intro to hemostasis,08!11!2008

Is the complex process by which the body spontaneously stop bleeding and maintains blood in the fluid state within the vascular compartment.

The major role of the hemostatic system is to maintain a complete balance of the body’s tendency toward clotting and bleeding.

State of fluid equilibrium within the blood vessels

Vessels

Platelets

Fibrinolysis/Inhibitors

Coagulation Proteins

Bleeding Clotting

Hemostasis

Normal Hemostasis :RapidLocalized

Vascular injury

Exposed sub endothelium

Thrombin

Adhesion

Aggregation

Plug formation

Consolidation

Fibrin stabilization

1-2 sec

10-20 sec

1-3 min

3-5 min

5-10 min

ADP, TXA2

Platelet release

Fibrin formation

Retraction

***

Primary Hemostasis :◦ Platelet adhesion to exposed collagen within the

endothelium of the vessel wall. Secondary Hemostasis :

◦ Enzymatic activation of the coagulation proteins to produce fibrin from fibrinogen stabilizing fragile clot formed during primary hemostasis.

Primary Hemostasis (platelet plug)

Secondary Hemostasis (Hemostatic Plug)

Hemostasis is achieved by highly integrated and regulated interaction of Blood vessels Platelets Coagulation proteins Fibrinolysis

Platelets



Vessels

Vascular SystemVascular System

Elastic fibers on the outer layer of the artery provide resistance against pressure, such as the powerful heartbeat. The middle layer consists of elastic tissue and smooth muscles, which are important with regard to the regulation of the amount of blood dispatched. The flat epithelial tissue in the inner layer has a smooth, literally polished surface that permits the easy flow of blood by reducing friction.

The arteries divide into arterioles before leading into capillaries. They serve as control valves in the sending of blood to the capillaries. They therefore possess

very special features: Thanks to their strong muscular walls, they control the blood flowing into the capillaries and prevent them from being damaged.

The capillaries are so narrow that they refuse to permit many substances to pass through. Even tiny red blood cells can pass

through only in single file or by changing their shape.

a red blood cell approaches the tissue cell in need of oxygen and deposits oxygen in it. With the carbon dioxide it has taken from the cell, its charge is

now different and it now sets out through the veins toward the heart.

Unlike the arteries, the veins are not subjected to strong blood pressure, and therefore have different structures. Thanks to their muscular walls, the veins can

store large quantities of blood by enlarging and constricting. Thus blood stored in the veins is available for immediate use in emergencies.

As a requirement of the work the veins perform in our bodies, they possess a different structure. Valves in the veins can open and close as deoxygenated blood is carried back to the heart. Thanks to them, blood is prevented from

flowing backwards and always forwarded to the heart. How do the veins know that they need to possess such valves? This example once again shows us the superior knowledge and creation of our Lord, Who

created us so perfectly.

Synthesis and secretion of a vasodilator prostacyclin (PGI2).

Secretion of tissue Plasminogen Activator (t-PA).

Inactivation and clearance of thrombin. Activity of the cofactor thrombomodulin in

the thrombin dependent activation of protein C.

Degradation of proaggregating substances such as ADP and vasoactive amines.

***

Platelets



Vessels

Disk-shaped “cells”produced in the megakaryocytes ofthe bone marrow

Mature Platelet

Megakaryocyte

BoneMarrow

Quantity - 150,000 - 400,000/mm3

Life Span - 10 days33%pooling

67%in the

circulation

Megakaryocyte Spleen

Roughly 2-4 Roughly 2-4 μμm in diameter.m in diameter.

Dense BodyADP SerotoninATP Calcium

Microtubular System

Glycogen

Exterior Coat(Glycocalyx)

Mitochondria

SurfaceConnecting SystemCytosol

Factor XIII

Alpha GranulesPF4 FibrinogenFactor V FibronectinFactor VIII R:ag Thromboglobulin

(vWF)

Plasma MembranePF3

Dense Tubular System

Microtubules. Microfilaments. Submembranous filaments.

Providing a negatively charged phospholipid surface for factor X and prothrombin activation.

Release of substances that mediate vasoconstriction, platelet aggregation, coagulation (thrombin generation), and vascular repair.

Providing surface membrane glycoprotein such as GPIb and IIIa to attach to other platelet via fibrinogen.

***

Aggregation

Coagulation

Shape Change Release

FibrinFormation

A

B

C

3 seconds

10 seconds

5 minutes

ADPRelease

Adhesion

The principle mechanism of platelet adhesion involves :◦ Plasma◦ Collagen fibres◦ Platelet membrane glycoprotein GPIb (the

receptor for von Willebrand’s factor/vWF)

Platelet form pseudopods organelles (α-granules, dense bodies) reorganized to the center contraction granules spill their contents into the open canalicular system (OCS).

Mitogen Soted in and secreted from the α-granules

***

Activation of second passenger pathways with the platelet leads to intracellular :◦ Biochemical changes.◦ Culminating in platelet activation events such as :

Shape change Secretion Cytoskeletal reassembly

◦ Platelet aggregation

Platelet aggregation (platelet-to-platelet interaction) is an energy dependent process requires ATP (primarily derived from glycolysis)

Inactive Platelets

platelet activation

4 specific proteins secreted from the α- granules :◦ β - Thromboglobulin.◦ Platelet Factor 4 (Pf-4).◦ Thrombospondin.◦ PDGF.

