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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
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
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
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
(+) 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
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
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
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
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)
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
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
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
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
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
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
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
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
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
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
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
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
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
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