Coagulation and platelet

Introductory lecture (Adapted from Dr. Aaron Auerbachs lecture) AFIP Please let me know if there is copyright conflict to any
parties , then I will delete this file.

Outline
1. Normal coagulation 2. Tests of coagulation 3. Hemorrhagic and thrombotic disorders

 Primary hemostasis
Platelet plug formation Adhesion and aggregation

Secondary hemostasis
Coag pathway, form fibrin

Tertiary hemostasis
Crosslinking of fibrin and fibrinolysis

COAGULATION
A complicated process that must prevent both excessive bleeding and excessive clot formation Three key components: blood vessels, platelets, and plasma components Three steps: adhesion, plts release granules, aggregation

First step--adhesion
You get an injury to a blood vessel and the endothelium is ripped apart There is circulating von Willebrands factor in the plasma, and there is also subendothelial vWF. The circulating vWF in the plasma attaches to the naked endothelium and subendothelium. Then the platelets attach to the vWF using the glycoprotein Ib/V/IX

adhesion

PLATELET GP Ib/IX/V

VWF

COLLAGEN EXPOSURE
--Damaged endothelium --Circulating vWF in plasma attaches to endothelium. --Then, platelets attach to vWF using GP Ib/V/IX

Platelets
Discoid shape

2 - 3 um

Then, the platelets release their granules and change shape

Alpha granule - thromboglobulin - P-selectin - PDGF - PF4 - platelet fibrinogen - Thrombospondin - VWF

Platelet alpha and dense granules

Dense (delta) granule -ADP dense granule which causes vasoconstriction - ATP - Serotonin - Ca++

Goldman: Cecil Textbook of Medicine, 21st ed.,

Aggregation
When the platelets change shape, they expose their fibrinogen receptors GP IIb\IIIa receptor binds fibrinogen And aggregation occurs and plugs the hole. This is the primary platelet plug (monolayer) GPIIbIIIa inhibitors (Abciximab-Reopro, Integrilin,
Aggrastat)

GP IIb/IIIa

PLATELET AGGREGATION

Platelet activation

VWF

Granule secretion

Primary hemostatic plug

Aggregation Endothelium

Adhesion

understand some diseases understanding this process will help us

Robbins 6 ed Fig 5-7

Secondary hemostasis (the dreaded coag pathway)

Formation of fibrin Laying down fibrin over plt plug Defects can result in hematomas, hemarthroses

www.azer.com/.../magazine/73_folder/ 73_photos/73_306.jpg

THROMBOPHILIA

BLEEDING

Enzymes that, when activated, catalyze activation of sequential steps that lead to fibrin formation
Bowen, D J Mol Pathol 2002;55:127-144

Mostly made by liver (except vWF, mostly made by endothelial cells)

Copyright 2002 BMJ Publishing Group Ltd.

2002 BMJ Publishing Group Ltd.

FVIII and V cofactors

Division into intrinsic and extrinsic based on lab tests, doesnt apply in body Intrinsic path activated by blood in contact with negative charged
glass

Extrinsic path activated by tissue factor (subendothelium) Early factors in intrinsic path (Kallikrein) usually do not cause bleeding, but deficiency causes PTT

Bowen, D J Mol Pathol 2002;55:127-144

Factor VIIa activates X directly, but also activates IX Tissue factor passway

Pathol 2002;55:127-144

Secondary hemostasis
Thrombin does many things It converts fibrinogen- fibrin Thrombin activates F5, F8, F11 F13

Tertiary hemostasis: FACTOR XIIIa cross links fibrin

Factor XIII

Thrombin

Factor XIIIa

Fibrinolysis

Plasmin dissolves fibrin

fibrinolysis

TPA cleavs plasminogen to plasmin Plasmin cleaves fibrin into fibrin split products Three inhibitors 1. Alpha two antiplasmin inhibits plasmin 2. PAI inhibits plasminogen 3. TAFI (thrombin activatable fibrinolysis inhibitor) inhibits binding of plasminogen and TPA to fibrin
Noble

