Homeostasis and Hemostasis Disorders

hemostasis and hemostasis disorder

may not be fully accurate; may be reproduced

mpajcayanan : 2D-Med : june 2012

a lecture by dra gacasan on june 11, 2012

HEMOSTASIS AND HEMOSTATIS DISORDERS

A Lecture by Dra. Gacasan on June 11, 2012

Taken from lecture and PPT copy given both by Dra. Gacasan.

HEMOSTASIS

the process by which the body simultaneously stops bleeding from

an injured site and maintains blood in fluid state

in order for bleeding to stop, there must be clot formation

o bleeding – occurs when there is an injury

o normal person – naturally reacts by stopping the bleeding

through making clots

the clot that you form will not stay in the blood vessel forever, it has

to be lysed – taken out of the particular area

o once the blood vessel wall is already repaired, you need to

get rid of the clot

so what happens?

o formation of clot if there is a need

o dissolution of a clot in order to maintain a fluid state

failure in hemostasis can result to extreme results:

o hemorrhage

extensive bleeding → hypovolemic shock → death

o thrombosis

↑ clot formation

several factors involved in the hemostasis and thrombosis:

o platelets

o WBCs - minor participation of the ff:

granulocytes

monocytes

o coagulation system

o fibrinolytic system

o natural anticoagulant system

o endothelium – lining epithelium of vascular tissue

PHYSIOLOGIC HEMOSTASIS

2 major parts

o cellular components

platelets

endothelial cells

neutrophils

monocytes

o plasma proteins

coagulation system

fibrinolytic system

anticoagulant system

ie serine-protease inhibitors

ENDOTHELIUM

o special type of cells that forms the lining epithelium of blood

vessels or vascular channels

o has glycosaminoglycans that can bind antithrombins

o has thrombomudulin – the locus of protein C activation

o secrete certain enzymes

ectonucleotidase, CD 39 – degrades ADP

o very impt role in hemostasis

secretes prostacyclin and nitric oxide - prevent

platelet activation

o binds plasminogen, plasminogen activator and single chain

urokinase (involved in fibrinolysis)

o particular parts of the blood vessels has a lot of roles with

the different substances that it induces or are in it

o during vascular injury, all these three work together in order

to seal off the injured area and stop bleeding:

initial reaction is vasoconstriction

usually affects only the small arterioles and

capillaries

contact activation of platelets

adhesion





aggregation

activation of the coagulation pathway

schematic diagram of an endothelium:

*endothelium lines the LUMEN of the blood vessel

PLATELETS

o highly complex cells

o anucleated

o disc-shaped

o came from the megakaryocytes (biggest cells you see in the

bone marrow

megakaryoblast → megakaryocyte → gets our of

blood → platelets

o 1-4 micra, smaller than RBCs

o lifespan is 10 days

o protect the vascular wall integrity

first cell to get into the site of injury to seal off the

injury

o involved in primary hemostasis

peripheral blood smear:

*if granules are orange – eosinophil

*smaller things in PBS – platelets

*with vessel wall injury, the collagen within the blood vessel wall is exposed

*”tuklap ang endothelium mo…”

*collagen is exposed →platelet will be called upon → adhere to the site of the

injury

*collagen gives the signal that there is a problem

*platelets go to the site of injury and adhere there with the help of:

*von Willebrand factor – one of the coagulation factors, binds with

collagen and glycoprotein 1b-IX-V complex

*vessel wall injury → collagen exposed → platelets are called upon →

platelets adhere to the site of injury → further activation of platelets to come

to the site of injury → stimulates platelets release contents of their granules

→signals more platelets to go to the site of injury

*thrombin is generated forming a hemostatic plug

Mark

Highlight

Mark

Highlight

Mark

Highlight





(+) vessel inury

involvement of:

o von Willebrand factor and adhesion

o release of granules → activation of more platelets

o involvement of substances (ADP, EP, collagen, PAF)

