Anatomia e Fisiologia

Joao Meirinho Moura, MD, PhD(c)

 Calendário aulas Teóricas (JM)

1. Sangue - Fisiologia Geral + Hematopoiese

2. Hemostase - Primária e secundária
3. Fisiologia do Glóbulo Rubro + Imunohematologia eritrocitaria
4. Imunidade Celular
5. Imunidade Humoral
6. Endocrinologia - Hormonas e Sistemas de Controlo Hormonal + Anatomia dos
orgãos endócrinos
7. Endocrinologia - Eixo Hipotálamo-Hipofisário + Eixo Tiroideu
8. Endocrinologia - Stress, Crescimento e Metabolismo Cálcico
9. Reprodução - Anatomia e Embriologia dos sistemas reprodutivos
10.Reprodução - Gametogénese e Fecundação
11.Fisiologia da gravidez e do parto
 Dúvidas?

1. Final da aula - 10 minutos para discussão/dúvidas

2. Email - joao.moura@hds.min-saude.pt
Anatomia e Fisiologia 2. Hemostase

Joao Meirinho Moura, MD, PhD(c)

joao.moura@hds.min-saude.pt
aíma + stasis
 Topics of today lectures: Hemostasis
❖ Meaning of hemostasis
❖ Mechanisms of hemostasis
- Vascular contraction
- Platelets plug
- Blood coagulation (clotting)

- Structure and functions of platelets

- Blood coagulation
- Mechanisms
- Clotting factors

- Prevention of clot formation inside normal blood vessels.

- Lysis of blood clots (Fibrinolysis)
 Meaning of Hemostasis

❖ It means prevention of blood loss from circulatory

system. Also it concerns with maintaining of blood in
the fluid state in the vascular compartment.
❖ Hemostasis consists of series of events responsible for the
rapid repair of any break in the vascular endothelium.
❖ Abnormality in hemostasis
system may result in
Hemorrhagic disorder or
intravascular clotting (Thrombosis)
 Haemostasis
The haemostatic response Blood vessels
to vascular injury has
three elements:

1. Vasoconstriction
2. Platelet aggregation
3. Formation of Clot Platelets Clotting factors
 Vasoconstriction after cut in blood vessels

❖ First response to damage or cut in blood vessels. It is localized response.

❖ It occurs almost immediately after cut.
❖ The degree of vascular constriction is directly proportional to severity of damage
(i.e the more a vessel is traumatized the greater the degree of spasm).
❖ The vasoconstriction may last for many minutes and may continue for hours.
❖ It narrows the lumen of the vessel to minimize the loss of blood.
❖ The aim of vasoconstriction to reduce or stop blood flow from ruptured vessel.
 Vasoconstriction

❖ Nervous reflexes - initiated by pain or other sensory impulses originate from the
damaged area. It starts almost immediately after the cut.

❖ Local myogenic spasm due to direct damage to vascular wall. The smooth muscle
in the vessel wall contracts and the diameter of the blood vessels decreases.

❖ Vascular constriction is also mediated by release of local humoral factors from

traumatized tissue. Blood platelets release thromboxane A2 and serotonin which
are vasoconstrictors.
Vasoconstriction
 Aggregation

Second mechanism of hemostasis is

formation of Platelets plug (platelets
Aggregation)
 Characteristics of Platelets
❖ Known also as thrombocytes.
❖ Smallest of the circulating formed elements
❖ Diameter 1 to 4 µm.
❖ Are anuclear bodies produced in bone marrow from the cytoplasm of Megakaryocytes.
❖ Normal concentration of platelets in circulation is 150000-300000/ µl. Spontaneous
hemorrhaging occurs when platelet count gets below 10 x 109/L (10000/µl)
❖ Have a life span of 8-12 days. Old platelets are removed by macrophages (mostly in spleen).
❖ 75% of platelets are in circulation and 25% in spleen
- no. of platelets in circulation is increased after splenectomy

❖ Production is regulated by thrombopoietin

 Platelets have many of the functional
characteristics of whole cells

• Contain contractile proteins – actin, myosin, thrombosthenin. These proteins enable

platelets to contract.
• Endoplasmic reticulum, Golgi apparatus – protein synthesis, calcium storage
• Mitochondria – ATP, ADP formation
• Enzyme systems for synthesis of prostaglandins
Platelets have many of the functional
characteristics of whole cells
 Platelets characteristics (cont.)