Normal fibrinolysis, plasmin dissolves fibrin clots

DD
E

fibrin split products

DD

D-dimers

In pulmonary embolism

Fragment E

Anticoagulation
Protein C Thrombomodulin binds to thrombin to activate protein C; with its carrier, protein S, activated protein C inhibits FV and FVIII

Antithrombin Liver-produced AT binds to heparin and inhibits conversion of II to IIa; also inhibits Xa action

Protein C, S Protein C, S measure level or activity best to measure functional activity As vitamin K-dependent factors, may be abnormal during warfarin treatment Both acute phase response proteins; may be falsely normal (in deficiency) with acute illness

Screening tests
CBC-platelet count PT aPTT

 1. CBC--platelet count --Put platelets through impedance counter, analyze particles <13 fL
--Tells you nothing about the functional ability of plts

2. bleeding time
-worthless test, used to tell if patient might bleed at surgery -Use blade on pts forearm, blot blood with filter paper time w stopwatch to see when bleeding stops -Each lab has different normals, -Does not predict functional bleeding. If you have a normal or abnormal bleeding time, it will not predict whether you bleed Platelet function - analyzer (PFA) is better

Activated Partial Thromboplastin Time (aPTT) <40 seconds

Negatively charged surface Phospholipid (partial thromboplastin)

PPP + calcium chloride

Measure time to clot

Measures the intrinsic and common pathway Measures all factors except 7 and 13

Prothrombin Time (PT) <15 seconds

-Tissue factor -Phospholipids

Citrated (blue top) Patients platelet-poor Plasma (PPP)

Calcium Chloride

Measure the time clot to clot

Measures the extrinsic and common pathway

INR This formula shows up on boards

Patient PT INR = Normal mean PT

ISI

ISI International Sensitivity Index; related to amount of tissue factor in reagent INR--Developed to compensate for reagent differences

Normal PT, aPTT, platelets

Mild vWD Uremia Surgery Inherited platelet D/O Vascular D/O Fibrinolytic D/O XIII deficiency Dysfibrinogenemia Mild factor deficiency (VIII, IX, XI)

PT, normal aPTT, normal platelets

F VII deficiency or inhibitor Coumadin Liver disease dysfibrinogenemia

Normal PT,
Heparin effect

aPTT, normal platelets

vWD VIII, IX, XI deficiency inhibitor Lupus anticoagulant

PT, aPTT, and normal platelets

Common Coumadin Heparin Liver disease Vitamin K deficiency DIC Dysfibrinogenemia Primary fibrinolysis

PT, aPTT,and low platelets

Common DIC Liver disease Heparin with thrombocytopenia

Nl PT, Nl aPTT,
Common Destruction Sequestration Decreased production Bernard-Soulier

Low platelets

Nl PT, Nl aPTT,

Low platelets

Myeloproliferative disorders

Additional tests
Thrombin time Reptilase time Mixing studies Platelet aggregation studies

Thrombin time
Exogenous thrombin + Patients platelet-poor plasma

Measure the time to clot

Measures common pathway TT measures conversion of fibrinogen to fibrin Doesnt need Ca or phospholipid paraprotein, amyloid, heparin, dysfibrinogenemia

Reptilase time
Bothrops atrox venom
Mixed with Patients platelet-poor plasma Measure time to clot

If a patient is on heparin, they will have an TT but their reptilase time will be normal Reptilase measures the conversion of fibrinogen to fibrin, but is insensitive to heparin Reptilase in dysfibrinogenemia heparinase + protamine will correct TT that is in a patient on heparin

Mixing Study
Patient plasma Mixed (1:1) with Pooled normal plasma Perform PT or aPTT -Initial -At 60 minutes
--mixing study w PTT correction = factor deficiency --mixing study w/o PTT correction = inhibitor (ex. lupus anticoagulant)
--Some inhibitors correct w 1:1 mix, so try a 4 (pt plasma) : 1 (nl plasma) mix. --Time dependent prolongation = F8 inhibitor will correct and them prolong after 1-2hrs, --Dysfibrinogen inhibitor and will only partially correct but hypofibrinogen will completely correct.

