1. HEMOSTASIS
Is a complex of homeostatic reactions, which result in arrest of bleeding from damaged blood vessels (hemo = blood, stasis = standing)
Is counter-balanced by reactions, which prevent blood coagulation in uninjured vessels and maintain the blood in a fluid state
Balance between procoagulants and anticoagulants
4 overlapping processes or stages
Local vasoconstriction
Formation of a platelet plug
Formation of a web of fibrin proteins that penetrate and surround the platelet plug – blood coagulation or clotting
Clot retraction.
HEMOSTASIS
Circulating in a high-pressure, closed system that communicates
with all tissues and cells in the body, blood exchanges
oxygen, nutrients, and wastes and provides necessary components
for host defense. This communication takes place largely
in the complex and dynamic networks of capillary beds that
provide oxygen to almost every cell in the body (only the
cornea and intervertebral disks are avascular; these tissues receive
oxygen by diffusion). Disruption of the integrity of the
fragile capillaries may result from minor tissue injury associated
with normal physical activity or from massive tissue
trauma as a result of serious injury or infection, and may
quickly lead to death. Any opening in the vascular network
may lead to massive bruising or blood loss if left unrepaired.
To minimize bleeding and prevent blood loss after tissue
injury, components of the hemostatic system are activated.
The components of this dynamic, integrated system include
blood platelets, endothelial cells, and plasma coagulation
factors. They may be activated on exposure to foreign
surfaces during bleeding, or by torn tissue at the site of
injury, or by products released from the interior of damaged
cells. Hemostasis can be viewed as four separate but
interrelated events:
• Compression and vasoconstriction, which act immediately
to stop the flow of blood
• Formation of a platelet plug
• Blood coagulation
• Clot retraction

2. LOCAL VASOCONSTRICTION
Results from
Release of vasoconstrictor substances (paracrine & autocrine agents) from
Platelets (i.e., serotonin & thromboxane A2)
Traumatized tissue
Local myogenic spasm initiated by direct tissue damage
Reflex vasoconstriction initiated by activation of nociceptors and other sensory endings
Effects
↓ blood flow and Pressure in the damaged area
Last for many minutes or even hours,
during this time the ensuing processes of platelet plugging and blood coagulation can take place
Physical Factors Immediately Act
to Constrain Bleeding
Immediately after tissue injury, blood flow through the disrupted
vessel is slowed by the interplay of several important
physical factors, including compression or back-pressure
exerted by the tissue around the injured area, and vasoconstriction.
The degree of compression varies in different tissues;
for example, bleeding below the eye is not readily deterred
because the skin in this area is easily distensible.
Back-pressure increases as blood which leaks out of the disrupted
capillaries accumulates. In some tissues, notably the
uterus after childbirth, contraction of underlying muscles
compresses blood vessels supplying the tissue and minimizes
blood loss. Damaged cells at the site of tissue injury
release potent substances that directly cause blood vessels
to constrict, including serotonin, thromboxane A2, epinephrine,

3. FORMATION OF A PLATELET PLUG (temporary hemostatic plug, white plug)
Damaged blood vessel wall
Collagen fibers are exposed to the blood and coated with vWF*
Adhesion of the platelets
Platelet release reaction & activation
Platelet aggregation & plug retraction
Local vasoconstriction
Intact blood vessel wall
Temporary hemostatic (platelet) plug
Secretion of prostacyclin & nitric oxide
+ stimulation
- inhibition + -
Factors that prevent/limit formation of a plug
1. Prostacyclin (prostaglandin I2).
Inhibits platelet aggregation; vasodilator
2. Nitric oxide* (NO).
Inhibits platelet adhesion, activation and aggregation and stimulates local vasodilation
In intact blood vessels platelets are repelled from each other and from the vessel wall due to glycoprotein coat and action of prostacyclin (a derivative of prostaglandins), which is produced by the endothelial cells.
Disruption of the endothelium at sites of tissue injury exposes
a variety of proteins in the subendothelial matrix,
such as collagen and laminin, which either induce or support
platelet adherence. Endothelial cells also rapidly deploy
cellular adherence antigens known as integrins on the
outer surface of their plasma membranes during wound
healing. These adherence antigens are deployed to the cell
membrane by cellular processes set in motion by factors
generated during coagulation or by factors released from
platelets during clotting. In turn, activated endothelial cells
release substances that participate in hemostasis. von
Willebrand factor, a protein synthesized by endothelial
cells and megakaryocytes, enhances platelet adherence by
forming a bridge between cell surface receptors and collagen
in the subendothelial matrix. The protein thrombin,
which is generated by the plasma coagulation cascade, is a
potent activator of platelet adherence and secretion. Ruptured
cells at the site of tissue injury release adenosine
diphosphate (ADP), which causes platelets to aggregate at
the damaged site. These aggregates effectively stop the
flow of blood from the ruptured vessels.
* Von Willebrand factor, a protein synthesized by endothelial
cells and megakaryocytes, enhances platelet adherence by forming a bridge between cell surface receptors and collagen in the subendothelial matrix.

