Institute of Pathophysiology LF MU1
Hemostasis disorders
2
Hemostasis
▪ Hemostasis is the process by which bleeding is
stopped after an injury by the formation of a clot,
while at the same time, maintaining blood in a fluid
state elsewhere.
▪ Injury to a blood vessel results in vasoconstriction
and temporary platelet plug formation.
▪ followed by a coagulation process which arrests
bleeding at the site of injury by forming a fibrin clot.
3
Classic theory of haemostasis - 1905
▪ 1860 – German pathologist Rudolf Virchow
described thrombi and their tendency to
embolize
▪ 1905 – P. Morawitz – four factor model of
hemostasis
▪ 1964 – cascade and waterfall model of
hemostasis
– Intrinsic – all the factors required for this
pathway were present in blood
▪ aPTT
– Extrinsic – requires tissue factor present in
subendothelial cell membranes
▪ PT
4
Factors of blood fluidity
▪ change or failure in any of
these factors (or a
combination disorder)
results in a failure
▪ physiological blood clotting (=
hemostasis)
▪ pathological blood clotting (=
thrombosis)
▪ increase the risk
▪ Spontaneous
normal blood flow
- sufficient circulation,
no stagnation in the
part of the circulation
undamaged vascular
wall
- preserved endothelium
and sufficient production
of its mediators
normal clotting
- balanced
regulation pro- and
anti-clotting
mechanisms
blood
fluidity
preserved
5
Endothelium
▪ endothelium normally prevents haemostasis
by secretion of platelet aggregation
inhibitors and coagulation
– NO
– prostacyclin
– thrombomodulin
– heparan-sulfate
– tPA
▪ when the endothelium is damaged, platelets
adhere to vWf expressed on the exposed
subendothelium through their receptors
(GPIb-IX)
▪ platelets are activated and their mediators
are released from the granules
– thromboxane, PAF, ADP, serotonin →
activation of other platelets (aggregation),
vasoconstriction
– Integrins expression (GPIIb/IIIa) → binding of
fibrin and formation of a definitive plug
▪ thrombocytes also involved in the activation
of secondary hemostasis
6
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author
2009. For permissions please email: journals.permissions@oxfordjournals.org
Platelet
7
Secondary haemostasis
▪ 2 types of activation:
▪ inner patway
▪ occurs after HMWK contact,
factors XII and XI with a
negatively charged surface, e.g.
▪ naked collagen in the subendothelial
layer of blood vessels
▪ lipoproteins (chylomikrons, VLDL)
▪ wall of bacteria
▪ external pathway
▪ Tissue factor (TF, fIII) released
from destroid cells – f VII co-
activator
inner path
external path
8
Limitations of cascade/waterfall
model
▪ good for describing the coagulation
process in vitro
– selectively activated intrinsic or
extrinsic pathways
▪ Several clinical observations:
– Factor XII deficiency
▪ do not suffer from bleeding in spite of
the requirement of this factor for
initiating the intrinsic pathway
▪ prolonged
– Deficiency of high molecular weight
kininogen and pre-kallikrein
▪ do not lead to a clinical bleeding
tendency
– Factor IX or factor VIII deficiency
▪ severe bleeding even though
extrinsic and common pathways are
normal and should be sufficient to
promote clotting
– Deficiency of factor VII
▪ also causes bleeding even though
the intrinsic pathway is intact.
