Hemostasis and Coagulation

Miklós Molnár

Definition of HEMOSTASISDefinition of HEMOSTASIS

The arrest of bleeding by repair of vessel wallMaintaining a balance

CoagulationFibrinolysis

Hypocoagulation: excessive bleeding (inherited or acquired)Hypercoagulation (thrombosis) inadequate activation of the fibrinolytic system

Systems Involved in Hemostasis

Vascular systemInjured vessel initiates vasoconstriction

Platelet SystemInjured vessel exposes collagen that initiates platelet aggregation and help form plug

Coagulation Systemprotein factors of intrinsic and extrinsic pathways produce a permanent fibrin plug

HEMOSTASISPrimary vs. Secondary vs. TertiaryPrimary vs. Secondary vs. TertiaryHEMOSTASISPrimary vs. Secondary vs. TertiaryPrimary vs. Secondary vs. Tertiary

Primary HemostasisPlatelet Plug FormationDependent on normal platelet number & functionInitial Manifestation of Clot Formation

Secondary HemostasisActivation of Clotting Cascade Deposition & Stabilization of Fibrin

Tertiary HemostasisDissolution of Fibrin ClotDependent on Plasminogen Activation

vasoconstriction (vascular system)platelet exposure to subendothelial connective tissue of blood vesselsPlatelet release of ADP, ATP, Thromboxane A2 (promotes vasoconstriction)Platelet aggregation, phospholipid provides site for fibrin formation

Primary Hemostasis

Platelet Origin & Development

Endomitosis Multiple mitotic division of DNA without cytoplasmic division

Largest cell in the BM low powerStem cell to mature platelet = 5 daysEach megakaryocyte can shed 500-4000 plateletsCytoplasm breaks apart along demarcation membranes

Megakaryocyte

Platelets forming from cytoplasm

Normal platelets and one giant platelet

Platelet StructurePlatelet Structure

Three zonesPeripheral zone (adhesion & aggregation)

glycocalyx, plasma membrane

Sol-gel zone (structure & support)microfilaments, thrombosthenin, open canalicular system, dense tubular system

Organelle zone (Secretion & storage)granules: alpha, dense, glycogenmitochondria, lysosomes

Glycocalyx

GlycoproteinsIb (GPIb)

Receptor site for vWF

IIb, IIIa (GPIIb/IIIa)Complex becomes receptor site for fibrinogen

Granular content

Dense granulesATPADPCalciumMagnesiumSerotoninepinephrine

Granular content (Alpha granules)

Hemostatic proteins

FibrinogenFactor VvWFPlasminogenPlasminogen activator inhibitor (PAI-1)α2-antiplasmin

Nonhemostatic proteins

β-thromboglobulin,Platelet factor 4Platelet derived growth factor (PDGF)Albuminfibronectin,

Formation of primary hemostatic plug

Platelets converted from inactive to active state

Adhesion to collagenTriggers platelet activationTromboxane A2 is synthesized from arachidonic acid and stimulates secretion

Aggregation of platelets to each otherprostacyclin (PGI2) inhibits platelet aggregation

Secretion (discharge of granule contents)

VON WILLEBRAND FACTORLarge Adhesive GlycoproteinPolypeptide chain: 220,000 MWBase structure: Dimer; Can have as many as 20 linked dimersMultimers linked by disulfide bridgesSynthesized in endothelial cells & megakaryocytesConstitutive & stimulated secretionLarge multimers stored in Weibel-Palade bodiesFunctions:1) Stabilizes Factor VIII2) Essential for platelet adhesion

VWF

VWF

VWF

VWF VWF VWF

collagencollagen

collagen

collagen

Blood Flow

Platelet adhesion

PlateletRolling

Stable adhesion

Platelet activation/ aggregation

Inactive Active

Secondary hemostasis

Intrinsic PathwayAll components required for initiating this pathway are circulating in the bloodtriggered by contact with collagen or glass

Extrinsic PathwayInitiated by the release of tissue thromboplastin and calcium from damaged tissue

Common PathwayLeads to clot formation including the platelet plug and fibrin produced