o there will be platelet aggregation

o leading to formation of hemostatic plug

at the same time, the coagulation pathway is stimulated to be able

participate forming now a more stable hemostatic plug

SCREENING STUDIES FOR PLATELET COUNT

platelet count

platelet aggregation test

platelet retention

beta-thromboglobulin and platelet F4

o found when there is ↑ turnover or platelets

because they should be confined within platelets

only, lumalabas lang pag nasisira ang platelets

o not usually seen in normal people

bleeding time

*platelet count and bleeding time are the two most usual tests for platelet

function

PLATELET COUNT

o examination in a stained blood smear

o quickest

o least reliable

factors that affect:

platelets adhere to the side of the collecting

test tube – make sure that you shake the

test tube so you can dislodge the platelets

that had adhered to the side of the tube

o blood sample → drop of blood in slide → spread → PBS →

stain → OIO viewing

o some would do direct smear, blood from patient → dropped

on the slide, better from collecting sample from the tube

o still is being done

o NV = 1 platelet/20 RBC; 7-20 platelets/field of OIO

thrombocytopenia - ↑ number in platelets

o ammonium oxalate – may be used as anti-coagulant

o hematologic analysers – electronic counters

EDTA – anti-coagulant

subject to error if:

WBC > 100,000 uL

if there is severe RBC fragmentation that

may be perceived as platelets

PBS is also done is there are too many

fragmented RBC

normal range is 150,000-450,000 uL, ave is

250,000 uL

Mark

Highlight





o ie some bleeding patients have platelet count of hundreds

only

PLATELET AGGREGATION MEASUREMENT

o assess the functional ability of platelets to aggregate to form

plugs

o they make use of platelet-rich plasma with known

aggregation inducers

ADP

collagen

EP

ristocetin

thrombin

arachidonic acid

o aggregometer – machine that measure aggregation

o patients are advised not to take any medications that could

affect the action/function of platelets for a week before

testing

ie aspirin

o performed within 3 hours of collection

o do not refrigerate samples – inhibit platelet function

o when requesting platelet concentrate, do not put in

refrigerator

o in labs – left outside in room temperature in agitator

o sodium citrate – anti coagulant

o you don’t use glass tubes – activates platelets

o normal = 60% or more platelets aggregate

BLEEDING TIME

o very commonly requested

o best indicator of functional platelet deficiency (<100,000 uL)

o prolonged in:

thrombocytopenia

von Willebrand’s disease

most dysfunctional conditions

after aspirin ingestion

advise patients not to take aspirin if will be

tested for bleeding time

o three techniques:

duke’s technique – normal = 2-4 mins

prick the finger → every 30 secs, blot the

blood in the finger in a filter paper → after 30

mins, blot again until there is coagulation

fingertip is pierced with lancet, earlobes in

babies

ivy method – normal = 3-6 mins

approx. 3mm-deep incision

template method – normal = 6-10 mins

spring-loaded blade used for pricking –

standard size and depth of cut/prick

more reproducible

both ivy and template

use volar aspect of the hand

constant pressure of 40mmHg

o use a bp cuff → raise to 40 mmHg

→ create the puncture wound →

blot , 30-sec interval until blood clots

COAGULATION PROTEIN SYSTEM

o formation of a more stable clot

o secondary hemostasis

o there is conversion of the soluble plasma protein fibrinogen

to the insoluble gel called fibrin through the action of

thrombin → establishes secondary hemostatis





o certain factors/substances in classification:

Surface-bound

zymogens

Vitamin K-dependent

zymogens

Cofactors / substrates

XII

Prekallikrein

XI

VII

IX

X

II

Protein C

High molecular

weight Kininogen

VIII

V

Fibrinogen

Protein S

COAGULATION FACTORS

Factor I Fibrinogen

Factor II Prothrombin

Factor III Tissue thromboplastin (tissue factor and phospholipid)

Factor IV Ionized calcium

Factor V Labile factor or proaccelerin

Factor VI Unassigned

Factor VII Stable factor or proconvertin

Factor VIII Antihemophilic factor (AHF)

Factor IX Plasma thromboplastin component (PTC), christmas factor

Factor X Stuart-Prower factor

Factor XI Plasma thromboplastin antecedent (PTA)

Factor XII Hageman factor/contact factor

Factor XIII Fibrin-stabilizing factor (FSF)