9
Cell based model
▪ Central point
– Formation of thrombin
from prothrombin
▪ Further reactions
precedes
▪ Three phases
– interconected
– overlaped
▪ Initiation phase
▪ Amplification phase
▪ Propagation phase
10
Initiation phase
▪ TF – expressed only after damage
– Subendothelial
▪ Smooth muscle cells, fibroblasts,
macrophages, endothelial cells
– In circulation
▪ Platelets – small amount
▪ On the surface of cells carrying TF
– Contact with factor VII, activation of
complex TF/VIIa
– activation of factors X a IX
– Conversion of prothrombin to thrombin
▪ At this stage, only a small amount of
thrombin is produced
– Inhibitory factors
▪ TFPI – inhibitory complex TFPI/Xa
▪ Antitrombin III
11
Amplification phase
▪ Takes place on the surface of the platelets
– Plates adhere to vascular and extravascular
structures
▪ Von Willebrand factor
1) TF activate platelets
– Activation includes
▪ Irregular shape, pseudopodia (surface
magnification)
▪ Expression of receptors and binding sites
▪ Release of serotonin, Ca, ADP, factor V, fibrinogen,
PDGF, vWF
2) Activation of factor V, VIII and XI
– Factor V is activated on the platelet surface
with thrombin from the initiation phase
– Thrombin activates factor VIII on platelets
▪ vWF separates from the complex and allows for
further adhesion
▪ Factor VIII remains on the surface of platelets
– Thrombin activates also factor XI
12
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author
2009. For permissions please email: journals.permissions@oxfordjournals.org
Platelet
13
Platelets
▪ Express glycoprotein receptors on membranes.
Gp Ib,IIb/IIIa
▪ Have three types of granules
– Alpha granules
▪Fibrinogen, fibronectin, factor V and VIII, PDGF,
TGFb
– Dense bodies or delta granules
▪ATP/ADP, ionized calcium, histamine, serotonin,
epinephrine
– Lysosomal granules
14
Amplification phase II
3) Tenase complex
– Activated factor IXa binds to VIIIa
– tenase complex (VIIIa/Ixa)
– Converts factor X to active Xa
4) Prothrombinase complex
- Activated factor Xa forms a
complex with Va
5) Thrombin formation
Prothrombinase converts
prothrombin to large amounts of
thrombin
300,000 more effective than factor Xa
15
Propagation phase
▪ Further production of
thrombin
▪ Thrombin converts
fibrinogen to fibrin
▪ Creation of a stable fibrin
network
– Participation of factor XIIIa
16
Thrombin function
17
Fibrinogen - fibrin
▪ 3 pairs of polypeptides ([A-α][B-β][γ])2 – 340kDa
▪
▪ thrombin (serine protease) breaks down
fibrinopeptides A and B and generates fibrin
monomers (α-β-γ)2
▪ monomers spontaneously aggregate and create a
fibrin network
▪ thrombin also activates f XIII (transglutaminase),
which forms transverse links between fibrin
polymers
18
Blood clotting control mechanisms
▪ blood flow rate
▪ concentration of inhibitory
factors
– (1) thrombin level control
▪ antithrombin III (and heparan-sulfate)
Inhibition of fVII, X, XI a XII
▪ a2-makroglobulin
▪ heparin cofactor II
▪ a1-antitrypsin
– (2) control at factor Xa level
▪ protein C + thrombomodulin
▪ protein S
▪ activity of fibrinolysis
19
Fibrinolytic system
▪ plasmin (serine protease)
circulates as an inactive
proenzyme (plasminogen)
– free plasmin rapidly inhibited α2-
antiplasmin
▪ Activation of plasminogen by tPA
(endothelial cells) and urokinase
(epithelial cells) to plasmin
▪ degradation of fibrin to
degradation products
▪ activity of tPA inhibited by PAI-1
20
Heparin vs. Warfarin
21
Future
22
Blood clotting disorders
▪ (A) hypocoagulation conditions (bleeding diathesis)
