Unit 5: Coagulation

Hemostasis

Body process controlling bleeding & maintaining blood fluidity
Blood vessel breach=clotting=bleeding controlled; blood flow reestablished during healing
Split between Primary & Secondary systems

Primary Hemostasis

Activated by small vessel damage consists of two minor systems: Vascular System & PLTs

Secondary Hemostasis

Activated by major trauma, surgery or hemorrhage, consists of: Clotting Factor System, & Fibrinolytic System (Breaks clots when blood loss is stopped & vessel is repaired)

Vascular System

Controls blood loss through Vasoconstriction, PLT Activation & Coagulation System Activation

Vessel Wall

Intact Vessel doesn’t interact w/PLTs, damage to it exposes blood to tissue, composed of Fibrous Tissue (Collagen & Smooth Muscle) & Endothelial Cells

Collagen

Vessel injury exposes collagen which reacts with PLTs via Von Willebrand Factor

Smooth Muscle

Provides vasoconstriction=reduced blood vessel size=decreased blood flow=minimized blood loss

Endothelial Cells

Cells containing procoagulant, anticoagulant, & fibrinolytic properties vital to hemostasis

Platelets (PLT)

150-450×10⁹/L circulating in normal people
7-10 day life span
Contains Dense & Alpha Granules released through a dense tubular system

PLT Function Phases

Adhesion, Shape Change, Granule Secretion, Amplification, Aggregation

Adhesion

Enabled by: Von Willebrand Factor (vWF) glycoprotein 1b (GpIb)
Occurs: In plasma/Tissue & PLT surface
vWF connects to collagen & G1b

Shape Change

Activated plt morphology= long tendrils, spiny sphere shape

Granule Secretion

Dense Granule: ADP, Serotonin, Ca²⁺, Thromboxane (TXA2)= more plts
Alpha Granule: Clot-Activating Factors= more adhesion

Amplification

Secretion of Thromboxane A2 (TXA2) recruits more PLT to aggregate

Aggregation

Enabled by Fibrinogen & Glycoprotein IIb/IIIa
PLT adhere to each other via Glycoprotein IIb/IIIa binding Fibrinogen

Coagulation Factors

Inactive proenzymes circulation in blood, change to active form during coagulation cascade

Coagulation Factors Types

Enzymes, Substrates & Cofactors

Coagulation Enzymes

All factors are serine proteases except Factor XIII (Transglutaminase)

Coagulation Substrate

Substance the enzymes act upon

Coagulation Cofactors

Accelerate enzyme activity, consumed during use of Factor V & VIII

Factor I (Fibrinogen)

Thrombin substrate & fibrin precursor, converted from soluble component to insoluble fiber= fibrin= clot

Factor II (Prothrombin)

Converted to Thrombin (IIa), cleaves fibrinogen, activates Cofactors V, VIII, Protein C & Factor XIII

Factor III (Thromboplastin/Tissue Factor)

Activates Factor VII when blood is exposed to Tissue Fluids

Factor IV (Ionized Ca²⁺)

Active form of Ca needed for thromboplastin activation & converting prothrombin to thrombin

Factor V (Proaccelerin/Labile Factor)

Accelerates the transformation of prothrombin to thrombin, consumed during clotting
20% found on PLTs

Factor VII (Proconvertin/Stable Factor)

Activated by tissue thromboplastin & activates Factor X, depends on Vitamin K

Factor VIII (Antihemophilic Factor)

Required for cleaving Factor X to Factor Xa, deficient in Hemophilia A

Factor IX (Plasma Thromboplastin Factor)

Combined w/ Factor VIII to activate Factor X, Deficient in Hemophilia B, Vitamin K dependent

Factor X (Stuart-Prower)

Merges Factor V to convert prothrombin to thrombin, independent activation by Russell’s Viper Venom

Factor XI (Plasma Thromboplastin Antecedent)

Essential to intrinsic pathway, Activates Factor IX

Factor XII (Hageman Factor)

Activated by exposed endothelial cells in combination w/PK & HMWK

Factor XIII (Fibrin Stabilizing Factor)