Platelets produce :

❖ Clotting factors like Fibrin Stabilizing Factor (clotting factor no. XIII) and von Willebrand
factor.

❖ Platelet derived growth factor (PDGF) - causes vascular endothelial cells, smooth muscles
and fibroblasts to grow and repair damaged vessel wall and stimulates wound healing.

❖ Serotonin

❖ Prostaglandines

❖ ADP

❖ Thromboxane A2
• Serotonin released by platelets contributes to VASOCONSTRICTION observed
immediately after a vascular injury.
• Platelets aggregate to PLUG the vascular injury.
• Platelets provide phospholipids which accelerate the process of CLOTTING.
• Contractile proteins of the platelets bring about CLOT RETRACTION.
• Platelets have a growth factor which stimulates mitosis in vascular wall leading to
REPAIR of damaged vessels.
• Platelets seem to be involved in all stages of HEMOSTASIS - from the initial
reaction till the final repair.
 Platelets Plasma Membrane

❖ Has Glycoprotein coat that repulses normal

endothelium while adhering to injured endothelium and
any exposed collagen.

❖ Contain receptors for collagen.

❖ Is rich in phospholipids that activate many steps of

clotting process.
Platelets Plasma Membrane
 Platelets production
❖ Megakaryocytes, giant cells in the bone
marrow, form platelets by pinching off bits
of cytoplasm and extruding them into the
circulation
❖ Colony stimulating factors regulate the
production of Megakaryocytes
❖ Thrombopoietin, a circulating protein factor
(produced in the Liver and Kidney) facilitates
maturation of Megakaryocytes. Provides a
feedback control for maintaining a normal
platelet count.
 Role of platelets in hemostasis
❖ Platelets contribution in hemostasis consists of many steps.
❖ Interact with injured vessel wall (adhesion)
❖ Interact with each other (mass of platelets)
❖ Produce platelets plug

❖ Platelet plug is
❖ Fragile
❖ Can easily be dislodged from the vessel wall
**The platelet plug is reinforced by fibrin fibers (clot).
 1. Platelet Adhesion
❖ Damage to endothelium exposes blood to the subepithelial tissue matrix (collagen)

❖ Injured endothelium releases a plasma cofactor protein (von Willebrand Factor) which
attaches to the platelet membrane glycoprotein receptor causing adhesion.

(Congenital absence of vWF causes bleeding disorder)

 2. Formation of Platelet plug

• Contact with damaged endothelium/collagen ! changes in platelets shape.

• Swelling, more irregular shape with multiple pseudopodia, forceful contraction
(due to calcium mediated contraction of actin and myosin) and degranulation
with release of multiple active factors
• Become sticky and adhere to collagen via von Willebrand factor
• Secrete large quantities of ADP and form thromboxane A2 from arachidonic
acid.
• The release of large amounts of ADP combined with thromboxane A2 amplifies
the initial aggregation of platelets (positive feedback ) into a large platelet mass
formation of the platelet plug.
The third mechanism of hemostasis

Mechanism of Blood Clotting

 Balance between anticoagulants and procoagulants

❖ Normally blood in blood vessel kept in fluid state because of

dominancy of anticoagulants factors.
❖ When blood vessel is traumatized, locally the procoagulants are
activated and clot develops

clot Fluid state

procoagulants anticoagulants
 Blood coagulation in ruptured BV

❖ Starts quickly
-20 seconds in severe
bleeding, and 1-2 minutes in
minor trauma.
❖ Interaction between substances
released from traumatized
blood vessel wall, platelets and
clotting factors.
 Cycle of blood clot
❖ Blood clot formed in the body follows one of these two courses:
1. invasion of the clot by fibroblasts starts few hours after clot formation.
It converts clot in the wall of the blood vessel into fibrous tissue
within 1-2 weeks.
*** platelets enhance this process through growth factor that they
secrete.