Coagulation Proteins

Zymogens enzyme precursors II, VII, IX, X, XI, XII, PrekallkreinWhen activated become serine proteases

CofactorsNonenzymatic V, VIII, HMWK, Tissue factor(thromboplastin)

Kinin factors prekallikrein, kallikrein, HMWK

Roles include coag activation as well as fibrinolytic activation

Coag factors (by group)Coag factors (by group)

Fibrinogen group: I,V,VIII,XIIImost labile, are consumed in coagulation, found on platelets

Prothrombin group: II,VII,IX,XVitamin K dependent, may be affected by coumarin,diet, antibiotics

Contact group: XI,XII,HMWK, Prekallikrein

initiate intrinsic path and fibrinolysis

Tissue DamageVessel wall

Cell particles

Tissue Factor (TF)

VIIa+ X Xa

II(prothrombin)

IIa(thrombin)

Initial Tissue Factor Pathway Activation of Hemostasis

Tissue factorpathway inhibitor

Positive feedback

IIaThrombin

XI XIa

VIII VIIIa

V Va

Pro-coagulant effects

Fibrinogen Fibrin

XIa

IX IXa

X Xa

II IIa

Fibrinogen Fibrin

FVIIIa/Ca2+/Phospholipid

FVa/Ca2+/Phospholipid

Precursor Enzyme

Reaction on Activated Platelets

FIBRINOLYTIC SYSTEMFIBRINOLYTIC SYSTEMFIBRINOLYTIC SYSTEMFIBRINOLYTIC SYSTEM

Definition: temporary fibrin clot systematically and gradually dissolved as the vessel healsKey components

Plasminogen (inactive form)Plasminogen activatorsPlasminFibrinFibrin Degradation Products (FDP)Inhibitors of plasminogen activators and plasmin

Activators of Fibrinolysis

Intrinsic activatorsFactor XIIa, XIa, kallikrein

Extrinsic activatorsTissue type plasminogen activator (t-PA)Urokinase type plasminogen acitvator

(u-PA)

Exogenous activatorsStreptokinase (derived from beta strep)

XII XIIa

XI XIa

IX IXa

X Xa

TF / VIIa

II IIa

Fibrinogen Fibrin

VIIIa+Ca+Pl

Va+Ca+Pl IIa/Thrombomodulin

interactionTFPI

Protein CProtein S

Protein S

Fibrinolysis

IIaThrombin

Anti-coagulant effects

thrombomodulin

IIa

VIIIa Va

Protein CActivatedProtein C

Protein S

Protein C Anticoagulant Pathway

Coagulation Regulatory MechanismsCoagulation Regulatory Mechanisms

Naturally Occurring Anticoagulants rapidly interact with components of coag cascade to avoid unabated clot formation

Protein C (PC) and Protein S (PS)deficiencies may be congenital or acquired

Antithrombin (AT) and Heparin Cofactor IIserine protease inhibitors (serpins)

Deficiency of inhibitors cause increased risk of thrombosis

Inhibitors of Fibrinolysis

Plasminogen Activator Inhibitors (PAI)α2 –antiplasmin

α2 -macroglobulin

Bleeding Bleeding ddisordersisorders

Disorders of Hemostasis

Vascular disorders Scurvy, easy bruising, Henoch-Schönlein purpura.

Platelet disordersQuantitative - ThrombocytopeniaQualitative - Platelet function disorders – Glanzmans

Coagulation disordersCongenital - Haemophilia (A, B), Von-WillebrandsAcquired - Vitamin-K deficiency, Liver disease

Mixed/Consumption: DIC

Tests of Hemostasis:

Screening tests: Bleeding.T 10m. Platelet & BV function Prothrombin.T Extrinsic, aPTT InstrinsicThrombin.T common path. (DIC)

Specific tests:Factor assays – hemophilia.Tests of thrombosis – TT, FDP, DDA, Platelet function studies:

Adhesion, Aggregation, Release tests.

Bone Marrow study

Bleeding: Clinical Features

Local-vs- General, spontaneous…Hematoma / Joint Bleeds- CoagSkin / Mucosal Bleeds – PLTwound / surgical bleeding – Immediate - PLT Delayed - Coagulation

PlateletCoagulation

Petechiae, Purpura Hematoma, Joint bl.