Stabilizes polymerized fibrin monomers in the initial clot when exposed to Ca
Only Factor enzyme made of Transglutaminase

High-Molecular-Weight Kininogen (HMWK)

Surface Contact Factor activated by PreKallikrein

Prekallikrein (Fletcher Factor/PK)

Surface to Contact Factor

Platelet Factor 3

Supports Common Pathway

Fibrinogen Group

Factor I, V, VIII, XIII

Prothrombin Group

Vitamin K dependent, made of Factor II, VII, IX, X

Contact Groups

Factors XI, XII, Preallikrein, HMWK

The Intrinsic Pathway

Intravascular, slower response, quantitatively more significant than extrinsic, contact activation

Intrinsic Cascade Reaction

1 Contact Factor XII activated by exposure to subendothelial basement membrane at tissue/vessel injury w/PK & HMWK→ Factor XIIa
2 Prekallikrein, HMWK & Factor XIIa activate Contact Factor XI→ XIa
3 Factor XIa + Ca²⁺ activate Factor IX→ IXa
4 Factor IXa + Factor VIII + Ca²⁺ activate Factor X→ Factor Xa on PLT phospholipid layer

The Extrinsic Pathway

Activated by Tissue Factor III released from damaged Extravascular cells & tissues, faster than Intrinsic Pathway

Extrinsic Cascade Reaction

1 Tissue Factor III released from cells at injury site → Tissue Factor IIIa
2 Tissue Factor IIIa + Ca²⁺ bind to Factor VII→ Factor VIIa
3 Factor VIIa + Ca²⁺ activate Factor X→ Factor Xa

The Common Pathway

Pathway common to both Intrinsic & Extrinsic Pathways, forms the fribin clot

Common Cascade Reaction

1 Factor X activated by intrinsic/extrinsic→ Xa
2 Xa +Factor V + Ca²⁺+ PF3 converts Prothrombin (Factor II) to active enzyme Thrombin (Factor IIa)
3 Thrombin cleaves Fibrinogen (Factor I)→ Fibrin monomer strands (Ia)= loose polymer clot
4 Thrombin + Factor XIII + Ca²⁺= Factor XIIIa
5 XIIIa crosslinks Fibrin covalently forming a stabilized fibrin clot
6 Factor IIa activates Factor V, VIII, XI as a +feedback loop to amplify clot formation

Inhibitor Characteristics

Systemic clot control, blocks activated coagulation factors, prevents widespread coagulation via limiting fibrinolysis & neutralizing activated circulating factors

Inhibitors

Regulatory inhibitor=soluble proteins in plasma & natural anticoagulants made up of:
Protease Inhibitors, Antithrombin (AT), Protein C/S Complex, Tissue Factor Pathway Inhibitor (TFPI)

Antithrombin (AT)

Serine Protease Inhibitor neutralizes Primary Factors IIa (Thrombin) & Xa, Secondary Factors IXa, XIa, XIIa
Requires heparin to function, made in liver

Heparin Inhibitor Role

Enhances Antithrombin’s inhibiting function, found on surface of endothelial cells

Protein C Inhibitor

serine protease
Zymogen=Inactive form
activated Thrombin-Thrombomodulin
Inactivates Factor V & VIII
Vitamin K & Protein S dependent

Protein S

Protein C Cofactor required for inactivating Factor Va & VIIIa, Vitamin K dependent, 40% circulate freely, 60% bound to C4b-binding protein

C4b Binding Protein (C4bBP)

Binds Protein S, produced in the liver

Tissue Factor Pathway Inhibitor (TFPI)

Inhibits TF(IIIa)-VIIa complex & Factor Xa

Fibrinolytic System

System that dissolves blood clots, plasminogen→ plasmin→ breaks down fibrin clot into split fibrin products

Plasminogen

Glycoprotein produced in liver, converts into plasmin via thrombin activation

Tissue Plasminogen Activator (tPA)

Produced in endothelial cells, activates plasminogen conversion to plasmin, used as a pharmaceutical product during stroke episodes