4. FORMATION OF A PLATELET PLUG (cont.)
Stage 1. Platelets adhesion
A. vWF - von Willebrand factor (soluble plasma protein) binds to collagen of subendothelial matrix
Failure of this step may be due to:
- Absence of von Willebrand factor
- Malfunction of collagen - Scurvy
B. vWF exposes multiple intrinsic binding sites for the platelet specific membrane glycoprotein Ib (GPIb)
vWF binds to glucoprotein Ib receptors of platelets and to collagen

5. FORMATION OF A PLATELET PLUG (cont.)
Stage 2-3 Platelets release reaction and activation.
Binding of the platelets to the collagen → Release of agents from secretory granules (degranulation) – serotonin, adrenaline, several clotting factors, thromboxane A2, tissue factor and ADP
Serotonin, adrenaline and ADP act locally → changes in the metabolism, shape, and surface proteins of the platelets.
Serotonin and thromboxane A2 stimulate local vasoconstriction

6. FORMATION OF A PLATELET PLUG (cont.)
Stage 4: Recruitment and loose platelets aggregation
ADP and thromboxane A2 stimulate adhesion of the next layers of platelets (recruitment) through a positive feedback mechanism and formation of a platelet plug inside the vessel
Platelets begin to swell, assume irregular form with numerous pseudopods. Contractions of the contractile proteins facilitate release of the secretory granules.
Failure of this step:
- Insufficient number of platelets
- Dysfunctional platelets (prior activation occurs during cardiopulmonary bypass, storage, exposure to aspirin, uraemia and acute and chronic alcohol exposure)

7. FORMATION OF A PLATELET PLUG (cont.)
Stage 5- irreversible platelet aggregation
Destruction of the platelets membrane (stimulated by thrombin) → release of BAS from thrombocytes → secondary vasoconstriction
Release of factor 3 (platelet thromboplastin) facilitates activation of blood coagulation
Stage 6. Plug retraction
Contraction of actin and myosin in the aggregated platelets → compression and strengthening of the platelet plug

9. BLOOD COAGULATION (CLOTTING)
Is the transformation of the blood into a solid gel (a clot or thrombus)
Occurs locally around the platelet plug; supports and reinforces the plug
Requires 12 plasma clotting factors and 12 platelet clotting factors
Involves a cascade of biochemical reactions in which each factor that is activated in turn activates the next factor
The fundamental reaction is conversion a soluble protein, fibrinogen to an insoluble protein, fibrin

10. PLASMA CLOTTING FACTORS
In coagulation a series of plasma proteins called blood-clotting factors play major roles.
Most of these are inactive forms of proteolytic enzymes.
When converted to the active forms,
their enzymatic actions cause the successive, cascading reactions of the clotting process.

11. 3 PHASES OF BLOOD COAGULATION
Formation of a complex of activated substances - prothrombinase (prothrombin activator)
Formation of active thrombin from prothrombin
Is catalyzed by prothrombin activator
Formation of insoluble fibrin from soluble fibrinogen
Is catalyzed by thrombin

12. PHASE 1 – FORMATION OF PROTHROMBINASE
Is divided into 2 pathways
Extrinsic pathway:
Starts with contact of blood with a traumatized vascular wall or extravascular tissues.
Requires a cellular element outside the blood – tissue factor (tissue thromboplastin).
Is fast and explosive (15 sec).
Intrinsic pathway:
Starts with trauma to the blood cells or blood contact with collagen of a traumatized vascular wall.
Blood contains all factors, which are necessary for coagulation.
Is slow (1-6 min).
Note that the two pathways merge at factor Xa. Formation of prothrombin activator is the rate limiting reaction in blood coagulation.
Prothrombinase