***

During thrombus formation :

-Thrombin

-Plasminogen

-T-PA

-Antiplasmin

Incorporated into the clot

PlateletsCoagulation Proteins


Vessels

Coagulation factors are designated by Roman numerals (I to XIII)

Activation of a particular factor is designated by a lower case “a” (X Xa)

Factor I FibrinogenFactor II ProthrombinFactor III Tissue ThromboplastinFactor IV Calcium IonsFactor V Labile Factor, ProaccelerinFactor VII Stable Factor, ProconvertinFactor VIII Antihemophilic FactorFactor IX Christmas FactorFactor X Stuart-Prower FactorFactor XI Plasma Thromboplastin AntecedentFactor XII Hageman FactorFactor XIII Fibrin Stabilizing Factor

Substrate Cofactors Enzymes

***

Fibrinogen Group Factors I, V, VIII and XIII

Prothrombin Group Factors II, VII, IX and X

Contact Group Factors XI, XII Prekallikrein (Fletcher Factor) High Molecular Weight Kininogen (Fitzgerald Factor)

***

Contact proteins Prothrombin proteins Fibrinogen or thrombin-sensitive proteins.

***

Extrinsic pathway Intrinsic pathway Common pathway

Decreased synthesis.Dysfunctional factor molecule(s).

Excessive destruction of factors.

Inactivation of factors.

Platelets



Vessels

Physiologic process of removing unwanted fibrin deposits.

Important to : Inflammation Vascular permeability Chemotaxis

Activated by coagulation & fibrinolytic system

***

Composed of approximately 22 serum proteins

Working together with antibodies and clotting factors,

Plays an important role as mediators of both immune and allergic reactions.

***

***

***

Screening assays in hemostasis Monitoring of anticoagulant therapy Disseminated Intravascular Coagulation Thrombophilia Inhibitor (Lupus Anticoagulant, Anti

Phospholipid Antibody)

1. Tourniquete Test (Rumple Leede)2. Bleeding Time3. Clot Retraction4. Platelet Count5. Clotting Time6. PT7. APTT8. TT9. Euglobulin Clot Lysis Time10. D=Dimer

-Vascular & Platelet-Platelet-Coagulation factors-Fibrinolysis

Platelet Rich Plasma (PRP)

Aggregating Reagent

AggregateClumping

Baseline Light Transmission

Increased LightTransmission

+

Typical Biphasic Pattern

100908070605040302010

0

PlateletRichPlasma

PlateletPoor

Plasma

Secondary Response(Release)

Primary Response

Injection Point

Lag

***

Cut 1 mm deep5 mm long

Constant Pressure

Expected Range 2 - 10 minutes

40 mm

Thromboplastinand Calcium

Patient’s Plasma

Factors

IIIVVIIX

Ca++

Patient’s Plasma

FactorsIIIV

VIIIIXXXIXII

Phospholipidand Activator

Highconcentrationof thrombin

1:10 dilutionpatient’splasma

Tim

e (

in s

e co

nd

s)

Fibrinogen in mg/dL

20 40 60 100 200 400 600

60

30

10

6

3

1:40 1:3

0 1:20

1:10

1:5

Low concentrationof thrombin

Undiluted patient’splasma

Screens foreffects of • Heparin • FDPs

Activators

Plasminogen Plasmin

Fibrinogen Fibrin

Degradation Products

R.E.S.

Antithrombin-IIIDecreased Levels

1. Congenital

2. Acquired – decreased synthesis

3. Acquired – increased utilization

4. Drug-induced

• Vitamin K-dependent plasma protein

• Inactivates Factors V and VIII

• Stimulates fibrinolysis

Protein CBiosynthesis: Liver, Vitamin K dependentMW: 56,000 daltonsPlasma Concentration: 3-5 mg/LIn Vivo Half-Life: 6-7 hours Pathology: Protein C deficiency, autosomal recessive (?)

* Requires Protein S for functional activity

Thrombo-modulin

FactorIIa

Protein C

ActivatedProtein C

Protein CInhibitor

Protein C

Inhibitor

ActivatedProtein

C*

Protein CActivation

Peptide

Activation

Procoagulant

Anticoagulant

Inhibition

I. Congenital Hereditary autosomal dominantII. Acquired

A. DICB. Liver diseaseC. During post-operative periodD. Anticoagulant therapy

• Superficial thrombophlebitis

• Venous thromboses in adolescents or young adults

• Arterial thromboses rarely observed

• Skin necrosis during onset of oral anticoagulant therapy

• Cofactor for Protein C

• Vitamin K-dependent protein

• Enhances binding of Protein C to phospholipid surfaces

PlasminogenActivity – Chromogenic Substrate

Antithrombin IIIAntigen – RID, ElisaActivity – Chromogenic Substrate

Protein CAntigen – EID, ElisaActivity – Chromogenic SubstrateFunctionality – Clot-based Assay

Thank you