Platelet aggregation studies

Tell you how platelets respond to certain chemicals to see if they will aggregate Use a platelet rich plasma (spin it down to get a good # of plts) Add aggregating agent i.e ristocetin Look for a change in light transmission We can look for in turbidity or an in transmission Curve is flipped

PLATELET AGGREGATION STUDIES

Platelet suspension

Aggregated platelets

Agent

Less light scattering: Low Optical Density

Optical 100 density (%)*

First phase of aggregation (ADP release)

50

Second phase of aggregation (more ADP release & TXA2 release)

3 Time (min)

*w/ aggregation, less light scattering, lower optical density)

What are the aggregating agents

ristocetin--physical clumping agent epinephrine collagen arachidonic acid (used for detecting aspirin defects)

Board high yield factoid

ADP and epinephrine are biphasic No secondary phase w epi & ADP in storage pool defects & aspirin

Platelet vs. factor bleeding

petechiae menorrhagia female no family history vWD, BS dz thrombocytopenia hematoma\hemarthrosis large bruises male + family history coag disorders

Congenital
Bernard-Soulier Disorder Glanzmann thrombasthenia May Hegglin

Bernard-Soulier
Adhesion problem (like in beginning of lecture) defect GP1b/V/IX (CD42) large giant platelet w pseudonucleolus thrombocytopenia Nl PT, PTT, bleeding time impaired ristocetin aggregation

If you add normal ptls + ristocetin, aggregation will be nl b/c the abnormality is on the patients plt.

Bernard-Soulier

VWF

GPIb adhesion, bleeding occurs

Bernard Soulier Disorder

Glanzmann thrombasthenia
Aggregation problem, plts cant bind fibrinogen abnormal GP IIb/IIIa normal plt count, morphology (pts dispersed); aggregation ADP, collagen, and EPI, but normal with ristocetin Dx: Clot retraction test- clot no retract test tube

Diff dx: Glanzmanns and afibrinogenemia both defect in fibrin:fibrin interactions Glanzmanns has nl PT + PTT, afibrinogenima has PT + PTT

Fibrinogen

No aggregation, bleeding

Nl adhesion
VWF

Glanzmann thrombasthemia

May Hegglin Anomaly

Mutation in myosin heavy chain 9 gene WBC inclusion (resembles Dohle body in RER + giant plt) Thrombocytopenia, but little bleeding Autosomal dominant PMN function is normal

Giant Platelets, high yield for boards

ITP, May Hegglin, gray platelet syndrome, Bernard Soulier, Montreal plt syndrome, Mediteranean macrothromocytosis. Sebastian, Fechner, Epstein, and Alport syndromes

Storage Pool Deficiency also always on boards, never in clinical practice

plt aggregation due to deficiencies in either dense granules/ alpha granule contents or both Nl morphology, no granules EM Plt agg studies: NO 2nd wave-ADP, EPI, collagen +AA, normal ristocetin ATP:ADP ratio

Storage pool deficiencies

Gray platelet syndrome No granules, Large gray plts, no granules From cardio pulmonary bypass Plt agg blunted with all agents except ADP/epi Quebec plt disorder No granules Wiscott Aldrich syndrome x-linked No granules EM Small granulated plts, like FeDa Thrombocytopenia, infection, eczema malignancy Chediak Higashi No granules EM Hermansky-Pudlak Syndrome No granules EM pigment reticuloendothelial cell Swiss cheese platelets AK, nevi, tumors, pulmonary fibrosis Puerto Rican/Swiss, vW Thrombocytopenia w absent radii

