Haemostasis
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Blood clotting system – essential for life
Ability of the body to prevent blood loss…… maintaining liquid blood volume in the intact blood
stream → → → → crucial for survival
•
In case of vascular wall damage /risk of loss of blood volume/ … immediately (fractions of
seconds)
haemostatic processes are initiated / vessel closure / VASOCONSTRICTION /
in the next stage (seconds, minutes)… transformation of liquid blood to blood clot…/BLOOD
COAGULUM/
after a certain period of time (hours, tens of hours) the clot dissolves /FIBRINOLYSIS/…the
original blood circulation is restored..
HAEMOSTASIS ….a number of contradictory processes..
specific proteins / circulating freely, bound to b. membrane endothelium/
adhesion molecules
many processes has not been fully explained (a complex system of positive and negative
feedbacks)
HAEMOSTASIS
physiology of blood clotting
Blood clotting – complex process, co-action of cellular and plasma systems in blood and vessel
wall
- activated very efficiently …response to vascular damage
/multiple specific cellular and protein interactions…/
-coagulum formation
- maintaining fluidity of blood
- intact (closed) bloodstream
coagulation response:
a) vessel wall (endothelium, subendothelian structures)
b) tissue component (tissue factor)
c) platelets
d ) plasma factors (activators, coagulation inhibitors, fibrinolytic factors)
e) complement proteins, lipoproteins, white and red blood cells…..
HAEMOSTASIS
–
/
/
normal activity of the anticoagulant system prevails
and to maintain blood in a liquid state
…constant specific cell and protein reactions
Due to the complexity and biological potential of the system….
!!!!! In case of their failure … the balance of pro- and anticoagulant forces is upset
….. Life-threatening event:
Primary plug
3-5 min.
Definitive plug
Restoration of flow
5-15 min.
3-48 hrs.
HAEMOSTASIS
At the site of injury
Remodeling of the
plug by overlay
of fibrin fibers
- strengthening
– VASOCONSTRICTION
– PRIMARY PLUG
(platelet stopper, 2-4 min., bleeding time)
Acute sealing of defect
in vascular wall
SECONDARY PLUG …. Resistance to increasing blood flow
FIBRINOLYSIS
COAGULUM REACTION,
ORGANIZATION OF CONNECTIVE TISSUE…
RECANALIZATION – restoration of blood flow
a) Vascular wall / wessels .. the largest endocrine organ 1000m2/
- active part, mainly endothelium and subendothelium
- function „mechanical„  VASOCONSTRICTION
„synthetic„  formation and depositing of substances involved in haemostasis
the most important physiological anticoagulant agent

   intact hydrophobic endothelium   
Endothelium
- nonthrombogenic barrierinhibits the interaction between blood components and subendothelial structures
- negative electric charge
- Expression of substances similar to thrombodulin a heparin (natural clotting inhibitors)
Endothelium
production of NO a prostaglandin I2
 increase in vascular diameter
 inhibition of platelet aggregation
-
The source of „tissue plasminogen activator „(tPA) 
 plasminogen  plasmin +++ FIBRINOLYSIS
X in the membrane, it does not contain „tissue facor „ (TF)  X COAGULATION
!!! After exposure to endotoxin, TNF, tumorous bb??? ……… possible TF expression !!!
/dangerous with contact with PLT , coagulant fa/!!!
Subendothelium
- highly thrombogenic,
- contact with blood physiologically only in case of injury
- procoagulant function: collagenous fibres, tissue factor
Re : - VASOCONSTRICTION
- absence of tissue factor x coagulation
- intact hydrophobic endothelium- non-thrombogenic barrier
- source of „tissue plasminogen activator„(tPA)
b) thrombocytes
 2/3 blood pool - unactivated 1,5 – 4x 1011/l
 1/3 splenic pool – ( in pathological state up to 90% )
active in all rections of haemostasis
- c. membrane
receptors called glycoproteinys(GP), - „ primary stopper “
fce - binding of platelets to subendothelium
- mutual cell interaction
phospoholipids (PF3
- binding of various factors to the surface of platelets
negatively charged surfaces
!!!!! Cave … Release of a large amount of PF3
…developement of DIC)
for coagulation reaction
Function of platelets:
1. formation of the “primary temporary blood stopper“ – involvement in coagulation reactions
2. called procoagulation, ei. Provision of surface = phospholipids to ensure proper run of coagulation
processes
3. release fa. involved in coagulation reactions (after activation) tromboxan…..