Vascular disorders:

Petechiae, purpura, ecchymosessenile purpuravitamin C deficiency (scurvy)Connective tissue disordersInfections – MeningococcusHenoch-Schonlein Purpura-Immu

Senile PurpuraSenile Purpura

Petechiae in Petechiae in VasculitisVasculitis

(Rocky Mountain Spotted (Rocky Mountain Spotted Fever)Fever)

Henoch-Schölein purpura

Immune disorderChildrenFollows infectionPetechiae with edema and itching.

Henoch-Schönlein purpura

20y Male, fever, painful symmetric polyarthritis for a day. During the next two days, edema and palpable purpura developed.

Platelet Disorders - Features:

Mucocutaneous bleedingPetechiae, Purpura, Ecchymosis.Spontaneous bleeding after traumaCNS bleeding (severe plt)Prolonged bleeding time (BT)

BLEEDING TIME vs. PLATELET COUNT

0

50

100

150

200

250

300

350

400

3,5 4 4,5 5 5,5 7 9 12 15 25 30

Minutes

Pla

tele

t cou

nt (x

100

0)

REDUCED PLATELET NUMBER:

THROMBOCYTOPENIA

Normal platelet count 140-400

Increased bleeding time <100

Spontaneous bleeding Spontaneous bleeding <20 <20

PLATELET FUNCTION DEFECTSProlonged Bleeding TimeProlonged Bleeding Time

CongenitalDrugsAlcoholUremiaHyperglobulinemiasFibrin/fibrinogen split productsThrombocythemiaCardiac Surgery

PLATELET FUNCTION DEFECTSPlatelet AdhesionPlatelet Adhesion

Bernard Soulier DiseaseAbnormal GPIb-IX ComplexReceptor for von Willebrand factorOnly adhesion mediator @ high shear stressTested by ability to aggregate platelets in presence of ristocetin

Von Willebrand diseaseReduced or dysfunctional von Willebrand factor

Von-Willebrand Disease:

Coagulation + PLT disorder:Congenital disorderDeficiency of vWF molecule Part of FVIII, Mediates platelet adhesionProlonged Bleeding timeLow Factor VIII & long aPTTMucocutaneous bleeding

Von-Willebrand Von-Willebrand DiseaseDiseasevWF: F-VIII & PLT function.Defective Platelet AdhesionSkin Bleeding Prolonged Bleeding time.Low Factor VIII levels.Prevalence: 0.8–2% (probable underestimate)

PLATELET FUNCTION DEFECTSPlatelet Release Defects - CongenitalPlatelet Release Defects - Congenital-storage pool disease

Failure to form dense granules (Hermansky-Pudlak syndrome)

Do not release ADP, serotonin, calcium on activationFail to recruit platelets for aggregation

Gray platelet syndromeFailure of packaging of α-granulesDo not release protein mediators of platelet aggregation

Decreased platelet aggregationMild bleeding disorder

PLATELET FUNCTION DEFECTSAggregation-CongenitalAggregation-Congenital

Glanzmann's thrombastheniaAutosomal recessiveLack of fibrinogen receptor, GP IIb/IIIa

Platelets cannot aggregate in responseto usual stimuliBleeding sometimes severe

Platelet Aggregation CurvesPlatelet Aggregation Curves

PLATELET FUNCTION DEFECTSAcquired - Drug InducedAcquired - Drug Induced

AlcoholProstaglandin Synthetase Inhibitors

AspirinNon-Steroidal Antiinflammatory DrugsPhenylbutazone

ADP receptor inhibitorsClopidogrelTiclopidine

Beta-lactam antibioticsHeparin

THROMBOCYTOPENIATHROMBOCYTOPENIADecreased productionDecreased productionDecreased megakaryocytes

Normal platelet life spanGood response to platelet transfusion

Neoplastic CausesLeukemiasAplastic AnemiaMetastatic CarcinomaDrugsRadiotherapy

Primary Marrow DisordersMegaloblastic AnemiasMyelodysplastic syndromesMyeloproliferative diseasesSome congenital syndromes