13. PHASE 2 – FORMATION OF ACTIVE THROMBIN
- Catalyses the formation of fibrin
- Activates factor XIII
- Promotes platelets activation and aggregation
- Activates protein C, which inactivates factors VIIIa and Va
- Synthesis in the liver requires vitamin K
Contributes to the activation of factors XI, VIII (intrinsic pathway) and V – positive feedback effect on its own formation
Prothrombinase

14. PHASE 3 – FORMATION OF FIBRIN
Thrombin catalyses release of 2 pairs of polypeptides from each fibrinogen molecule and formation of fibrin monomers
Ca++ and platelet factors are also required
Monomers join together to form insoluble fibrin polymers – a loose mesh of stands
Stabilization of fibrin – formation of covalent cross-bridges, which is catalyzed by factor XIII (+ Ca++)
In the early stages of polymerization, monomers molecules are held together by weak non-covalent hydrogen bonds and fibers are not cross-linked with one another → weak reticulum of fibers can be easily broken.

15. FINAL EVENTS OF HEMOSTASIS
Fibrin forms a meshwork, which supports the platelet plug
Clot occludes the damaged blood vessel and ↓ or stops bleeding
Retraction of the clot due to contraction of fibrin fibers and contractile proteins of the platelets
↑ clot density
Occlusion of the damaged vessel
Bringing the edges of wound together → facilitation of wound heeling
Fate of the blood clot
Invasion by fibroblasts → formation of connective tissue through the clot
Fibrinolysis and destruction of the clot
Note that a clot consists of fibrin (the most essential component of the clot), platelets, RBC and WBC. Note that further heeling of the wound involves proliferation of fibroblasts, formation of connective tissue, scar formation and regeneration of the endothelium.

16. Overview of Hemostasis and Tissue Repair
Damage to
wall of
blood vessel
Tissue factor
exposed
Intact blood
vessel wall
Reinforced
platelet plug (clot)
Fibrin slowly
dissolved by
plasmin
Clot dissolves
Collagen
Platelets aggregate
into loose platelet
plug
Temporary
hemostasis
Cell growth and
tissue repair
Vasoconstriction
Platelets
adhere and
release
platelet
factors
Thrombin
formation
Coagulation
cascade
Converts
fibrinogen
to fibrin
Figure 16-10

17. ROLE OF VITAMIN K IN CLOTTING
Vitamin K acts as a cofactor of the enzyme γ-glutamyl carboxylase
Is required for γ carboxylation in the liver of
Prothrombin and factors VII, IX and X
Proteins S and C (natural anticoagilants)
γ carboxylation (introduction of a carboxylic acid group) of certain glutamate residues in target clotting factors → binding sites for Ca++ and PF3
most of clotting factors are synthesized by the liver. Therefore, liver diseases (i.e., hepatitis, cirrhoses, atrophy) depress the clotting system. Decreased dietary intake of vit K has limited consequences on blood clotting because Vit K is continuously synthesized by the intestinal flora. Note that Vit K is fat soluble and requires fats for absorption. Lack of the bile decreases fat digestion and absorption.
Note that most of clotting factors are synthesized by the liver. Therefore, liver diseases (i.e., hepatitis, cirrhoses, atrophy) depress the clotting system. Decreased dietary intake of vit K has limited consequences on blood clotting because Vit K is continuously synthesized by the intestinal flora. Note that Vit K is fat soluble and requires fats for absorption. Lack of the bile decreases fat digestion and absorption.

18. ROLE OF Ca++ IN COAGULATION
Ca++ is required for all steps of coagulation (except first 2 steps of the intrinsic pathway)
↓ in the plasma [Ca++] below the threshold level for clotting → ↓ blood clotting by both pathways

19. ROLE OF THE PLATELETS IN COAGULATION
Activated platelets
Display specific plasma membrane receptors that bind several of the clotting factors → several cascade reactions take place on the surface of activated platelets
Display phospholipids (platelet factors), which act as cofactors of the bound clotting factors

20. ROLE OF THE LIVER IN BLOOD COAGULATION
Synthesis of the plasma clotting factors
Synthesis of the bile salts, which are required for intestinal absorption of lipid soluble vitamin K
Note that most of clotting factors are synthesized by the liver. Therefore, liver diseases (i.e., hepatitis, cirrhoses, atrophy) depress the clotting system.