Metabollism FA --- the most important metabolism - arachidonic acid
ARA- lipoxygenase….leukotrienes
cyklooxygenase… prostaglandins, prostacyclins tromboxanes..
….inhibítoxy cox….inhibits fce PLT for whole life..bleeding events…(cave .. surgery)
, synthesis in liver, the normal concentration in plasma is 1,5 - 3 g/l.
synthesis in liver, concentration in plasma is about 0,150 mg/ml.
(inaccurately tissue thromboplastin ), glycoprotein present in
cytoplasmatic membranes of subendothelium cells
called labile factor
– not known (previously referred to as activated factor V)
called stable factor
–
in the absence (???deficiency- nedostatečnost)
heamophilie A develops.
antiheamophilic factor B , in the absence (???deficiency)
heamophillie B develops
- PTA
, plasmatic predecessor of thromboplastin
, contact factor
–
is a tetramer composed of two A chains and two B chains,
the factor creates cross-amide bonds between fibrin molecules contributing to formation of insoluble
clot
Process resulting in formation of primary plug
Closes damaged vascular wall and in this way stops bleeding ..(vascular wall and platelets )
Stages
 vascular (parietal)
 platelet

(intact endothelium forms a continuous layer to which, when blood flows, platelets do not
adhere
• Normal endothelium cells, by means of production of platelet adhesion and aggregation
inhibitor - /prostacyclin PGI 2/.. Prevents interaction of platelets and vascular wall
 platelets adhere via receptors of glycoprotein nature (GP) to
collagen fibres (GPIa/IIa/Iib,GPIb/V/IX)
/adhesive proteins (von Willebrand factor – vWF, fibronectin)/
 by adhesion the shape of platelet changes and together with
activation of GP nature receptors the cascade of biochemical processes is initiated
 activation of platelets
   !!!!!! Release of highly active proagregation agents
( AD P, t h r o mb o x a n e A2 – T XA2 , f i b r i n og e n – F b g e t c.) /
…..formation of temporary plug
at platelet shape change → → → expression of phospholipids on the platelet membrane surface
/transfer of phospholipids (phosphatidyliserine, phosphatidylinositol) from the inner membrane bilayer into the
outer membrane structures / …. flip - flop phenomenon/
..
Other GP receptors are exposed – GP IIb/IIIa and through bivalent proteins (Fbg, vFW,
vitronectin) mutual bonding of platelets occurs
• white thrombus is formedprimary platelet plug.
…….formation of insoluble

….cascade of reactions during which inactive plasma coagulation factors (enzymes)
activated and then activate other enzymes …(more than 40 substances---from plasma, platelets and tissues)
(r. is catalyzed by thrombin. ….)
…. Is made from PROTROMBIN
due to
extrinsic tenase
amount of thrombin
(Fa Xa, Fa Va ,PL Ca2+)
can be activated by one of the two systems intrinsic or
…coagulation system ..activated by revert mechanisms , it produces already sufficient
necessary for the initiation of the process ….formation of a small amount of
THROMBIN required for intrinsic tenase system
Accessory pathway
Primary pathway
FXI,FIX, FVIII
PF 3 (PL) Ca 2+
TF (III), FVII, PF3, Ca 2+
Intrinsic tenase
FIXa, FVIIIa,PI, Ca2+
FXa
Extrinsic tenase
FVIIa, TF, PI, Ca2+
Prothrombinase FX, FV, PL, Ca2+
TROMBIN
FIBRINOGEN
FIBRIN MONOMERS
FIBRIN
The organism is protected against uncontrolled action of
hemocoagulation factors by
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•
•
TFPI (Tissue factor pathway inhibitor)
Protein C system
Antithrombin (AT)
Heparin cofactor II (HC II)
– activated simultaneously with formation of extrinsic tenase complex
(simultaneously with release of TF)
– synthesis is induced by Fxa level rise
(activated FX a binds to TFPI … FXa is blocked by this binding)
– Resulting complex
…
- - inhibits extrinsic tenase pahtway
- - blocks TF activity
(can bind to LDL .. participation in formation of atherosclerotic plaques)
-produced THROMBIN /FIIa/ binds to transmembrane glycoprotein
THROMBODULIN /TM./
- in complex /TM. FIIa /--- thrombin becomes coagulation -negative
--- activates protein C (APC)
-- inhibits F VIIIa, F Va
-- inhibits intrinsic tenase system
(prothrombinase, thrombin formation..)