Prekallikrein Fletcher factor

HMWK Fitzgerald factor

vWF von Willlebrand factor

coagulation pathway

o intrinsic pathway

beginning with factor XII

o intrinsic pathway

beginning with factor VII

o common pathway

factor X

o endpoint is formation with fibrin which when bound with

platelets, forms the more stable clot

all coagulation factors are produced in liver EXCEPT:

o III – tissues

o VIII – liver endothelial cells and megakaryocyte

o vWF- endothelial cells

o XIII – liver or platelets

all factors are present in normal, fresh plasma

all are stable except V and VIII

o so pag nastore na ang blood, wala ka ng factors V and

VII

TESTS OF COAGULATION SYSTEM

lee-white clotting time

activated coagulation time

activated partial thromboplastin time (PTT) – commonly requested

prothrombin time (PT) – commonly requested

thrombin clotting time (TCT)

fibrinogen measurement

fibrinopeptides and fibrin monomer

LEE-WHITE CLOTTING TIME

o oldest and least accurate

o 3 tubes incubated at 37oC containing 1 mL of blood → every

30 secs, tilt until clotting occurs

o NV = 4-8mins

o extremely sensitive

o used to monitor heparin therapy before

double its baseline (significant)





ACTIVATED COAGULATION TIME

o monitors heparin therapy in extracorporeal circulation

bypass surgery

hemodialysis

hemapheresis

o whole blood is collected into tube celite (clay)

o NV = <100sec

PARTIAL THROMBOPLASTIN TIME (PTT)

o anti coagulant is citrate (citrated blood. 3.2gm% citrate)

o mixture:

negatively charges surface

phospholipid

patiet’s plasma

o you take note of the time it takes for a clot to form

o NV = “activated” PTT = 28-40secs

o looks into the intrinsic coagulation and common pathway

you look into the different factors in the intrinsic and

common pathways that affects the length of partial

thromboplastin time

PROTHROMBIN TIME (PT)

o asses extrinsic and common pathways

o most frequently performed coagulation test

o tissue thromboplastin + patient’s plasma + CaCl2

o NV = 11-13sec or within 2secs of control

o International Normalized Ratio:

𝐼𝑁𝑅 =𝑃𝑇 𝑜𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡

𝑃𝑇 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑚𝑒𝑎𝑛

o used to standardize anticoagulant intensity

how much anticoagulant do you need to administer

o only useful in prescribing oral anti-coagulants

o (+) recommended INR for different conditions

to interpret:

PT prolonged

PTT normal

VII deficiency

Deficiency of common pathway factors (fibrinogen,

prothrombin, V or X)

liver disease

warfarin

most probably extrinsic p/w

PT normal

PTT prolonged

assoc with bleeding: VIII, IX, XI defects

not assoc with bleeding: XII, PK, HMW kinninogen

most probably intrinsic p/w

PT prolonged

PTT prolonged

medical conditions

patients under anticoags

DIC

liver disease

vit K deficiency

massive transfusion

rare dysfibrogenemias, X, V and II defects





PT normal

PTT normal

XIII deficiency

THROMBIN CLOTTING TIME

o measures the time taken for a citrated blood to clot after

Ca++ and known amt of thrombin is added

o direct measure of fibrinogen function

o remember: thrombin converts fibrinogen to fibrin

o prolonged in hypofibrinogenemia and dysfibrogenemia

o NV = 10-15secs or within 1.3 times as long as control

FIBRINOLYTIC SYSTEM

o after coagulation, clot doesn’t stay in vascular channel

forever

o while clot is being formed, repair of the wall of blood vessel

is setting in

o injury →tissue repair

o formation of clot – part of orchestration of repair

underneath the clot, there is repair

if repair is already done, you have to get rid of the

clot because it will hinder smooth flow of blood in the

vessels

this is done through fibrinolytic system

o the clot is removed

o what are involved here:

tissue plasminogen activator (tPA)

single chain urokinase plasminogen (ScuPA)

two-chain urokinase plasminogen activator (TcuPA)

all these three converts plasminogen to plasmin

role of plasmin – digests fibrin → fibrin

degradation products (FDP) → phagocytic

system → restore smooth flow of blood

TESTS OF FIBRINOLYTIC PATHWAY

thrombin time

fibrinogen degradation products (FDPs)