21. contact activation pathway
formerly known as the intrinsic pathway
tissue factor pathway
formerly known as the extrinsic pathway

22. FIBRINOLYTIC SYSTEM
Fibrinolysis - dissolution or disposal of blood clots
Fibrin is digested by an enzyme, plasmin (fibrinolysin) into fibrin degradation products
Plasmin also degrades factors Va, VIIIa and GPIb
In the blood, plasmin is present as an inactive precursor, plasminogen
Plasminogen is activated by plasminogen activators
Adrenaline, urokinase, thrombomodulin-thrombin complex, kallikrein, tissue plasminogen activators (t-PAs)
t-PAs are secreted by the endothelial cells
urokinase is produced by kidney.
Plasminogen activators
Plasminogen
Plasmin
Fibrin
Soluble fibrin fragments
Note that urokinase is secreted by the epithelial cells in the kidneys. Note that plasmin also digests fibrinogen, factors V, VIII & XII and prothrombin.
The fibrin formed within blood vessels is gradually dissolved to restore the fluidity of the blood.
The process of liquefaction or lysis of the fibrin is called fibrinolysis
There are two types of plasminogen activator
Vessel activator.
Some clotting factors in the intrinsic pathway, such as XIIa, XIa, prekallikrein , HMW kininogen, IIa (thrombin) et al.
Tissue activator.
Released by the injured tissue and endothelium
tissue-type plasminogen activator (t-PA)
urokinase synthesized by the kidney
Plasmin is a most powerful proteolytic enzyme.
digest the fibrin, fibrinogen, Factor V, VIII, prothrombin and Factor XII

23. Fibrinolysis Clinical application-
Human t-PA is produced by recombinant DNA technology and available for clinical use.
lyses clots in the coronary arteries if given to patients soon after the onset of myocardial infarction.
Streptokinase(from bacteria-streptococcci) and urokinase are also fibrinolytic enzymes
used in the treatment of early myocardial infarction

24. ANTICLOTTING MECHANISMS
Removal of activated clotting factors from the blood by the liver
Factors that reduce the adhesiveness of platelets
The smooth lining of the intact vessel walls
Mucopolysaccharides on the surface of endothelial cells (glycocalyx) – repulsion of clotting factors and platelets
Circulation of the blood
Antiplatelet-aggregation effect of the prostacyclin by the intact endothelial cells
Note that blood does not normally coagulate in undamaged blood vessels. This is due to the action of intravascular anticlotting mechanisms.

25. ANTICLOTTING MECHANISMS: Natural anticoagulants
Antithrombin III (antithrombin-heparin cofactors)
Is a plasma α globulin
its binding to heparin increases its activity.
Inactivates thrombin and some other clotting factors (IX, X, XI, XII)
Heparin
Is produced by the mast cells and blood basophils
By itself, it has little or no anticoagulant property,
but when it combines with antithrombin III, it increases a hundred-fold the effectiveness of antithrombin III
Activated protein C
Inactivates factors Va and VIIIa and activates plasminogen

26. NATURAL ANTICOAGULANTS (cont.)
Thrombin/thrombo-modulin/protein C pathway
Endothelial cell
Thrombomodulin
Thrombin
Protein C
Activated Prot C
Protein S
VIIIa VIII
Inactivation of inhibitors of plasminogen activator
Va V
Plasminogen Plasmin
Thrombin Fibrinolysis
Thrombomodulin is a thrombin-binding endothelial cell receptor
Binds thrombin and inactivates it
Complex of thrombin+thrombo-modulin binds protein C and activates it
Protein C in collaboration with protein S inactivates factors Va and VIIIa and activates plasminogen and fibrinolysis

27. ANTICLOTTING MECHANISMS: SUMMARY
Tissue factor pathway inhibitor
AT III-Heparin
Proteins C & S

28. DRUGS THAT INHIBIT BLOOD CLOTTING (ANTICOAGULANTS)
Heparin
Coumarin derivatives (i.e., warfarin)
Block stimulatory effects of vitamin K on synthesis of clotting factors II, VII, IX, and X by the liver (inhibit epoxide reductase which activates vit K in the liver: K → K1)
Aspirin
Low doses inhibit prostaglandins and thromboxanes synthesis by the platelets → inhibition of platelet release reaction and platelet aggregation
Is effective in preventing of heart attack and reduction of the incidence of sudden death
Note that some rat poisons contain Warfarin.