efficacy of protein C je potentiated.. . by cofactor protein S
-- --profibrinolytic activity !!!!!
increases the release of tissue plasinogen activator (t-PA)
from binding to its inhibitor (PAI-1)
• time rate of formation of the complex /AT: serum protease / significantly
potentiates
HEPARIN /- its binding to AT is reversible/
- increases effectiveness of AT a thousandfold
- to inactivate thrombin .. high molecular weight heparin is required
( low molecular weight heparins FIIa do not inhibit, they have
anti Xa activity only )
activity directed solely against THROMBIN
local character
Generally : deficiency of natural coagulation system inhibitors..
…. risk factor for thromboembolic disease !!!!!
• Responsible for the dissolution of stable fibrin clot
• After vessel injury … fibrin coagulum ….
significantly restricts blood flow
(recanalization is necessary)
• very similar to the coagulation system (proenzymes, enzymes, inhibitors)
•
BASIC COMPONENT …
(precursor of active enzyme - serine protease
)
Simultaneously with the activation of the coagulation system
• PLASMINOGEN is activated by its activators to plasmin after binding to stabile
fibrin coagulum
(at sites of bound plasminogen there are also binding sites for its activators)
PLAZMINOGEN activation :
- through blood ( FXIIa, kallikrein, thrombin)
- through tissue activators (tPA) /intrinsic pathway/
- exogenous substances - urokinase uPA /extrinsic pathway/
streptokinase (therapeutically)
tPA – is released from endothelial cells
- as an impact of thrombin, stress, physical strain
- lungs, uterus, kidneys
/activation is significantly accelerated by fibrin, (cofactor in plasminogen
activation)/
acts under physiological conditions proteolytically only
at the site of fibrin coagulum (is generated only on the coagulum surface)!!!
.
PLASMIN … endopeptidase with activity
is not specific for fibrin molecule but can cleave also fibrinogen and other factor
F VIII, F V and the like ..…. /very effective/
Fibrinogen cleaves, splits into products called fibrinogen degradation
products ( FDP).
•
•
FDP- may have antithrombogenic action
capable of inhibiting platelet aggregation.
Fibrin cleavage takes longer than fibrinogen cleavage and the specific fibrin
cleavage products are called D- dimery.
• Increase in fibrinolytic activities also off-site the vascular injury can
result in bleeding !!!!! /PLASMIN overproduction/
• In deficiency of fibrinolytic system
uncontrolled vessel closure may occur resulting in a serious thrombotic
complication !!!!!!!
a
• belongs among serine protease inhibitors
• Fce: maintaining the localization of fibrinolysis at the side of fibrin coagulum and
preventing the spread of fibrinolytic processes in the surrounding area.!!!!
• PAI-1 together with fibrinogen, CRP …. Acute phase proteins. …
(its increase indicates damage to endothelium)
• Plays a key role among procoagulation, inhibitory and fibrinolytic systems
• TAFI activation is as well as protein C influenced by the complex [TM . FIIa]
• By hydrolytic cleavage in the end positions of fibrin chain reduces the number of binding
sites for plasminogen and t-PA.
Complexity and biological potential of haemostatic system
   !!! Necessary strict regulations!!!   