– primary hemostasis defect
▪ vascular wall disorders (senile purpura)
▪ thrombocytopenia and thrombocytopathies
▪ von Willebrand disease
– coagulopathies
▪ hemophilia A and B
▪ Chronic liver disease
▪ (B) hypercoagulation states (thrombophilia)
– congenital
▪ activated protein C resistance (APCR)
– acquired
▪ (C) combined
– Syndrome of disseminative intravascular coagulation (DIC)
23
Primary haemostasis defects
▪ symptoms: petechiae, purpura, epistaxis, bleeding
from the gums or git, hematuria, menorrhagia
▪ (1) vascular wall disorders (vasculopathy)
– congenital
▪ telengiectasia hereditaria (m. Rendu-Osler)
AD, weakening of the walls of the vessels → telengiectasia
(skin, mucosa, lungs, urogenital tract)
▪ Ehlers-Danlos and Marphan syndrome
Defect of connective tissue structure (collagen)
– acquired
▪ senile purpura
▪ bacterial toxins (scarlet fever, measles)
▪ deficit of vit. C (scorbut)
▪ immunocomplex (Henoch-Schönlein purpura)
▪ (2) thrombocytopenia
▪ (3) thrombocytopathies
▪ (4) von Willebrand disease
24
Thrombocytopenia a thrombocytopaties
▪ Platelet count 150 – 400 000/μl (1.5–41011/l)
▪ In circulation survival approx. 8-10 days
▪ (A) thrombocytopenia = reduction in number
▪ <50 000/μl – increased risk of bleeding
▪ <20 000/μl – significant risk
▪ <5 000/μl – extremely high risk
– Primary or secondary
– Etiology
▪ Reduced production
 aplastic anemia (e.g., Fanconi’s)
 myelodysplastic syndrome
 Myelofibrosis
 Hereditary (e.g., May-Hegglin, Wiscott-Aldrich, Bernard-Soulier)
 Acute leukemia
▪ destruction
 autoimmune - idiopathic thrombocytopenic purpura (ITP)
 drugs
 hypersplenism
▪ Increased consumption
 DIC
 thrombotic thrombocytopenic purpura (TTP)
▪ (B) thrombocytopathies = impaired function
– Aggregation and adhesion defects
▪ Bernard-Soulier syndrome (disorder of receptor GPIb-IX)
▪ Glanzmann thrombastenie (disorder of receptor GPIIb-IIIa)
– Degradation disorders
▪ Heřmanského-Pudlákův syndrome
▪ Chédiak-Higashiho syndrome
25
About Thrombotic
Thrombocytopeneic Purpura (TTP)
▪ Disorder of systemic platelet aggregation in
microvasculature
▪ Stimulus: unusually large vWf
▪ In children: likely to be deficiency in vWf
metalloproteinase to break down vWf
▪ In adults: vWf metalloproteinase inhibited by
autoantibodies
▪ Low PLT count, intravascular hemolysis, RBC
fragmentation, high LDH
26
von Willebrand's disease
▪ the most common congenital coagulation
disorder
▪ group of states leading to a reduction in
plasma vWf level
– thrombocyte adhesion disorder
– vWf is also plasma carrier of fVIII (without
vWf is unstable and rapidly degraded) → (
i.e. secondary hemostasis)
▪ several types of vW disease
– type 1 (~75%) – reduction of concentration
in Wf
– type 2 (~20%) – normal concentration of
malfunctioning vWf
▪ plate binding failure (type 2A)
▪ disorder of binding to collagen subendothelial
layer (type 2B)
▪ fVIII transport failure (type 2N)
– type 3 – absolute deficiency of vWf
(homozygots)
27
Defects of "secondary hemostasis"
▪ typical tissue hemorrhage (hematomas), e.g. joints,
muscles, brain, retroperitoneum, no petechiae and purpuras
▪ (A) congenital disorders
– hemophilia A (Xq-linked) – defect of fVIII
▪ fVIII is a cofactor of fX activation to fXa in response to catalyzed fIXa
▪ reduction of concentration up to 25% of normal does not cause
coagulation disorder, decrease to 25-1% mild form, <1% severe form
▪ >150 point mutations in the fVIII gene – large phenotypic variability!!!