Urokinase-like Plasminogen Activator (UPA)/Urokinase

Serine protease secreted by kidneys, activates plasminogen conversion to plasmin, minimal effect in clot dissolution, used in stroke, heart attacks & other thrombotic episodes

Plasminogen Activator Inhibitor-1 (PAI-1)

Inhibits tPA function, secreted by endothelial cells during injury

Alpha-2-Antiplasmin (α2AP)

Inhibits Plasmin by preventing its binding to fibrin= lysis prevention= negative feedback, most important inhibitor in fibrinolytic system

Prothrombin Time (PT)

Measures extrinsic pathway, assists w/common pathway, sensitive to early changes in Factor VII, affected by decreases in Common Pathway Factors (X, V, II, I)
Monitors PO anticoagulants Warfarin/Coumadin

PT Reagent

Made of Tissue Factor (Factor III), PLT phospholipids & Ca, added to citrate anticoagulated plasma, clot formation measured by automated device

Activated Partial Thromboplastin Time (aPTT)

Measures intrinsic pathway, assists w/common pathway, most sensitive to Factors VIII, IX, XI, & XII, will detect abnormal X, V, II, I
Monitors Heparin anticoagulant therapy

aPTT Reagent

Based on plasma test w/standardized amount of PLT-like phospholipid & an activator of the contact factor

Factor Assays

Assess the percent activity of a clotting factor, commercially prepared known factor deficient plasma mixed w/pt plasma

Abnormal PT/Normal PTT

Extrinsic Pathway issue detecting possible Factor VII Deficiency

Normal PT/Abnormal PTT

Intrinsic Pathway issue, detects possible deficiency of Factor XII, XI, IX, & VIII

Abnormal PTT & PT

Common Pathway Issue, possible deficiencies of Factor X, V, II, I

Abnormal Factor Assay

Mixing study suggesting pt is missing a specific factor or more, resulting in ↑aPTT time
Can be run specifically or in a panel

Normal PLT Count

150-450×10⁹/L, adequate PLT present for normal coagulation creating a PLT plug or stimulating a solid fibrin clot formation

Decreased (DEC) PLT Count

May cause abnormal bleeding or prolonged bleeding, PLT count 60×10⁹/L= bleed in surgery
PLT <10×10⁹/L= gingival bleeding, epistaxis, ecchymosis or risk bleeding into the CNS

DEC PLT SxS

Gingival Bleeding- Gum Bleeding
Epistaxis- Nose Bleeding
Ecchymosis- Excessive bruising
Petechiae- Small hemorrhage spots
Purpura- Hermorrhage into skin/mucuous membranes

DEC PLT Count Causes

Bone Marrow Aplasia (Lack of MEGA-K)
Cancer (Blasts crowd out MEGA-K)
Chemotherapy destroying MEGA-K

PLT Consumption & Causes

Excessive clot formation all over the body via: Idiopathic (immune) Thrombocytopenia Purpura
Thrombotic Thrombocytopenic Purpura
Hemolytic Uremic Syndrome

Altered PLT Distribution

Enlarged spleen traps large quantity of PLTs

Pre-Analytical Coagulation Study Factors

Improper collection of coagulation samples drawn into 3.2 Sodium Citrate (blue top) tubes, Proper Collection:
Blood:Anticoagulant ratio-9:1, 90% filled tube
Must be properly mixed, small clots may form
No Hemolysis nor traumatic sample collection

Platelet Satellitism

PLT forms rosette around SEGs, MONO, & Bands due to IgG Ab directed against Gp IIb/IIIa in EDTA, binding PLT to WBCs= Falsely low PLT count
Seen in CBC/Peripheral Smears

PLT Satellitism Corrective Action

Redraw blue top tube to resolve it & cycle through automated hematology counter for more accurate results

Drug-Induced Immune Thrombocytopenia

Drug ingestion causing Ab formation against drug via binding to PLT glycoprotein, RES removes coated PLT from circulation
Drug+ Carrier Protein= Antigen→ Body creates Ab against antigen that targets PLT to be destroyed by Spleen

