Ústav patologické fyziologie LF MU1
Hemostasis disorders
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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.
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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
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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
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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
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author
2009. For permissions please email: journals.permissions@oxfordjournals.org
Platelet
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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
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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.
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Cell based model
▪ Central point
– Formation of thrombin
from prothrombin
▪ Further reactions
precedes
▪ Three phases
– interconected
– overlaped
▪ Initiation phase
▪ Amplification phase
▪ Propagation phase
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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
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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
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author
2009. For permissions please email: journals.permissions@oxfordjournals.org
Platelet
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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
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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
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Propagation phase
▪ Further production of
thrombin
▪ Thrombin converts
fibrinogen to fibrin
▪ Creation of a stable fibrin
network
– Participation of factor XIIIa
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Thrombin function
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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
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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
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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
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Heparin vs. Warfarin
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Future
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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)
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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
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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
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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
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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)
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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
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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
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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
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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)
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Deep vein thrombosis and
subsequent pulmonary embolism
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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.
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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.
▪
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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.
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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).
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37
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).
37
38
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
38
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
39
Dose/anticoagulant effect of warfarin
39
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
40
CYP2C9 ACTIVITY
40
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.
41
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
41
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
42
Interaction with drugs metabolised
and/or reacting with CYP2C9
42
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
43
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 %.
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