Hemostasis/Thrombosis I - Columbia University to Hemostasis Tuesday, February 24, 2004 – 10:00 am...

Platelets/Introduction to Hemostasis Tuesday, February 24, 2004 – 10:00 am

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Hemostasis/Thrombosis I

Normal Hemostasis/Thrombosis; Assessment of Clotting System

HEMOSTASIS & THROMBOSIS

• Platelets• Coagulation Cascade• Regulation of Coagulation


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CIRCULATORY SYSTEM• Low volume, high pressure system• Efficient for nutrient delivery to tissues• Prone to leakage 2º to endothelial surface

damage• Small volume loss large decrease in

nutrient delivery• Minimal extravasation in critical areas

irreparable damage/death of organism

HEMOSTASISPrimary vs. Secondary vs. Tertiary

• Primary Hemostasis– Platelet Plug Formation– Dependent on normal platelet number & function– Initial Manifestation of Clot Formation

• Secondary Hemostasis– Activation of Clotting Cascade Deposition & Stabilization of

Fibrin• Tertiary Hemostasis

– Dissolution of Fibrin Clot– Dependent on Plasminogen Activation


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HEMOSTATIC DISORDERSSuspicions

• Spontaneous bleeding• Prolonged or excessive bleeding after

procedures or trauma• Simultaneous bleeding from multiple sites

HEMOSTATIC DEFECTS

B l e e d i n gP r o b l e m

P l a t e le tD i s o r d e r

C o a g u l a t i o nA b n o r m a l i t y

P e t e c h ia e + -

B le e d in gS i t e s

M u c o u sM e m b r a n e

D e e p T is s u e

T im e o fo n s e t o fb le e d in g

I m m e d ia t e D e la y e d

E c c h y m o s e s/H e m a t o m a s

+ +


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HEMOSTATIC ABNORMALITIES"Screening Tests" (History)

• Dental Extractions• Prior Surgery/Biopsy• Easy bruising• Heavy menses• Deliveries• Blood transfusion

PLATELETS• Anucleate cellular fragments; multiple granules, multiple

organelles• Synthesis controlled by IL-6, IL-3, IL-11, &

thrombopoietin• Circulate as inactive, non-binding concave discs• On stimulation, undergo major shape change• Develop receptors for clotting factors• Develop ability to bind to each other & subendothelium


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PLATELET ACTIVATION

vWFRestingPlatelets Platelet

Adhesion

Initial Thrombin Formation

VIIa-TF

X XaII IIa

IIaTR


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PLATELET ACTIVATION

PlateletActivation

VIIa-TF

X XaII IIa

Platelet Release

TxA2, ADP, Serotonin, Fibrinogen, Thrombospondin

VIIa-TF

X XaII IIa


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PLATELET PLUG FORMATION


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COAGULATION CASCADEGeneral Features

• Zymogens converted to enzymes by limited proteolysis

• Complex formation requiring calcium,phospholipid surface, cofactors

• Thrombin converts fibrinogen to fibrin monomer

• Fibrin monomer crosslinked to fibrin• Forms "glue" for platelet plug


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COAGULATION CASCADE

THROMBIN• Serine protease• Cleaves fibrinogen to fibrin• Activates V to Va, VIII to VIIIa• Activates platelets-cleaves thrombin receptor• Activates XIII to XIIIa• In presence of thrombomodulin activates

Protein C to APC


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COAGULATION CASCADE

Platelet Release

TxA2, ADP, Serotonin, Fibrinogen, Thrombospondin

VIIa-TF

X XaII IIa

VIIa-TF

IX IXa

VIII VIIIa

V Va


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Tenase/Prothrombinase complex assembly

VIIIa RVIIIa/IXa

X Xa

Va/XaVa R

II IIa

FIBRIN FORMATIONA

B

A

B

F XIIIa

T


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VITAMIN K DEPENDENT CARBOXYLASE• Post-translational modification• Factors II, VII, IX, X; proteins C & S• Converts 1st 7-12 glutamic acids toγ-carboxyglutamic acid