29. IN VITRO INHIBITION OF BLOOD CLOTTING
Keeping of blood in seliconized containers – prevention of contact activation of platelets and factor XII
Substances that bind ionized calcium to produce un-ionized calcium compound or to form insoluble salts with calcium
Sodium citrate or oxalate
Ammonium or potassium citrate
EDTA (ethylenediaminetetraacetic acid)
Is ability to "sequester" di- and tricationic ions (Ca2+ & Fe3+)
Is widely used as an anticoagulant for blood samples for complete blood count/full blood examination
Heparin
Note that citrate anticoagulants have an important advantage over the oxalate anticoagulants – they are less toxic. Therefore, moderate amounts of citrate can be injected intravenously without causing toxic effects. Citrate is rapidly removed from the blood by the liver and metabolized for energy or for glucose synthesis. In case of the liver damage, the citrate ions may not be removed from the circulation quickly enough and they can depress calcium level in the blood → tetany and convulsive death.

30. PROTHROMBIN TIME (protime, PT test)
Measures the clotting time of plasma from the activation of factor VII, through the formation of fibrin clot
Assesses the integrity of the extrinsic/tissue factor pathway and common pathways of coagulation (factors VII, X, V, II, I)
The PT test is widely used to monitor patients taking anticoagulants as well as to help diagnose clotting disorders
The PT test is widely used to monitor patients taking anticoagulants as well as to help diagnose clotting disorders.

31. PROTHROMBIN TIME (cont.)
Depends on [prothrombin] in the blood
Normal range 12 – 14 sec
Increased
↓ prothrombin (less than 10% of normal)
Deficiency of fibrinogen or factors V, VII, or X
Therapeutic anticoagulants (i.e., heparin, warfarin, aspirin), some drugs (i.e., antibiotics, anabolic steroids, estrogens, etc.)
Liver diseases
Vit K deficiency
Disseminated intravascular coagulation
Decreased
Vit K supplementation
Thrombophlebitis
Newborns normally have prolonged PTs in comparison with adults. However, newborns and infants do not normally experience bleeding, because a balance between procoagulants and natural anticoagulants is maintained. A PT time that exceeds approximately two and a half times the control value (usually 30 seconds or longer) is grounds for concern, as abnormal bleeding may occur.

32. ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT)
Assesses the integrity of the intrinsic and common pathways of coagulation
Measures the clotting time of plasma, from the activation of factor XII by a reagent through the formation of fibrin clot
Normal range 25 – 38 sec
Prolonged time
Use of heparin
Antiphospholipids antibodies
Coagulation factors deficiency (intrinsic and common pathways; i.e., hemophilias)
Apart from detecting abnormalities in blood clotting, it is also used to monitor the treatment effects with heparin. Heparin or warfarin causes both the PT and aPTT to become prolonged because both drugs inhibit factors in the final common pathway.

33. 2 TYPES OF ABNORMALITIES OF HEMOSTASIS
Excessive bleeding (hemorrhagic disease) caused by deficiency of a clotting factor/s or platelets
Excessive clotting: thrombosis, embolism, disseminated intravascular coagulation

34. CONDITIONS THAT CAUSE EXCESSIVE BLEEDING
Vitamin K deficiency
Deficiency of clotting factors (i.e., hemophilia)
Deficiency of thrombocytes – thrombocytopenia
Deficiency of von Willebrand factor

35. VITAMIN K DEFICIENCY Results from
↓ intestinal absorption of fats due to ↓ bile secretion (i.e., liver disease or obstruction of the bile ducts)
↓ dietary intake of vit K (limited importance)
Results in
↓ hepatic gamma carboxylation of
Prothrombin
Factors VII, IX and X
Protein C and S
Bleeding tendency
Prolonged prothrombin time and partial thromboplastin time
Normal platelets count and serum fibrinogen split products
Decreased dietary intake of vit K has limited consequences on blood clotting because Vit K is continuously synthesized by the intestinal flora. Note that Vit K is fat soluble and requires fats for absorption. Lack of the bile decreases fat digestion and absorption. Note that Vit K is often given to patients with liver diseases before performing a surgical procedure. Note that without gamma carboxylation clotting factors are unfunctional.