▪ prevalence in the male population from 1:5,000 to 1:10,000
– hemophilia B (Xq-linked) – defect of fIX
▪ prevalence 10 times less than hemophilia A
▪ >300 point mutations in the fIX gene (85% point, 3% short deletion
and 12% extensive deletion)
▪ defects of other factors
▪ rare, mostly autosomal recessive, clinically manifest disorders only in
severe deficiency
▪
 afibrinogenemia (defect of fI)
 hemophilia C (defect of fXI) – Ashkenazy Jews
 Other
▪ (B) acquired disorders
– hepatic insufficiency/failure
– vitamin K deficiency (disorder of fat resorption in the intestine)
– DIC
28
DIC (consumptive coagulopathy)
▪ initially excessive coagulation (thrombotic condition), then depletion of the coagulation
factors (bleeding state)
▪ coagulation in DIC is locally unlimited and is not primarily a reaction to vessel damage
▪ pathogenesis
– TF is not normally present in circulation!!!
▪ endothelium or blood cells do not produce TF on their surface
▪ in some pathologies TF occurs and activates factor VII
▪ TF pathological resources
 cells of other tissues – e.g. fetus cells during childbirth, extensive injuries, soaking of tumor cells during surgery, etc.
 pathological blood elements expressing TF – e.g. in myelo- and lymphoproliferative diseases
 pathologically activated endothelia and monocytes that begin to express TF in the membrane – e.g. endotoxin in sepsis
 TF from erythrocyte cytoplasm released under hemolysis
▪ Consequences
– Stage 1 - formation of microthrombi in microcirculation
– ischemia to gangrene
– Stage 2 - hypo- to afibrinogenesis, trombocytopenie
▪ bleeding into the organs
– pathologically escalated fibrinolysis
29
Hypercoagulation conditions
▪ lead to an increase in risk or even spontaneous and often repeated venous thrombosis and
thromboembolism (to the lungs most often), or complications of pregnancy and infertility
▪ (A) congenital thrombophilia
– (1) Disorders of the formation of clotting inhibitors
▪ Defect of AT III (AR)
▪ Defect of protein C and S (AD)
▪ syndrome of fV resistance to activated protein C (APCR)
 most common congenital disorder (“Leiden” mutation of fV)
▪ mutation of the prothrombin gene (promotor → quantitative effect)
▪ hyperhomocysteinemia (mutations in the gene for MTHFR)
▪ antiphospholipid syndrome
 Anti-cardiolipin antibodies, lupus anticoagulant, …
 unclear pathophysiology
– (2) fibrinolysis disorders
▪ LP(a)
▪  PAI-1 (promotor → quantitative effect)
▪ (B) acquired thrombophilia
– (1) clinical situations and complications of treatment
▪ immobilization
▪ hyperestrogenic conditions (pregnancy, oral contraceptives, HRT)
– (2) Pathologies
▪ Atherosclerosis
▪ Obesity ( PAI-1)
▪ Hyperviscose syndrome
 polycytemia vera, thrombocytemia, sec. polyglobulia, gamapathy)
▪ tumors
▪ heart failure
▪ hyperlipidemia, nephrot. syndrome
▪ venous insufficiency
30
Hyperhomocysteinemia
▪ homocysteine is an intermediate product of the
transformation of methionine in the methionine cycle
– is either further metabolized to cysteine
– remethylated back to methionine (in the folate cycle)
▪ the presence of several enzymes and their cofactors
(vitamins of group B, folic acid)
▪ the reason for the metabolism of homocysteine and
subsequent HHcy may be the genetic and nutritional
factors
– mutations in enzyme-coding genes
– decreased intake of vitamin B6, B12 and folic acid
▪ HHcy =pathological increase in plasma homocysteine
concentrations
▪ HHcy is an independent risk factor for atherosclerosis and
thromboembolism, fertility disorders and certain
developmental and neurological abnormalities (cleft spine
defects)
▪ homocysteine causes endothelial dysfunction and initiates
apoptosis
▪ (A) monogenic homocystinuria
– cystathionin--synthase deficiency leads to a significant
elevation of plasma Hc
– relatively rare disease
▪ (B) „mild hyperhomocysteinemia“
– polymorphism in the methylenetetrahydrofolatereductase
gene (MTHFR)
31
Deep vein thrombosis and
subsequent pulmonary embolism
32
Understanding tests
▪ By mixing the examined plasma, tissue
thromboplastin and calcium ions, the outer part of the
coagulation cascade is triggered.