• Confers calcium binding and lipid binding on these proteins

• Without vitamin K, secrete des-γ-carboxyglutamic acid containing proteins(inactive in coagulation)

γ -CARBOXYGLUTAMIC ACID

γ-carboxy Glu

COO-COO-

COO-

CH

CH2

CHNH3+

COO-

COO-

CH2

CH2

CHNH3+

Glu

O2,CO2

Vit KCarboxylase


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PLATELET FUNCTION STUDIES

• Bleeding Time• Platelet Count• Platelet Aggregation Studies

BLEEDING TIME vs. PLATELET COUNT

050

100150200250300350400

3.5 4 4.5 5 5.5 7 9 12 15 25 30

Minutes

Plat

elet

cou

nt (x

100

0)


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COAGULATION CASCADE

Clotting Tests• Recalcification times• Blood collected in sodium citrate, a weak

calcium chelator• Amount designed to drop calcium

concentration to < 1 mM• At that level, blood won’t clot spontaneously• RBC’s & platelets centrifuged off, leaving

plasma (unclotted liquid portion of blood)


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Prothrombin Time

• Mixture of: – 50% patient’s platelet poor plasma– 25% Mixture of Tissue Factor & phospholipid

species– 25% Calcium chloride (to bring final calcium

concentration to c. 3-5 mM)– Time to clot formation measured

COAGULATION CASCADE

Va/Xa/PLVa/Xa/PL

VIIa/TFVIIa/TF

TF FVII

FGF

FVIIaCa+2

V VaT

Ca+2FXFXa Ca+2

Ca+2PTTCommon Pathway

Middle Components

EXTRINSIC PATHWAY

ProthrombinTime (PT)


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aPTT (activated partial thromboplastin time)

• 50% patient’s platelet-poor plasma• 25% mixture of phospholipid & surface active

agent (Celite, Kaolin)• 25% Calcium Chloride to bring calcium

concentration to 3-5 mM• Time to clot formation measured

COAGULATION CASCADE

Va/Xa/PLVa/Xa/PL

VIIIa/VIIIa/IXaIXa/PL/PL

FXIIFXIIa

FGF

HMWKFXIFXIa

Ca+2FIXFIXa Ca+2

VIII VIIIaT

V VaT

Ca+2FXFXa Ca+2

Ca+2PTTCommon Pathway

Middle Components

Surface Active Components

INTRINSIC PATHWAY

aPTT


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COAGULATION CASCADEContact Phase

F XII

F XIIa

HMWK

F XI

F XIa

Prekallikrein

Kallikrein

COAGULATION CASCADEIntermediate Phase

IXa/VIIIa/PL

VIIa/TF/PLF IX

F IXa

Ca+2

Ca+2

Ca+2

XIa

F VII/TF

F VIIa/TF

Ca+2

VIII VIIIa +T

F XF Xa

Tenase Complex


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COAGULATION CASCADECommon Pathway

IXa/VIIIa/PL VIIa/TF/PL

F Xa

F XF X

V Va + Xa/Va/PL

PT

TFibrinogen

Fibrin monomer

Prothrombinase ComplexCa+2

Ca+2 Ca+2

CLOTTING ASSAYSSpecific factors/Inhibitors

• Deficiency states of every clotting protein described• 50% of any clotting factor will yield normal PT

and/or aPTT• Mix 1/2 patient plasma with 1/2 factor deficient

plasma; then perform PT and/or aPTT• Factor deficient plasma with missing factor will

prolonged clotting time; all others WNL• If more than one clotting assay prolonged, implies

inhibitor to clotting protein

Hemostasis/Thrombosis I - Columbia University to Hemostasis Tuesday, February 24, 2004 – 10:00 am...

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Transcript of Hemostasis/Thrombosis I - Columbia University to Hemostasis Tuesday, February 24, 2004 – 10:00 am...