THROMBOCYTOPENIATHROMBOCYTOPENIAIncreased DestructionIncreased Destruction

Shortened platelet life span Increased megakaryocytesMacroplateletsPoor response to platelet transfusion

THROMBOCYTOPENIAIncreased Destruction - CausesIncreased Destruction - Causes

Immune ITP Lymphoma Lupus/rheumatic diseases Drugs

Consumption Disseminated intravascular coagulation Thrombotic thrombocytopenic purpura Hemolytic/uremic syndrome

Septicemia

IDIOPATHIC THROMBOCYTOPENIA PURPURA

(ITP)

Acute - children (post infection)Chronic - adults ( females,20-40 yrs)IgG autoantibodies bound to plateletsPlatelets removed by macrophagesAntibodies can act on marrowNo good diagnostic testTreatment - Inhibit macrophage clearance

CorticosteroidsHigh dose gamma globulinSplenectomy

HIV-ASSOCIATED THROMBOCYTOPENIA

Early Immune mediatedOften in absence of AIDSRemainder of marrow WNLTreatment - Antiretroviral therapy

LateUsually marrow infiltrationOften pancytopeniaOften associated infection or neoplasmPoorly responsive to all treatments

Coagulation disorders:

Deficiencies of Clotting factorsOnset - delayed after traumaDeep bleeding

Into joints - HemarthrosesHemarthroses Into deep tissues – HematomaHematoma large skin bleed – EcchymosesEcchymoses

Coagulation DisordersLaboratory findings:Laboratory findings:

Normal bleeding time & Platelet countProlonged prothrombin time (PT)

deficiencies of II, V, VII, X

Prolonged time (aPTT)all factors except VII, XIII

Mixing studies - normal plasma corrects PT or aPTT

Factor VIII Deficiency Classic hemophilia (Classic hemophilia (HHemophilia A)emophilia A)

X-linked disorder (affects 1º males)Prevalence is 1:5,000 malesMost common - severe bleedingSpontaneous hematomasAbnormal aPTT – Intrinsic path.Diagnosis - factor VIII assayTreatment - factor VIII concentrateCryoprecipitate (less desirable)

Factor IX DeficiencyChristmas disease (Hemophilia B)Christmas disease (Hemophilia B)

X-linked recessive disorderPrevalence is 1:30,000 males Indistinguishable from classic hemophilia (F VIII)Requires evaluation of factor VIII and IX activity levels to diagnoseTreatment - factor IX concentrateCryoprecipitate if factor IX unavailable

FACTOR XI DEFICENCY ((HHemophilia C)emophilia C)

Inherited form transmitted as an autosomal recessive trait.Prevalence is 1:100,000Increased prevalence in Ashkenazi Jewish population (in Israel, estimated at 8%) A clinically mild bleeding problemProlongs only the PTTMost often associated with liver disease

Secondary Hemostatic Disorders

Vitamin K deficiencyVitamin K deficiency

Neonates - decreased intestinal flora and dietary intakeOral anticoagulants (coumadin)Fat malabsorption syndromesRequired for factors II, VII, IX, - Prolonged PT and aPTT

Combined Primary and Secondary Hemostatic

Disorders Disseminated Intravascular Coagulation Disseminated Intravascular Coagulation

(DIC)(DIC) Major pathologic processes -obstetric complications, neoplasms, infection (sepsis), major traumaPrimary - platelet consumption

( bleeding time, platelets)Secondary - factor consumption

( PT, aPTT)

Combined Primary and Secondary Hemostatic

DisordersSevere Liver DiseaseSevere Liver Disease

Primary - dysfunctional platelets and/or thrombocytopenia (BT)Secondary - decrease in all coagulation factors except vWF (PT, aPTT)Vitamin K will promote synthesis of factors II, VII, IX, X

Summary

Symptom Platelet Coagulation Petechiae Yes No

Sites Skin & Mucosa

Deep Tissue

Time Immediate Delayed

Ecchymoses/Hematomas

Yes Yes