(a set of control mechanisms including a number of positive and negative feedbacks…)
- Balance between susceptibility to bleeding (hypocoagulation) and increased coagulation
(hypercoagulation)
Anticoagulation system activity normally prevails and blood is maintained in a liquid state
XXX injury at the site of vessel damage ….. activity of procoagulation factors is more significant
• formation of blood clot, which is by means of most sophisticated mechanisms limited solely to the
site of injury
• Simultaneously fibrinolytic system activation… also limited only to the injury site , respectively fibrin
coagulum site
In case of failure of these control mechanisms   
life –threatening event, when the balance of pro – and anti-coagulation forces
is disrupted
resulting in profuse bleeding or for instance thrombosis also in intact vessels
and often with a development of DIC
DISSEMINATED
INTRAVASCULAR
COAGULATION/ COAGULOPAHTY
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the most common syndrome (Naun,1873)
part of the systemic inflammatory response of the organism /SIRS/
development of multiorgan dysfunction syndrome /MODS/
a life-threatening syndrome, difficult to define  „ death is coming „
is not a separate unit  it accompanies secondarily other serious clinical
conditions
defibrination sy, defibrinogenation sy, consumption coagulopathy
consumption thrombohemorhagic sy, disseminated intravascular formation of fibrin“
•
1/ 3147 newborns
1/ 867 other cases
1/ 1000 admitted to hospital
the most frequently – infection
- obstetric complications
- is not a separate nosological unit
- always a secondary state, accompanying other disease
(all states disturbing haemocoagulation balance)
HAEMOSTASIS - highly effective response to vascular damage
- strict regulation  balance between hypocoagulation and hypercoagulation
….. damage to endothelium ….. blood clot formation at the wound site
X
failure of control mechanisms

balance of pro- and anti- coagulation forces is disturbed

PROFUSE BLEEDING, THROMBOSIS

 excessive inadequate intravascular activation of blood coagulation of complex charact
 loss of “localization character“ (many places in blood stream especially microcirculation
 damage to microcirculation (formation of thrombi)microvascular obstruction/microthromb
 formation of focal necrosis  tissue perfusion defect  organ dysfunction
 excessive clot formation leads to consumption of coagulation system components
 consumption of plasma coagulation factors and platelets
 tendency to bleeding / bleeding
 reactive fibrinolysis – massive  tendency to bleeding / bleeding
 formation of biologically active cleavage products of blood clotting proteins
(anticoagulant properties)
Definition???
DIC – a dynamically developing pathological process,
which is characterized by intravascular activation of
coagulation and subsequent formation of multiple
thrombus
consumption of haemostatic factors ---- it leads to a
secondary activation of fibrinolysis, which further
deepens the emerging bleeding event, /Klener, 003, Marek 2005/
Pathophysiology of DIC syndrome is complex…
• MECHANISMS inducing DIC .. ALSO TYPICAL OF HAEMOSTASIS
OCCURRING PHYSIOLOGICALLY
• In case of DIC syndrome, however, adaptive mechanisms do not sufficiently
compensate for the condition…INSULT ACTS FOR A LONG TIME AND
EXCESSIVELY
• ACTIVATION OF ALL HAEMOSTATIC SYSTEMS :
excess of THROMBIN and PLASMIN circulation (normally they are absent)
• DIC sy is most commonly caused by an inflammatory reaction
accompanying
or massive tissue
(may also act concurrently)
Patophysiology of DIC : aktivation all systems of haemostasis
- fluctuation of haemocoagulations balance to: hypo- hyperkoagulation
TF
basic disease

TROMBIN
activation of coagulations system( coagul. Fa , PLT)
FIBRINOGEN  fibrin fibrinemia
a microvascular trombosis
Tissue rich of tPA
consumption of platelets,
PLAZMIN
coagulations fa., inhibitors
activation of fibrinolysis
biodegradation ff. a inhibitors
( MULTI) ORGANS FAILURE
MULTIPLE BLEEDING DIATESIS
-
disturbance of a relatively stable hemocoagulation balance upon a significant impulse
Imbalance between pro-thrombotic and anti-thrombotic activities
Etiology :
Over-limit??? uncontrolled activation of plasmatic
coagulation system /PKS/ …„dysregulation of thrombin activity „
1. Release of a lager amount of TF
2. Activation of platelets
3. Disorders / damage / vessel wall
1.