D-dimer assay

o found in >90% of patients with thrombotic and

thromboembolic d/o

euglobin lysis time

o 2-6 hours

o very rarely used

other tests:

o plasminogen activator inhibitor – 1

o alpha-2 anti-plasmin assay

o plasminogen

ANTICOAGULATION PROTEIN SYSTEM





o kung bakit hindi nagcoclot yang mga blood na yan…

o two major system:

protein C/protein system

plasma serine protease inhibitor system

(antithrombin)

tissue pathway factor inhibitor

o they check and balance to maintain blood in a fluid state

PLATELET DISORDERS

quantitative

o thrombocytopenia - ↓ number of platelets

o thrombocytosis – , ↓ number of platelets, excessive

production of platelets

qualitative – dysfunctional platelets

o inherited disorders of platelet function

o acquired disorders of platelet function

THROMBOCYTOPENIA

o ↓ number in platelets

o congenital thrombocytopenia

may-hegglin anomaly, fetchner syndrome, Epstein

syndrome, sebastian syndrome

Bernard-soulier syndrome, digeorge.velocardiofacial

syndrome, paris-trosseau thrombocytopenia,

mutations in transcription factor GATA1

wiskott aldrich syndrome

o immune thrombocytopenic purpura – WBCs

o drug-induced thrombocytopenia

heparin

quinidine

gold

sulfa antibiotics

o heparin-induced

o thrombotic thrombocytopenic purpura

THROMBOCYTOSIS

o ↑ number of platelets

o benign - reactive process

not over 1000x 10^9/L

o myeloproliferative d/o (malignancy)

>1000 x 10^9/L

INHERITED DISORDERS OF PLATELET FUNCTION

o d/o in platelet adhesion

bernard soulier syndrome

autosomal recessive

abnormal in platelet GP-Ib-IX complex

abnormalities of GP Ia/IIa and VI

von Willebrand disease

autosomal dominant

three types:

prolonged bleeding time (deficient vWF)

prolonged PTT (low VIII)

BT varies

normal platelet count and CRT





failure of platelet aggregation with ristocetin

failure of platelet adhesion when passed

through column of glass beads

acquired von Willebrand disease:

o >40 yo

o no previous manifestations nor

family Hx of bleeding

o assoc with other hematologic d/o

lymphoproliferative d/o

plasma cell proliferative d/o

myeloproliferative diseases

reactive thrombocytosis

o some assoc with autoimmune d/o

and medications

o d/o in platelet aggregation

glanzmann thrombasthenia

autosomal recessive

impaired or absent platelet aggregation

prolonged BT

more mucocutaneous bleeding

quanti/quail defects in GPIIb/IIIa complex

absent/decreased with all agonists excepts

ristocetin

o opposite characteristic with von

Willebrand disease

o d/o in platelet secretion and signal transduction

deficiency in granule stores

storage pool deficiencies/SPD – def.

granules and alpha-granules

deficiency in platelet signal transduction

o defects in cytoskeletal regulation

wiskott-aldrich syndrome

X-linked d/o

involving T lymphos

thrombocytopenia

immunodeficiency

eczema

o d/o in platelet procoagulant activites

scott syndrome

Xa binding sites

IXa and VIII are diminished

ACQUIRED DISORDERS OF PLATELET FUNCTION

o myeloproliferative d/o

bleeding tendencies

thromboembolic complications

qualitative defects

o acute leukemias and myelodysplastic syndromes

majority thrombocytopenia but there is also

dysfunction

AML is more common

o dysproteinemias

multiple mechanisms

o uremia

unclear

o acquired storage pool disease

dense granule

o antiplatelet antibodies and platelet function

accelerated destruction, activation, lysis etc

o drugs that inhibits platelet functions

NSAIDS





aspirin

COAGULATION DISORDERS

hereditary coagulation protein defects

o hemophilia

x-linked – walang babaeng affected, carrier lang;

