HOMEOSTASIS
Ideal balance of several systems:
• endothelium of vessel wall
• collagen below endothelium
• tonus of the vessels
• number and quality of platelets
• clotting and fibrinolytic systems
• character of blood flow in the vessel
prevents bleeding on one side and intravascular blood
clotting on the other side.
HEMOSTASIS (blood clotting, stop of bleeding)
= set of mechanisms which prevent bleeding on one
side and stop already existing bleeding on the other
side.
• Reaction of vessels
• Actions of platelets
• Blood clotting
REACTION OF VESSELS
Vasoconstriction.
Vasoconstriction depends on the severity of vascular
injury.
Serotonin (granules in platelets).
Adrenalin.
Fibrinopeptides.
PLATELETS (THROMBOCYTES)
Nucleus-less, colorless, granulated, the smallest formed
elements in blood.
Origin: megakaryocytes of bone marrow under the effect of
colony stimulating factors – interleukins (IL-1, IL-3, IL-6) and
granulocytes and macrophages stimulating factor (GM-CSF)
Number: 200 000 – 500 000 in µl, one third in lien and two
thirds in peripheral blood
No age and gender differences in platelet count.
Trombocytosis – after splenectomy.
Size: 2 – 4 µm in diameter, 0,5 – 1 µm thickness, 4 – 8 fl
volume
Shape: smooth, round discs
The shape is kept by cytoskeleton (disk of microtubules
around the periphery, invaginated membrane, canalicular
system connected to extracellular space).
Membrane: contains receptors for adhesion to certain
surfaces, e.g. collagen, von Willebrand factor, fibrinogen
Cytoplasm: contains actin, myosin, glycogen, lysozomes
and
Granules: dense granules (non-protein substances –
serotonin, ADP, adenonucleotides) and α granules
(protein substances - clotting factors, platelet derived
growth factor – PDGF)
Glycocalyx: 10 – 50nm, mixture of proteins and
mucopolysaccharides (clotting factors, ions, amino acids,
histamin, drugs…)
Life span: 9 – 12 days, biological half-time – about 4 days
Structure of trombocyte
Jurk K, Kehrel BE: Platelets: Physiology and biochemistry. Seminars in Thrombosis and Hemostasis
2005, 31(4):381-392.
Function of platelets
• Protection of organism from blood loss
• Keeping the integrity of vessel wall and healing of the
ruptured vessel (PDGF from α-granules)
• Inflammatory reactions, changes in permeability of
capillaries, removing of xenogenous substances, viruses,
bacteria, graft rejection …
• Carrier for many substances absorbed to platelets surface
HEMOSTASIS I. – white clot
Adhesion (exposure of the vessel wall – collagen – receptors for collagen on
platelet, laminin, von Willebrand factor).
Activation and change of shape – collagen, ADP, thrombin. Glycoprotein
IIb/IIIa receptors.
Secretion (degranulation):
Stimulation of aggregation – ADP
Stimulation of adhesion – vWF and fibronectin
Vasoconstriction – serotonin, tromboxane A2
mitogenic effects – growth factor (PDGF)
activation of platelets and phagocytes – PAF (cytokine, G-coupled receptor,
phospholipase C, DAG, increase of intracellular Ca2+concentration, phospholipase A2 –
arachidonic acid – thromboxane A2)!!! Therapeutic use of acetylsalicylic acid!!!
Aggregation.
Vasoconstriction.
Convolution of inner layer of vessel wall (at the place of rupture).
ADP
Ca++
Serotonin
Vasoconstriction
Noradrenalin
Dense granules Alpha granules
Fibrinogen
PDGF atherosclerosisvWF
V XIII
vWF
Collagen
Endothelium
Thromboxan A2
AA
COX-1
PGH2
Aspirin
Vasoconstriction
Activation
Inhibition
Ticlopidine
ADHESION
Inhibition
Fibrinogen
Receptor IIb/IIIa
Abciximab
vWF
AGGREGATION
Aggregation – an example of positive
feedback
Ligands and receptors involved
in adhesion of platelets
Broos K, De Meyer SF,
Feys HB,
Vanhoorelbeke K,
Deckmyn H: Blood
platelet biochemistry.
Thrombosis
Research 2012,
129(3):245-249.
Hemostasis - white thrombus –
overview and connections
HEMOSTASIS II. – red clot
Prothrombin (factor X) – thrombin.
Fibrinogen – fibrin monomer – fibrin polymer (factor III, Ca2+).
Intrinsic pathway – extrinsic pathway of factor X activation.
Intrinsic pathway
- Factors IXa, Xa, and
thrombin proteolytically
cleave Factor VIII to
form VIIIa, which is the
co-factor of the next
reaction.
- VIIIa, together with IXa,
calcium ions (from the
platelets) and negatively
charged phospholipids,
form the trimolecular
complex of tenase
- Tenase converts factor
X to Xa.