36. HEMOPHILIA Is a hemorrhagic disease that results from deficiency of
Factor VIII (the smaller component) - hemophilia A or classical
Factor IX – hemophilia B, Christmas disease
Factor XI – hemophilia C
Is a genetic disease
Hemophilia A and B are sex linked (X chromosome)
Occur in males
Females are hemophilia carriers
Results in ↑ aPTT (PT, thrombocytes count, fibrin split products are normal)

37. THROMBOCYTOPENIA Petechiae – small punctate hemorrhages(1-3 mm)
Low thrombocytes count (below /m l) → poor plug formation, deficient clot retraction, deficient platelet phospholipids, poor constriction of ruptured vessels → bleeding tendency from many small venules and capillaries
Multiple hemorrhages in the skin and mucous membranes – thrombocytopenic purpura
Petechiae – small punctate hemorrhages(1-3 mm)
Echymoses - large hemorrhages (bruises)
Other causes of purpura
↓ plasma level of 1 or more clotting factors
↑ fragility of capillary walls (congenital, Vit C deficiency, adrenal failure, toxins, drugs, allergic reactions)
Thrombocytopenia can be treated with fresh whole blood transfusion and splenectomy.

38. von Willebrand’s disease
Is the most common genetic bleeding disorder
Results from defect in vWF ( quantitative or functional)
Results in combination of
Platelet function abnormality (vWF) - impaired adhesion
Clotting factor deficiency (factor VIII) - ↑ aPTT (PT is normal)
Remember:
vWF is normally produced by endothelial cells and megakaryocytes 38

39. THROMBO-EMBOLIC CONDITIONS
Thrombosis - blood clotting within the CVS which obstruct the blood flow through the CVS (Should be distinguished from extravascular clotting, clotting in wounds and clotting that occurs in the CVS after death). Thrombosis is rather a pathological condition.
Common causes
Roughened endothelial surface (i.e., atherosclerosis, infections, traumas)
Slow blood flow
Hypercoagulobility
Acquired refers to transient or acquired conditions that increase the tendency to clot. This might include antiphospholipid antibodies or a temporary hypercoagulable state such as pregnancy. Also, advanced carcinomas of the pancreas or lung may produce a hypercoagulable state.
Congenital refers to hereditary conditions that increase the tendency to clot. These include Factor V Leiden, prothrombin ,protein C, protein S and antithrombin deficiencies

40. Consequences
Formation of emboli (thromboembolism) – braking down of the thrombus and spreading of its particles particles throughout the CVS
Thrombosis in the left side of the heart and large arteries → emboli in the brain, kidneys, etc
Thrombosis in the venous system and in the right side of the heart → emboli in the pulmonary circulation

41. DESSIMINATED INTRAVASCULAR COAGULATION
Reasons
Large areas of necrotic tissue (release of tissue factors into the blood)
Septicemia (activation of clotting by circulation bacteria and bacterial toxins)
Consequences
Consumption coagulopathy
↓ fibrinogen, thrombocytopenia
↑ fibrin split products
↑ PT and PTT

42. CHALENGE YOURSELF
A baby is born prematurely at 28 weeks gestational age with a birth weight of 1200 g. A few weeks after birth his mother noticed a bleeding tendency in the infant. Blood test revealed a low prothrombin level. Which vitamin can be given to the baby to reduce or to prevent the bleeding tendency?
a. Vitamin B12
b. Vitamin B6
c. Vitamin K
d. Folic acid
e. Vitamin A C
Taking aspirin every day can reduce the risk of heart disease because
a. it is a powerful vasodilator
b it stimulates fibrinolysis
c. it prevents atherosclerosis
d. it loosens atherosclerotic plaque on arterial walls
e. it prevents platelet aggregation E