manifestations are usually seen in men

hemophilia A

classic hemophilia

deficient VIII

1/5k-10k

hemophilia B

christmas disease

deficient IX

1/25k-30k

mixing studies, specific factor assays, molecular

genetic analysis can distinguish between type

manifestations:

excessive bleeding after obvious trauma

painful, long-term bleeding in joints

(hemarthroses) with sequelae of joint

deformity

o deficiencies of other coagulation factors

rare except factor XI deficiency

mild bleeding d/o

acquired coagulation protein deficiencies

o DIC – disseminated intravascular coagulation

usually assoc with inflammation process

activation of coagulation and fibrinolytic systems

formation of thrombin and plasmin

clot-lyse-clot-lyse, a combination of clot and

lyse

consumption of coagulation factors and inhibitors

at the beginning you have many fibrin

formation until you consume all coagulation

factors, all platelets → bleeding since all

coagulation factors and consumed

prolonged PT and PTT, thrombocytopenia

due to sepsis, malignancy, OB complications,

massive tissue injury

o liver disease

prolonged PT and PTT

remember: most of the coag factors are produced in

liver so if you have d/o in the liver, ↓ coagulation

factors → problems in hemostasis

↓ CHON synthesis and abnormal CHONs produced

prekallekrein – 1 of the first to ↓

fibrinogen – 1 of the last to ↓

o vit K deficiency

seen in very ill patients in parenteral nutrition

alcoholics

affects II, VII, IX, X, proteins C, S and Z

o massive transfusion





replacement of >1.5L of blood volume with 24 hrs

in practice today, we don’t usually give

whole blood, we give components, in

massive bleeding, take note of the

coagulation factors you have to give the

patient too – ie whole blood or RBC + frozen

plasma. if small amt of blood is lost, you can

give components only.

prolonged PT and PTT

↓ fibrinogen, thrombocytopenia

o acquired coagulation CHON inhibitors

against VIII – most common, severe

systemic amyloidosis – acquired deficiencies of X or

XI

multiple myeloma and waldentrom’s

macroglobulinemia

lupus anticoagulant or antophospholipid antibody

acquired hypercoaguable states

o antiphospholipid syndrome (APS)

one of most commonly acquired risk factors for

thrombosis

major cause of pregnancy loss

female patient always having miscarriage,

maybe of APS

(+)antiphospholipid antibodies (IgG, IgM, IgA)

multifactorial mechanisms

thrombomodulin or protein S inhibition

platelet activation

endothelial cell activation

lupus coagulant

persistence assoc with 30% risk of

developing symptoms – just like APS

because of heterogeneity characteristic,

most of the time this is not detected unless

you aid it with PTT and dilute russel viper

venom time (DRVVT)

o mahirap sya i-distinguish sa APS

because they have the same clinical

mani

o hyperhomocysteinemia

homocysteine – AA

↑ levels (genetic or acquired) – assoc with ↑ risk of

thrombosis → atherosclerosis

cyastathionine beta-synthethase (CBS) gene

homozygous state leads to sever thrombosis and

atherosclerosis

thrombophilic CHONS or factors:

o antithrombin – def assoc with deep vein thrombosis in lover

extremities and pulmo embolisms

o protein C def

o protein S def

o activated protein C resistance and factor V

o prothrombin-20210 – 2-fold inc. risk

o elevated coagulation factor levels – VII, VIII, IX, XI,

fibrinogen

SUMMARY

hemostasis

endothelium → natuklap → collagen expose → signals platelets to

go to site of injury → platelets in site of injury releases

factors/granules to recruit more platelets → adhesion →aggregation

→ primary hemostatic plug (unstable)

at the same time, coagulation pathway is also working

final product is fibrin

thrombin – converts fibrinogen to fibring

fibrin binds to platelet → formation of more stable, secondary

hemostatic plug





at the same time, repair is going on → if repair is done, fibrinolytic

system will degrade the clot → using plasmin → clot is broken down

to FDPs → phagocytosis → restoration of smooth flow of blood

in normal persons, you have the natural anti-coagulant system that

keeps on working at the different parts of the coagulation pathway to

prevent formation of clots

if you have problems in any of these areas, two extremes could

happen:

o bleeding

o thrombosis