▪ The cascade ends with the formation of a fibrin clot.
▪ The result of the test is the time from mixing the
mentioned substances to the formation of a clot.
33
Activated partial thromboplastin
time (APTT)
▪ information on the functionality of the inner part of the coagulation
cascade.
▪ Unlike Quick's coagulation cascade test, kaolin (Activator) triggers a
negatively charged surface of an injured vessel.
▪ For some reactions of the inner part of the coagulation cascade
(especially to activate factor X), the presence of phospholipid - platelet
factor 3 (PF3) from activated platelets is required.
▪ The plasma used in the APTT test does not contain platelets,
phospholipid should be added to the reaction. Instead of PF3, tissue
phospholipid kefalin (Partial Thromboplastin) is used.
▪
34
APTT test value
▪ indicates the time from the start of the coagulation
cascade with calcium ions to the formation of a fibrin
clot.
▪ Normal values range between 25-39 s.
▪ For example, APTT prolongation is due to
– lack of coagulation factors of the inner part of the cascade
(mainly VIII and IX),
– heparinem therapies,
– significant overdose of warfarin.
▪ In heparinized patients, the recommended APTT is
1.5 to 2.4 times the norm.
35
Quick test (prothrombin time, PT)
▪ the rate of conversion of prothrombin into thrombin by the action of
tissue thromboplastin.
▪ Tissue thromboplastin consists of a lipoprotein component (so-called
tissue factor) and a phospholipid component (also formed in tissues).
▪ This test determines the activity of the so-called prothrombin
complex and functionality of the outer part of the coagulation
cascade.
Most often used in testing the effectiveness of anticoagulant treatment
of vitamin K antagonists (warfarin).
36
Tests
37
38
P450
▪ CYP3A4 – 50% metabolized drugs
▪ CYP2D6 – 20%
▪ CYP2C9 + CYP2C19- 15 %
▪ CYP2D6, CYP2C9, CYP2C19 and CYP2A6 have been
demonstrated as functionally polymorphic
– E.g. affects the metabolism of warfarin, acenocoumarol
and other drugs (phenotyoin, tolbutamide, glipizide and
other oral antidiabetic drugs of the type sulphonylurea).
38
39
Cytochrome P4502C9 (CYP2C9)
▪ two allelic variants of the CYP2C9 gene 1, 2
– CYP2C9*2
▪ C430T replacement in exon 3 leads to substitution Arg144Cys
– CYP2C9*3
▪ replacement of A1075C in exon 7 leads to substitution Ile359Leu
▪ CYP2C9*1 is normal in vitro, while the CYP2C9*2 variant shows less
and CYP2C9*3 has significantly less enzymatic activity 3, 4
▪ phenotypical manifestation is a reduction in the clearance of CYP2C9dependent
drugs
39
1. Stubbins MJ et al: Pharmacogenetics 1996; 6:429-329 3. Rettie AE et al: Pharmacogenetics 1994; 4:39-42
2. Veronese ME et al: Biochem J 1993; 289:533-8 4. Haining RL et al: Arch Biochem Biophys 1996; 333:447-58
40
Dose/anticoagulant effect of warfarin
40
12. Alving BM et al: Arch Intern Med 1985; 145:499-501
13. Oldenburg J et al: Br J Hematology 1997; 98:240-4
polymorphism in
cytochrome P450
CYP2C9*2
CYP2C9*3
rezistence to
warfarin
- receptor
for warfarin
mutation in FIX
genetic factors environmental factors
relation dose / reaction
to warfarin
12
13
41
CYP2C9 ACTIVITY
41
Prescribed Daily Warfarin Dose and CYP2C9 Genotype
Warfarin Dose* Genotype
5.63 (2.56) *1/*1
4.88 (2.57) *1/*2
3.32 (0.94) *1/*3
4.07 (1.48) *2/*2
2.34 (0.35) *2/*3
1.60 (0.81) *3/*3
*Data presented as mean (SD) daily dose in mg
From: Higashi MK, et al. Association between CYP2C9 genetic variants and anticoagulationrelated
outcomes during warfarin therapy. JAMA 287:1690-1698, 2002.