Release of a lager amount of tissue factor "/TF
-
TF from the extravascular tissue (trauma, surgical, obstetric and gynaecological
surgical procedures) lungs, pankreas,brest are rich on TF
TF produced by tumour cells (on surface)
TF of subendothelium ( hypoxic- reperfusion injury)
TF expressed on endothelium and monocytes in the development of SIRS (of
inflammatory/non-inflammatory origin - G+,G-…)
TF from cytoplasm of hemolyzed erythrocytes
( trauma, high temperatures, malaria, viruses….)
broken cells of ascites
amniotic fluid, dead fetus syndrome..
cell lysis during anti-cancer treatment
-
-2.
Activation of platelets
viremia
septicemia
snake poisons, immune-complexes
3. Vascular wall disorders : activation of haemostasis on endothelium – after activation of endothelium
- in case of damage to endothelial
- Damage to the vessel wall …. Exposure of subendothelium …. Massive coagulation activation
-
( TF, by collagen)
HEMANGIOMAS
TELANGIECKTASIA
MICROCIRCULATION DISORDERS ( SHOCK STATES)
IMMUNE -COMPLEXES, METABOLIC DISORDERS /ALKALOSIS, ACIDOSIS/.
DAMAGE TO ENDOTHELIUM IN VASCULITIS .. .thrombocyte aggregation…. coagulation activation
• endotoxins , exotoxins released during sepsis
……EXCESSIVE CYTOKINE RELEASE
( tumour necrosis factor α (TNF-α), interleukin 1 (IL-1) a interleukin (IL-6)
these mediators are not inhibited by their natural inhibitors ….
→ → THEY INITIATE INFLAMMATORY ACTIVATION OF ENDOTHELIUM (excessive
spilling)
• activated endothelium..releases : tissue factor /TF/
tissue plasminogen activator (t-PA)
and their inhibitors: TFPI, PAI-I
• activated leukocytes .. release : platelet activating factor
elastase , heparanase
→ → disruption of the protective layer / proteoglycans on the vascular surface
→ activation of platelets, coagulation factors,, failure of antithrombotic function of endothelium
→ excessive uncontrolled formation of THROMBIN– deficiency of its inhibitor
(neiseria meningitis., pseudomonas aeruginosa , staphylococcus, E. coli, klebsiela.,
viral ( HPV,CMV, ebola, varicela zoster)
•
• In case of massive tissue damage uninhibited TF
thromboplastic active phospholipids are released from
damaged cells into the circulation
• “in microcirculation – immediate formation of a large amount of
thrombin“
( via activation of coagulation TF)
• Release of thrombin also from hematoma
(here, thrombin is bound to fibrin, therefore protected from inhibition)
polytrauma
extensive surgical interventions
surgeries at sites of tissue rich in TF a tPA /lungs, prostate, surgeries in tumoural
sites /
burns… CAVE – loss of proteins through the burned area !!!
Crush syndrome
TRAUMA – the most common cause of death 1- 44 years of age
- 37% bleed to death,
LTB…(life-threatening bleeding)
bleeding
ACIDOSIS
HYPOTHERMIA
temperature, pH, fibrinogen, ery???….
COAGULOPATHY
• DIC syndrome in cancer of prostate, pancreatic, brest, lung
ovary and acute promelocytic leukaemia ..
….. expression of a large amount of tissue factor TF
• tumour cc. produce enzymes similar to tissue thromboplastin
• They can secrete intracellular proteins which activate factor X
directly
– prevalence of DIC caused by a snake bite
amounts to 52 %
• some snake poisons contain enzymes similar to thrombin,
• or substances specifically activating factor X or prothrombin
• In pregnancy , generally, the disposition to thrombophilia is physiologically
increased (organism maintains the integrity of placenta)
• under the influence of pregnancy hormones( especially estrogen),
…increase in fibrinogen level and fa V, VII, VIII a von Willebrandova fa..