Drug-Induced Immune Thrombocytopenia Drug Types

Quinines, NSAIDs, Heparin, Sulfonamides, Diuretics

Idiopathic Thrombocytopenic Purpura (ITP) Pathology

DEC PLT count due to immune destruction via IgG against GP IIb/IIIa or GP Ib-Factor IX

Idiopathic Thrombocytopenic Purpura (ITP) SxS

Epistaxis, Petechiae, Purpura

Acute Idiopathic Thrombocytopenic Purpura (ITP)

Age: Peds
Prior Infection: Hx of Viral Illness
PLT Count: <20,000
Duration: 2-6wks
Therapy: Usually none

Chronic Idiopathic Thrombocytopenic Purpura (ITP)

Age: Adults
Prior Infection: No Prior Hx
PLT Count: 30,000-80,000
Duration: Months to Years
Therapy: Steroids, Splenectomy

Thrombotic Thrombocytopenic Purpura (TTP) Pathology

ADAMTs-13 cleaves von Willebrand Factor (vWF) into monomers
ADAMTs-13 deficiency= ↓cleavability= larger vWF w/more binding sites=excess PLT clots in circulation

Thrombotic Thrombocytopenic Purpura (TTP) Lab Results

PLT <20×10⁹/L, PT/PTT within reference range, Microangiopathic Anemia (MHA)- SCHISTO
Increased LDH, Decreased Haptoglobin, Hemoglobinuria

Thrombotic Thrombocytopenic Purpura (TTP) SxS

Fever, Neurological Symptoms, Renal Disease

Hemolytic Uremic Syndrome (HUS) Pathology

produced by E. coli O157:H7 Shiga Toxin, usually affects peds 6mo-4yrs

Hemolytic Uremic Syndrome (HUS) Lab Results

PLT count <20,000, Microangiopathic Hemolytic Anemia (SCHISTO)

Hemolytic Uremic Syndrome (HUS) SxS

Mimic TTP but w/o neurological symptoms, bloody diarrhea, renal failure, enterotoxin damage

Von Willebrand’s Disease (vWD)

Autosomal Dominant Hereditary disorder, qual/quantitative defect in vWF, affects 1-3% world pop, most prevalent inherited PLT adhesion disease/bleeding disorder

Von Willebrand’s Disease Lab Results

PLT count normal, decreased vWF antigen, decreased Ristocetin co-factor activity= single best predictive assay

Von Willebrand’s Disease Symptoms

Ecchymosis, Epistaxis, Menorrhagia in females, excess bleeding after tooth extraction/dental procedures

Bernard Soulier Syndrome Pathology

Autosomal Recessive, rare PLT adhesion defect involving GPIb/IX complex interfereing w/vWF mediated collagen binding, Thrombin Binding, & PLT shape regulation

Bernard Soulier Syndrome Lab Results

Thrombocytopenia, freq. Giant PLTs, absent Ristocetin aggregation, normal aggregation w/epinephrine, collagen, & thrombin

Bernard Soulier Syndrome SxS

Epistaxis, Gingival bleeding, Menorrhagia, Purpura

Glanzmann’s Thrombasthenia Pathology

Rare autosomal recessive aggregation disorder causing an abnormality to the GPIIb/IIIa Complex

Glanzmann’s Thrombasthenia Lab Results

Normal PLT count & Morphology, Normal Ristocetin aggregation, abnormal aggregation w/epinepherine, collagen, & thrombin

Glanzmann’s Thrombasthenia SxS

Purpura, Umbilical cord, gingival bleeding, slow bleeding from minor cuts

Drug-related PLT Abnormalities

Often w/Aspirin, inhibits production of PLT aggregator thromboxane A2, prolonged bleeding time

Extrinsic PLT Abnormalities via Multiple Myeloma & Waldenström’s Macroglobulinemia

Excess Ig production, hyperviscosity & paraproteinemia, PLT circulating high number of abnormal proteins=unable to participate in activating coagulation factors & forming fibrin

PLT Function Analyzer

Utilize cartridges w/small collagen/epi or collagen/ADP membrane coated aperture, PLT adhere to membrane & plug aperture (Closing Time)