2. Dissolving of the clot.

 General mechanism of blood clotting

** Three essential steps:

❖ When blood vessel is injured a cascade of
complex chemical reactions results in
formation of activated substances
collectively called prothrombin activator.
❖ Prothrombin activator converts
prothrombin into Thrombin
❖ Thrombin converts fibrinogen into Fibrin
fibers
 Prothrombin (clotting factor II)
❖ Plasma protein of globulin type.
❖ MW= 68000
❖ Produced by liver. It must be produced continuously because it is continuously
consumed by the body.
❖ Its production needs vit. K.
❖ Plasma concentration 15 mg/dl.
❖ When activated, it produces thrombin which acts on fibrinogen.
❖ Prothrombin attaches to platelets on the site of injury.
 Fibrinogen (clotting factor I)

❖ MW=340000
❖ Plasma concentration 0.1-0.7 g/dl
❖ Formed in the liver.
❖ Thrombin acts on fibrinogen producing fibrin monomers which polymerize into long
fibrin fibers.
❖ Fibrin fibers form the meshwork of the clot.
❖ The meshwork of the fibrin fibers is strengthened by fibrin stabilizing factor (XIII).
 Fibrinogen (cont.)

❖ 3 stages of conversion of fibrinogen to fibrin

❖ Proteolysis
❖ Thrombin cleavage of fibrinogen results in fibrin monomers
❖ Polymerization
❖ Spontaneous self-assembly into fibrin polymers
❖ Stabilization
❖ Introduction of covalent bonds into fibrin polymers by XIIIa
 Formation of prothrombin activator (Clotting Cascade**)

❖ Has two semi-independent pathways

❖ The intrinsic pathway has all of its components within the blood. It begins in the blood
itself.
❖ The extrinsic pathway is triggered by extravascular tissue damage; this pathway is
activated by exposure to a tissue factor (trauma to blood vessel wall and surrounding
tissues)
❖ Both pathways result in activation of prothrombin (factor II)
❖ The intrinsic pathway is relatively slow (1-6 minutes)
❖ The extrinsic pathway is quite fast (15 seconds)

** In Clotting Cascade, Each coagulation factor is converted to an active form

by the previous factor in the cascade
CLOTTING FACTORS
 EXTRINSIC PATHWAY

Tissue factor (tissue The extrinsic pathway is

thromboplastin) : initiated when there is an
injury to a blood vessel
Phospholipid from
wall and release of
damaged tissues.
tissue factor

Note: Prothrombin
activator is a complex
consists of Xa, Va, Ca+
+ and platelet
phospholipid
 INTRINSIC PATHWAY
Starts when blood exposes
to rough surface (collagen in
blood vessel wall. Exposure
of blood to collagen
activates factor XII and
causes release of
phospholipids from platelets.
Contact with
collagen
 Important points in Blood clotting
1.All factors or procoagulants present in blood in
INACTIVE forms.
2.INJURY to tissue, blood vessel wall, cells, platelets is
essential for initiation of blood clotting.