42
Clinical manifestations of CYP2C9
polymorphism
▪ overdose at initiation of anticoagulation by standard regimens 14, 16, 17,
18, 19
▪ maintenance dose required to achieve and maintain the therapeutic
range of 11, 15, 16, 18, 19
▪ higher risk of overdose with interactions with drugs metabolised and/or
reacting with CYP2C9 17, 21
▪ instability of anticoagulant therapy 15, 16
▪ prolonged anticoagulant effect after discontinuation or reduction in the
dose of warfarin
42
14. Aithal GP et al: Lancet 1999; 353:717-9 16. Higashi HK et al: JAMA 2002; 287:1690-8
15. Taube J et al: Blood 2000; 96:1816-9 17. Verstuyft C et al: Eur J Clin Pharmacol 2003; 58:739-45
43
Interaction with drugs metabolised
and/or reacting with CYP2C9
43
Competition for
substrate
Enzyme inductor Enzyme inhibitor
ASA a většina NSAID rifampicin fluvoxamin (ostatní SSRI slabí)
fenobarbital, fenytoin fenobarbital, fenytoin omeprazol
S-warfarin karbamazepin inhibitory HMG-CoA reduktázy
losartan tolbutamid
tolbutamid cimetidin (slabý)
sulfonamidy, dapson azolová antimykotika (slabá)
diazepam, tenazepam ritonavir
fluoxetin, moclobemid desethylamiodaron
zidovudin
17, 20, 21
20. Topinková E et al: Postgrad Med 2002; 5:477-82
21. Naganuma M et al: J Cardiovasc Pharmacol Ther 2001; 6:636-7
44
Metabolic rate
▪ According to the activity of the enzyme, the population can be divided into four
main groups - slow metabolizers (PM), intermedial metabolizers (MS), effective
metabolizers (EM) and ultra-fast metabolizers (UM).
▪ The majority of the Cucasian population individuals are among the so-called
extensive metabolizers (EM) in which drugs are metabolized at an estimated rate.
▪ 5-10% of individuals are genetically identified as slow metabolizers (PM) who have
slowed breakdown of metabolized substances and are at higher risk of a higher
incidence of adverse reactions.
▪ Intermediate metabolizers (MI) are represented in 10-15% and are comparable to
the PM group with long-term treatment.
▪ In ultrafast metabolizers (UM), metabolism takes place more intensively and does
not respond clinically to normal doses of medicines and is represented in 5-10 %.
45
46
• To demonstrate the role of the following common situations in medicine (Virchow's triad)
a) blood stasis or its slower circulation,
b) increased coagulation or decreased fibrinolytic potential,
c) endothelial dysfunction or damage
as precipitating factors for venous thrombosis.
• To elucidate the effect of exogenous heparin (a homolog of endogenous antithrombin III
cofactor) as a powerful anticoagulant substance with broad therapeutic and prophylactic
potential in the model of experimentally induced thrombosis.
• To explain principles of routine coagulation tests.
Aim of practical
47
Practical part
anaesthesia
1) preparation v. jugularis - application
2ml hypotonic solution
2) laparotomy – ligation segment of
abdominal aorta and v. cava inf.
3) thoracotomy – punction of left
ventricule – blood take
4) excision of ligated segment
+ heparin 4U/kg
1) preparation v. jugularis - application
2ml hypotonic solution
2) laparotomy – ligation segment of
abdominal aorta and v. cava inf.
3) thoracotomy – punction of left
ventricule – blood take
4) excision of ligated segment
1) Weight of thrombus
2) aPTT
1) Weight of thrombus
2) aPTT