase), UPA.

What are Fibrin(ogen) Degradation Products (FDP/FSP)?

Fragments produced when plasmin degrades fibrin or fibrinogen: X fragment (D-E-D), then Y fragment (D-E + D), then separate D and E fragments (D + E + D).

List the 5 fibrinolysis inhibitors and their functions.

PAI-1 (primary inhibitor of tPA and uPA; from endothelium/monocytes), PAI-2 (inhibits uPA only; high in pregnancy), TAFI (activated by thrombin/thrombomodulin; cleaves fibrin binding sites), α₂-antiplasmin (inhibits free plasmin), α₂-macroglobulin (backup inhibitor if α₂-antiplasmin depleted).

What is D-Dimer, what does it indicate, and what is its reference range?

A specific fibrin degradation product (cross-linked fragment).

Marker for DIC, PE, DVT, VTE. (Increase in clot forming and lysing)

Reference Range: <400 ng/mL FEU (or <0.4 µg/mL FEU).

What are causes of INCREASED D-Dimer?

Pregnancy, inflammation, malignancy, trauma, postsurgical treatment, liver disease, heart disease, DIC, burns, infections.

What are FALSE POSITIVE causes of D-Dimer?

Lipemic specimens, Rheumatoid Factor.

What is the FDP/FSP Assay principle?

Latex particles coated with anti-D and anti-E fragment antibodies are mixed with diluted serum. Antibodies react with D & E fragments to form macroscopic clumps. Reference Range: <10 µg/mL.

What conditions cause INCREASED FDP?

DIC, pregnancy complications, MI, PE, DVT, liver disease, glomerulonephritis monitoring, post-fibrinolytic therapy, extensive tissue damage.

What anticoagulant tube is used for coagulation testing and what is the blood-to-anticoagulant ratio?

3.2% buffered sodium citrate (blue top tube); 9:1 ratio of blood to anticoagulant.

In what order should coagulation tubes be drawn?

Coagulation samples should be drawn FIRST (or after a non-additive red top tube). A discard tube may be needed beforehand.

What is Platelet Poor Plasma (PPP)?

Plasma obtained by centrifugation at 1500g for 10–15 min at ambient temperature, yielding a platelet count low enough for coagulation testing.

How does a CLOTTED sample affect coagulation results?

Falsely prolongs PT, APTT, and Thrombin Time; decreases fibrinogen.

How does a SHORT DRAW affect coagulation results?

Falsely prolongs PT, APTT, and Thrombin Time; underestimates fibrinogen (excess anticoagulant relative to blood).

How do HEMOLYZED samples affect coagulation results?

RBC membranes contain phospholipids; can either prolong or shorten results.

How do ICTERIC and LIPEMIC samples affect coagulation results?

Interfere with photo-optical clot detection methods; prolong results.

What is the sodium citrate correction formula for high hematocrit patients (HCT >55%)?

C = (1.85 × 10⁻³) × (100 – HCT) × Vol, where C = volume of sodium citrate needed (mL), HCT = patient's hematocrit, Vol = volume of whole blood (mL).

What is the specimen stability for PT vs. non-heparinized PTT at room temperature?

PT: stable for 24 hours. Non-heparinized PTT: stable for 4 hours.

What are CLIA QC requirements for coagulation testing?

Two controls (normal and abnormal) every 8 hours and at every reagent change. Manual: all controls and patients in duplicates.