Alpha Granule Deficiency

agranular gray colored platelets

Acquired Disorders of Platelet Function

Drugs Uremia
abnormal adhesion, abnormal aggregation (ADP, epi, collagen)

Myeloproliferative disorders Cardiopulmonary bypass

Transient plt activation, granules

Aspirin/NSAIDS

platelet function by acetylation of platelet cyclo-oxygenase thromboxane formation platelet aggregation studies 2nd wave to ADP and epi, absent response to collagen and arachidonic acid

Other plt type bleeding disorders

Hereditary Telangectasia Osler-Weber-Rendu Oral Telangectasia FeDA Ehlers Danlos, Type 4 Connect tissue disorder plts cant stick, bruising and prupura, loose skin Hemoch-schonlein vasculitis, prupura, thrombocytopenia Arteriovenous malformation, plts Scurvy, Vit C, bad teeth, perifollicular petechiae Marfans, osteogenesis imperfecta, fabry syndrome Amyloidosis-- F10 Platelets dont stick, endothelium coated, lambda light chains

Von Willebrand factor amd disease

vWF binds to F8 Consists of large multimers which are cleaved by ADAMTS-13 deficiency of ADAMTS-13 associated with TTP vWD most common genetic plt type bleeding disorder 1-2% prevalence worldwide. vWF-blood type O, old blood sit Quant or qual deficiencies Secondary vWF deficiency -Wilms tumor, congenital heart disease, hypothyroid

VWD diagnostic tests

1. Platelet count Plts sometimes, screening test 2. FVIIICo measure F8 activity 3. VWF Ag measures vWF quantity blood add F8 measure vWF 4. vWF:Rcof measures vWF activity Pt plasma + nl plts + ristocetin, then measure time to aggregation very specific & most sensitive test Quantitate pt vWF activity (%) using ristocetin, standard curve with quantitative endpoint 5. RIPA Ristocetin induced platelet aggregation pts plasma + pt plts + low dose ristocetin = time to aggregation all or none, does pt aggregate w ristocetin 7. platelet agg studies 8. VWF multimers 9. mixing study

vWD multimers analysis

vWD Type I
associated with low quantity, normal multimers, functionally normal Most common type Sometimes all tests are normal Only type treated w DDAVP

vWD type 2a and 2b

Type 2a large and medium sized multimers a disproportionately low vWF:RCo (quality) relative to vWFAg (quantity)

Type 2b large multimers affinity HMW multimers for GP1b leads to increase clearance of vWF RIPA, No DDAVP Type 3 Autosomal recessive, most AD Severe marked deficiency Absence of vWF, F8 also low But may have nl coag parameters vW multimer test, all multimers low No DDAVP

vWD multimers analysis

vWF 2M and 2N
Type 2M Defect in GP 1b binding vWF made but doesnt work, vWFRco Sometimes nlRIPA, multimers, vWF Ag, F8 Suspect when vwf:Rcof < vwf Ag

Type 2N (Normandy) Defective F8 binding Vwf decreased affinity for F8 Hemophilia-like (but AR), women w low F8, think of this dz

Tests: VIII:C levels Normal RIPA, vWFAg, multimers, nl ristocetin

Pseudo VWFplatelet type

affinity plt GP1b for plasma vWD receptor Plts bind large VWF multimers There are less large vWF circulating Abn RIPA studies Spontaneous aggregation with risto Needs a higher concentration of ristocetin than 2B. Plt agg w cryoprecipitate, unlike 2B Plt agg studies show large VWF multimers The abnormality is only on the pt plts, not w vWF.

Factor deficiencies
Bleeding into joints PT, PTT or both Often nl bleeding time Hemophilia A factor VIII Hemophilia B factor IX Symptoms rare if factor level > 15-20%; severe disease if levels < 1% Chr (Xq28), 50% intron 22 inversion

HEMOPHILIA B (Christmas Disease)

Less common than Hemophilia A (1/20,000) Male patients/ X linked recessive. Severely affected patient are less common than in Hemophilia A.

