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 ml, one third in lien and two
thirds in peripheral blood
No age and gender differences in platelet count.
Trombocytosis – after splenectomy.
Size: 2 – 4 mm in diameter, 0,5 – 1 mm 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 a 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 a-granules)
• Inflammatory reactions, changes in permeability of
capillaries, removing of xenogenous substances, viruses,
bacteria, graft rejection …
• Carrier for many substances absorbed to platelets surface
Immune function of platelets
Platelets and inflammation
Trombocytes in hemostasis
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
Hemocoagulation as a part of hemostasis
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
Cell model
Cell model
prothrombinase comple
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.
Endothelium nad hemostasis
FIBRINOLYSIS AND TROMBOLYSIS
Inactive plasminogen.
Active plasmin (fibrinolysin).
Activators of plasminogen.
Inhibitors of plasminogen.
Fibrinolysis
Native machinery of plasminogen (plg) activation and fibrinolysis and its modification by vascular endothelial cells (VECs) and
platelets. (1) The upper right panel shows native machinery of plg activation and fibrinolysis. VECs secrete and retain tissue-type
plasminogen activator (tPA) and maintain adequate fibrinolytic potential on their surfaces. When fibrin is generated, tPA and plg
accumulate on fibrin surfaces through C-terminal lysine residues bound to the lysine binding sites (LBSs) of plg, which trigger plg
activation and fibrinolysis. (2) The lower right panel illustrates the inhibitory function of VECs on fibrinolysis. When thrombin is
generated adjacent to thrombomodulin (TM)-expressing VECs, TM-bound thrombin effectively generates the carboxypeptidase B,
thrombin-activatable fibrinolysis inhibitor (TAFIa), which specifically cleaves C-terminal lysine residues and inhibits fibrinolysis. (3)
The lower left panel shows the inhibitory function of platelets on fibrinolysis. Activated platelets secrete plasminogen activator
inhibitor type 1 (PAI-1) and the FXIIIa catalytic A subunit, the latter of which cross-links α2AP to fibrin to stabilize thrombus. The
mechanisms shown in (2) and (3) are coordinated to protect hemostatic thrombi from immature lysis. (4) In plasma, free tPA and
free plasmin are effectively inhibited by PAI-1 and α2AP, respectively. PAI-1 also inhibits tPA activity on VECs by facilitating the
dissociation of the membrane-bound tPA on the surface of VECs and regulates plg activation potential (upper left panel).
https://onlinelibrary.wiley.com/doi/full/10.1111/jth.14157
The role of antifibrinolytic proteins in the
fibrinolytic process. The fibrin component
of the thrombus is degraded by plasmin,
generated by tissue plasminogen activator
(tPA) activation of plasminogen. Antifibrinolytic
protein plasminogen activator
inhibitor-1 (PAI-1) binds tPA, preventing
plasminogen activation. Alpha-2
antiplasmin (α2AP) forms a stable complex
with plasmin in the circulation or becomes
cross-linked into the fibrin clot by activated
FXIII (FXIIIa), which makes the clot more
resistant to fibrinolysis. Thrombin
activatable fibrinolysis inhibitor (TAFI) is
activated by thrombin (IIa) in complex with
thrombomodulin (TM). Activated TAFI
(TAFIa) cleaves off lysine residues (Lys)
from the fibrin surface therefore decreasing
plasminogen and tPA binding, thus
reducing plasmin generation. TAFI, as well
as plasminogen activator inhibitor-2 (PAI-
2), can also be cross-linked into the fibrin
clot by FXIIIa. Complement C3 is bound
and cross-linked to the fibrin clot by FXIIIa,
causing prolongation of fibrinolysis.
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