• concurrently - hemodilution
- increase in natural inhibitors‘ activity
(TFPI, a2-makroglobulin a a1-antitrypsin)
………….therefore it DOES NOT RESULT TO THROMBOTISATION
• easy decompensation !!!!
…… by uninhibited release of a larger amount of tissue factor (TF)
• The most common causes: placental abruption, oxytocin hyperstimulation
AMNIOTIC FLUID EMBOLISM / fatality rate up to 80%/
dead fetus syndrome , uterine rupture , amniocentesis
• Acute, chronic form
( the classification is rather theoretical, the patient may be anywhere in between
these two extremes, one can pass into the other …)
(decompensated …….“ overt DIC“)
• dramatic changes in coagulation which the organism is not able to compensate
• Typical of :sepsis, shock states, metabolic disruptions, obstetric complications,
poisoning and complications accompanying surgeries
( compensated or non-overt form )
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•
•
•
•
inducing cause acts moderately or intermittently
production and consumption of coagulation factors, platelets ..balanced
disease often goes on unrecognized !!!
often diagnosed only on the basis of dynamics of laboratory parameters !!!
Intrauterine foetal death, adenocarcinoms and other neoplasms, for hepatopathy,
hemangiomas, vascular aneurysms some types of vasculitis
• Clinical manifestations vary:
- according to the stage of DIC syndrome (hypercoagulable,
hemorrhagic)
- according to the cause inducing DIC syndrome
- according to the nature of the disease
• Hypercoagulable stage
• may occur latently, the patient may not have any problems
• laboratory evidence of hypercoagulation
• In clinical manifestation:
• clinical picture of systemic inflammatory response of the organism
(temperature above 38 °C, tachycardia> 90/min, tachypnea > 20/min, leukocytes
> 12 x 109 /l or < 4 x 109 /l
• Abnormal function of renal, respiratory and central nervous
system
• bleeding is not typical of this stage!!!
Haemorrhagic stage
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•
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Characteristic bleeding events ..!!!
bleeding into subcutaneous tissue in the form of petechiae, suffusions,
Bleeding into internal organs
Typical manifestations include haematemesis, melaena, hemorrhage,
metrorrhagia, hematuria
• Acute form
• Bleeding from gums, around invasive entries, petechiae, suffusions, epistaxis,
gastrointestinal bleeding, hematuria and pulmonary bleeding
• Haemorrhages, conjunctiva bleeding, acral cyanosis
• Chronic form
• Intermittent or persistent surface or also extensive ecchymosis
• Recurrent episodes of epistaxis and mucosal bleeding
• In malignancies… migrating thrombophelebitis / atypical sites .v. subclavia
• Medical history (risk conditions)
• Clinical picture ( underlying condition, thrombi, bleeding,… indistinct
symptoms…)
•
Laboratory: - there is not a specific test
HGB, HCT, PLT, PT, APTT, fibrinogen, antithrombin, FDP, Ddimery, pH, totaly proteins
• Non-haemotological examinations: additional examination of the
underlying disease by liver tests, renal function examination, ABR,
biochemistry, iontogram… acute condition , there is no time !!!
• Thromboelastogram… shows the clot formation time and clot
firmness
Tromboelastogram
is a fragile balance between haemostatic and
fibrinolytic processes
• this balance under normal conditions prevents thrombosis and in
case of a light injury enables bleeding to stop
•
•
In excessive insult / traumatic, non-traumatic origin/
… disruption o this sensitive balance
….hyper.. hypo coagulation state.. ,
….consumption??? nebo depletion??? of haemostatic factors..
…..despite of timely initiation of an adequate treatment the mortality
of syndrome patients is often very high !!!
….. Problem areas : obstetrics, traumatology, malignancy..
(prevention necessary !!)