Extrinsic pathway
- Initiated by factors
outside of the vascular
system
- Expression of tissue
factor outside the vessel
- It is a receptor for
plasma protein - factor
VII
- Activation - VIIa
- Together with calcium
ions, the formation of a
trimolecular complex,
which resembles tenase
- Proteolytic activation of
factor X
Common pathway
- Initiated by factor Xa
- Subsequent activation
of Factor Va
- Creation of the
trimolecular complex
(Xa, Va, calcium ions
together with PL) =
prothrombinase
- Conversion of
prothrombin to thrombin
- Conversion of fibrinogen
to fibrin
Thrombin
- Thrombin catalyses the conversion of proteolysis of fibrinogen
- Fibrin monomers spontaneously polymerize and form gel - capture of blood elements
- Activation of factor XIII and formation of polymer network
- Thrombin catalyses the formation of further thrombin, and Va and VIIIa - positive feedback
- Paracrine action of thrombin - endothelial cells release NO, prostaglandin I2, ADP, vWF, TPA - thrombocytes
(PAR-1) - thrombocyte association with coagulation cascade
Modern concept - phases of
coagulation
1. Initiation phase
• = extrinsic pathway, exposure of TF and subsequent cascade
2. Amplification phase
• Slow accumulation of thrombin
• Recruitment of other thrombocytes at the site of the vessel injury
• Creation of Va and aplification of prothrombinase activity
3. Promotion phase
• On the surface of procoagulant phospholipids - platelets
• Cascade with the formation of thrombin, fibrin and its
polymerization - crosslinking
Modern concept
Thrombin – an example of positive
feedback
Thrombin
- very small amount of thrombin is insufficient to activate fibrinogen
- four major feedback mechanisms
1
2
3
4
Silverthorn, D. U. Human
Physiology – an
Integrated Approach.
6th. edition. Pearson
Education, Inc. 2012.
SUMMARY
INTRAVASCULAR COAGULATION
Damage of epithelium caused by:
1) Atherosclerosis (myocardial infarction, stroke)
2) Inflammation (venous thrombosis, pulmonary
embolism)
THROMBOSIS
EMBOLISM
damaged endothelium
damaged endothelium
Thrombus
Embolus
Example:
MI
Stroke
Example:
Pulmonary embolism
Antithrombotic drugs?
• We influence function of thrombocytes, not number of
throbocytes!
• Primary and secondary prevention of atherothrombosis
– Acute Coronary Syndromes (ACS)
– Cerebrovascular Ischemic Attack
– Peripheral arterial disease (PAD)
• antiplatelet agents?
• Inhibitors of cyklooxygenase/inhibitors of thromboxane A2 synthesis
or antagonists of the receptors
• Inhibitors of ADP receptors (P2Y12)
• Antagonists of protease-activated receptors (PAR-1)
• Antagonists of surface glycoproteins (GP IIb/IIa)
• Blockage of serotonin pathway
• Other mechanisms
CONTROL OF HAEMOCOAGULATION
Clotting is counteracted by anti-coagulating mechanisms:
Non-humoral control:
Endothelial surface factors.
Blood stream: restriction of increase of clot, dilution and
removal of clotting factors.
Interaction between thromboxane A2 and prostacycline.
Humoral control:
Fibrin: binds thrombin strongly – „antithrombin“
Antithrombin III: circulating inhibitor of proteases (active
forms of factors IX, X, XI, XII), binding of proteases of clotting
system is facilitated by heparin from mast cells (co-factor of
heparin)
Thrombmodulin: thrombin binding protein, produced by
endothelial cells.
Thrombin + Thrombmodulin = activator of protein C
Protein C: inactivation of factors V and VIII
Inhibition of the inhibitor of activator of tissue
plasminogen (= more plasmin – degradation of fibrin)
Plasmin (fibrinolysin): active part of fibrinolytic system.
Precursor: plasminogen, catalyzed by thrombin and tissue
activator of plasmin (TPA) – use in therapy of myocardial
infarction!!! Streptokinase.
Kauskot A, Hoylaerts
MF: Platelet receptors.
Handbook of
experimental
pharmacology
2012(210):23-57.
Ezihe-Ejiofor JA, Hutchinson N:
Anticlotting mechanisms 1: physiology
and pathology. Continuing Education in
Anaesthesia, Critical Care & Pain
Advance Access 2013
FIBRINOLYSIS
Inactive plasminogen.
Active plasmin (fibrinolysin).
Activators of plasminogen.
Inhibitors of plasminogen.
Thrombolysis
UPA, urokinase plasmin activator tPA, tissue plasmin activator
PAI, plasmin acivator inhibitor alpha2PI-Plasmin, complex
ANTI-CLOTTING TREATMENT
Defibrination: removal of fibrin (substances from snake
poisons) – in vitro
Decalcification: binding or removal of calcium ions (sodium
citrate, potassium or ammonium oxalate) – in vitro
Heparin: natural anticoagulant, mast cells, active only in the
presence of antithrombin III, used also in vivo
Cumarin derivatives (dicumarol, warfarin): inhibition of
effects of vitamin K in liver – disorders of factors II, VII, IX, X,
protein C, protein S (facilitates activation of Va and VIIIa via
protein C)
Hirudin: obsolete, salivary glands of leech (Hirudo
medicinalis)
Anticoagulants
Heparin antithrombin III
test: aPTT,
antifactor Xa level
Inhibition
Activation
In vivo:
Coumarin (warfarin)
Vitamin K Hepar prothrombin
Liver
In vitro: Heparin antithrombin III
Sodium citrate Ca++
test: PT
INR=PTpatient/PTnorm
warfarin INR 2-3
aPTT: activated partial thromboplastin time PT: prothrombin time
HMWK, high-molecular-weight-kininogen PK, prekallikrein F, factor
Tests aPTT and PT
Prothrombin time test
Activated Partial Thromboplastin Time test
STREPTOKINASE
CLOTTING DISORDERS
Clotting diseases = disorders, in which blood clotting starts
either spontaneously or after inadequately small stimulus.
Blood clotting disorders caused by diseases of vessels
Disorders of platelets:
1)thrombocytopenia
2)thrombocytopathy
Coagulopathy – loss or lack of plasmatic clotting factors:
1)Disorders of synthesis: hereditary (haemophilia),
attained (hypo-vitaminosis K, therapy with derivatives of
cumarin)
2)Disorders of metabolism:
•consumptive coagulopathy and hyperfibrinolysis
•repeated transfusions
•immunocoagulopathy
•therapy by heparin
•paraproteinemia