Inhibitor
Inhibitors may develop after treatment, requiring huge amounts of factors Inhibitors dont correct after mixing studies F8 inhibitor corrects initially prolongs after 1hr. Bethesda unit is a measure of the strength of the inhibitor 1 Bethesda unit = 50% activity 2 Bethesda units = 25% activity 3 Bethesda units = 12.5% activity 20% of patients with Hemophilia A. 1-3% of patients with Hemophilia B.

Other factor deficiencies

F9 deficiency F9 inhibitors can get anaphylaxis when treated Tx: F9 concPTcomplex concentrate FFP, whole blood, NO cryoprecipate F8-100% dose recovery, t1/2 12hrs F9-30% dose recovery, 1/1/2 8 hrs FX11 deficiency hemophilia C, Jews, mild bleeding, Autosomal recessive unlike other hemophiliac Cant activate TAFI FXII deficiency prekallikrein, HMWF, PTT, but no bleeding. F12-Hageman-PTT, but thrombosis mixing study prolonged F13 deficiency nl PT, nl PTT, cant crosslink fibrin Clot stability test-clot cant stop 5M urea from dissolving clot < 24hrs, nl>24hrs 1% monochloracetic acid

Fibrinogen problems
50% no symptom, 25% bleed, 25%clot Hereditary ADrare Acquired-liver disease Afibrinogenemia Quantitative, AR mixing study corrects Dysfibrinogenemia AD, thrombophilic, qualitative Mixing study partial corrects-inhibitor

Reptilase time, Thrombin time Platelet Agg Tests -- dec ADP, EPI & AA
Fibrinogen--acute phase response

Vit K deficiency
Inc Pt, PTT, nl TT, carboxylation of glu F2, 7, 9, 10 vitK-malabsorbtion, antibiotics, breast milk, anticonvulsant, biliary obstruction Hemorrhagic disease newborns

2 antiplasmin deficiency

Patient presents w bleeding, nl PT, nl PTT, euglobulin lysis time--measures the time to dissolve a clot Euglobulin lysis test <2hrs also in fibrinogen, DIC, tPA

Thrombocytopenia
Plt type bleeding production
marrow suppression, drugs, congenital (Fanconis, Wiscott Aldrich)

destruction
ITP, DIC, TTP

Pseudothrombocytopenia

Plt clumping in blood prep w EDTA antibodies to GPIIb/IIIa causes plts to clump Platelet count in sodium citrate anticoagulant usually normal Platelet satellitism --in vitro platelet adherence to leukocytes in EDTA anticoagulated blood

Plt clumping

Plt satellitism

Disseminated Intravascular Coagulation

Patient bleeding, thrombosing or both, typically with progressive organ dysfunction Acute bleed, chronic clot Peripheral smear anemia w schistocytes
Lab findings PT, PTT, TT, plt count, fibrinogen, ATIII, plasminogen prot C, S, ATIII, 2antitrypsin FDPs sensitive not specific D-dimer specific Protamine sulfate +

TTP/HUS-- a board favorite

Clinical manifestations: hemolytic anemia w schistocytes, plts, fever, neural probs, renal dysfunction, abdominal pain Microthrombi in different organs TTP Big vWF multimers, cant cleave, shear RBC causing MAHA HUS normal vWF cleavage protease HUS--Shiga toxin, salmonella, E.coli 0157:H7 Labs: same as DIC except fibrinogen normal, (DIC) Kids present w renal failure Adults present w neuro symptoms

Tx TTP: FFP + steroids +Ivig + splenectomy., dont give platelets HUS: supportive + antibiotics HUS + TTP are treated differently.