3.Ca++ is required for all steps of intrinsic and extrinsic

mechanisms EXCEPT for converting XIIa and Xla.
4.Clotting promotes more clot formation (vicious cycle -
positive feed back).
5.Most of clotting factors are produced in liver.
6.Formation of clotting factors II, VII, IX and X in the liver
needs presence of vitamin K.
 Summary of sequence of events after vessel injury

- Vasoconstriction- Controlled by vessel smooth muscle; enhanced by

chemicals secreted by platelets
❖ Platelet adhesion
❖ Adhesion to exposed subendothelial connective tissue
❖ Platelet aggregation
❖ Interaction and adhesion of platelets to one another to form
initial plug at injury site
❖ Fibrin-platelet plug
❖ Coagulation factors interact on platelet surface to produce
fibrin; fibrin-platelet plug then forms at site of vessel injury
❖ Fibrin stabilization
❖ Fibrin clot must be stabilized by Factor-XIIIa
Why blood in the vessels does not normally clot?
1.Endothelial surface factors:
a. The smoothness of intact endothelium of the blood vessels.
This prevents activation of intrinsic pathway.
b. a layer of glycocalyx on endothelium that repels the clotting
factors.
c. presence of protein called thrombomodulin which binds
thrombin (removing thrombin). The complex of thrombin-
thrombomodulin activates protein C which inactivates the
activated form of factors V and VIII.
2. The rapid rate of blood flow in the blood vessels, preventing
stagnation of blood.
thrombin-thrombomodulin and protein C

thrombin-thrombomodulin

Tissue plasminogen activator

 Why blood in the vessels does not normally
clot ?(cont.)
3. The presence of the natural anticoagulant substance,
heparin.

4. Formation of an antithrombin (known as Antithrombin

III) which removes free thrombin. The effectiveness of
antithrombin III as anticoagulant is increased greatly by presence of
heparin. The complex of heparin-antithrombin III can remove other
activated clotting factors (XII, XI, X, IX).

*** HEPARIN: Is a negatively charged conjugated polysaccharide,

produced by basophil and mast cells. It is used in medical practice
to prevent intravascular clotting).
PREVENTION OF BLOOD CLOTTING IN THE NORMAL VASCULAR SYSTEM

❖ Antithrombin action of Fibrin and

antithrombin III.
❖ When a clot is forming 85 - 90% of thrombin
formed from prothrombin becomes adsorbed to
fibrin fibers.
❖ Antithrombin III combines with any thrombin
which is not adsorbed to fibrin and blocks its effect
on fibrinogen and inactivates the bound thrombin.
 Lysis of blood clot
Tissue plasminogen activator (t-
PA) produced by injured tissues
❖ Blood contains protein called and endothelium. It coverts
plasminogen which entrapped in
plasminogen which can be the clot into plasmin which digest
activated into proteolytic fibrin fibers.
enzyme called PLASMIN.
❖ Plasmin digest fibrin fibers (so
causes lysis of the clot) it also
digests fibrinogen,
prothrombin, factors V, VIII and
XII (It sometimes decreases the
coagulability of the blood).
 Lysis of blood clots – Plasmin

Significance of plasmin system:

The lysis of blood clots allows slow clearing (over a period of

several days) of extraneous clotted blood in tissues and sometimes
allows reopening of clotted vessels.

It helps to remove minute clots from the millions of tiny peripheral

vessels.

[this is the basis for using t-PA or streptokinase for treating intravascular
clots in pulmonary or coronary artery]
 LABORATORY EVALUATION OF HEMOSTASIS
DISORDERS

❖ Platelets functions evaluation

❖ Peripheral blood smear evaluation

❖ bleeding time (N=1-6 min)

❖ PFA

❖ Clotting factors evaluation

❖ Clotting time (N=6-10 min)

❖ Prothrombin time (PT) –measures the extrinsic and common pathways (N=11-13 sec).

❖ Activated partial thromboplastin time (APTT) –measure intrinsic pathway (N=30-40 sec).

❖ Global Coagulation assays

❖ Rotem / TEG
Dúvidas?
Dúvidas?
Próxima aula …
Fisiologia GV
Ciclo celular da respiração
Imunohematologia e Transfusão
O Hemograma Normal
Anatomia e Fisiologia 2. Hemostase

Joao Meirinho Moura, MD, PhD(c)

joao.moura@hds.min-saude.pt