TTP

CD 61

THROMBOPHILIA

BLEEDING

Thrombophilia
Predisposition to thrombosis from familial or acquired disorders of hemostasis Inherited disorders of thrombosis
Resistance of activated protein C 6% caucasions Prothrombin G20210A mutation 2% caucasions Protein C deficiency Less common Protein S deficiency Antithrombin II deficiency Hyperhomocysteinemia

Acquired disorders of thrombosis

Lupus anticoagulant Heparin Induced Thrombocytopenia

Activated Prot C resistance

Most common cause of thrombophilia usually due to point mutation in factor V, which prevents inactivation by protein C, so prothrombin production is unchecked and you get fibrosis

Factor V gene Mutation: 1. Arg for gln at position 506 (Factor V Leiden) 2. Arg for threonine at position 306 (Factor V Cambridge)

Dx
1. Functional assay-(PTTresistance ratio) Screening test Normal Add ProtC should PTT >2:1 APCR add Prot C, PTT <2:1 2. PCR for Factor V Leiden mutations

Protein C Deficiency
neonatal fulminans, homozygous Heterozygous
Type I: reduced protein Type II: defective protein

Venous thromboembolism Coumadin skin necrosis Laboratory diagnosis:

Patient must be off Coumadin

Protein S def
Prot S is a cofactor to Prot C 60% Protein S binds to C4b binding protein Only 40% is free, that is the functional protein S C4b is an acute phase reactant, C4b, Protein S Thus Prot S during stress Labs: 1.total protein S (worst test) 2. total protein S (seond best test) 3. protein S activity (best test) Autosomal dominant

Antithrombin III deficiency

Antithrombin binds to heparin to inhibit factors II and X. In ATIII deficiency, patient presents w response to heparin Acquired- liver, nephrotic dz, DIC,

Prothrombin G20210A Mutation

Point mutation leads to high prothrombin levels Guanidine to adenine substitution in the 3 untranslated portion of prothrombin gene on Chr 11

Anti-phospholipid syndrome
antibodies against phospholipids - most commonly to b2 glycoprotein, also anticardiolipin, anti lupus anticoagulant

Clinical features:
Systemic Lupus Erythematosis (15-30% of cases) Venous thrombosis Peripheral arterial thrombosis Myocardial infarction Stroke or ischemic attacks (<55 years) Recurrent fetal loss Thrombocytopenia platelets but thrombosis

Antiphospholipid antibody tests

1. PTT 2. Dilute Russell viper venom test (AKA strypven test) Russell Viper venom + Factor V, phospholipid and calcium activates Factor X. Plasma containing lupus antibodies prolongs PTT. 3. Lupus anticoagulant antibodies PTT (staclot), fails to correct w mixing study, but partial correction w adding phospholipid 4. Anticardiolipin antibody--ELISA. NO PTT 5. 2 Glycoprotein I antibodies--immunoassay

tests
6. platelet neutralization procedure PTT fails to correct w mixing study, but PTT corrects when you add hexagonal phase phosphatidyl ethanolamine
7. Kaolin clotting time 8. Nontreponemal VDRL/RPR falsely positive in LA

Heparin induced thrombocytopenia and thrombosis

IgG Ab to heparin PF4 complex in granules Occurs in 8% unfractionated heparin, much less in LMW or porcine heparin platelets and thrombosis plts usually after 5-8 days

Dx: Numerous assays; patient blood add heparin look for coagulation
serotonin release assay Measure plt granule release Pt given low dose heparin, radioactive labeled serotonin released Pt given high dose heparin,immune complexed destroyed, so no serotonin released PF4 ELISA-best test, measures Abs rapid turnaround, but often false +

Tx: -Stop heparin/dont give plts, no warfarin - thrombin inhibitors, monitored by PTT,

Homocysteine
Sulfhydryl amino acid needed for the formation of cysteine and methionine level associated with venous thrombosis/atherosclerosis

Homocysteinemia
AR, lens dislocate, MR, peripheral neuropathy, sometimes folate Dx: 1. serum levels 2. Methyl tetrahydrofolate reductase mutation 3. Mutation in CBS cysathionine synthase