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Learning Objectives

Define Hemostasis and describe its components


Describe the major events of coagulation
Identify the physiologic inhibitors of coagulation
Describe common clinically significant disturbances of hemostasis and their clinical manifestations
Enumerate the laboratory tests for evaluating hemostasis (bleeding disorders) and state their principles
HEMOSTASIS
Haemostasis or Hemostasis
(Greek: aimstasis, from ama "blood" + stsis "stagnation")
Functions of Hemostasis
1. Arrests bleeding
2. Keeps blood in fluid state
3. Repair and re-establish the blood flow through the injured vessels
Remove hemostatic plug
*If any of the 4 functions of hemostasis is exaggerated or impaired it will cause either thrombosis or hemorrhage respectively;
COMPONENTS of HEMOSTATIC SYSTEM
1. ENDOTHELIAL CELLS
2. PLATELETS
3. CLOTTING FACTORS
1. INTACT ENDOTHELIAL CELLS
SMOOTH surface is not conducive to clot formation
A layer of glycocalyx that repels clotting factors and plateletsors and platelets
Has display membrane proteins (thrombomodulin) that inhibit clotting
store von Willebrand factor in cytoplasmic granules
make prostacyclin ~ inhibits platelet aggregation
SUBENDOTHELIAL CELLS
Contain membrane proteins and extracellular matrix proteins (collagen) that normally do not contact blood
When exposed after injury, platelets aggregate at the site by mediation of von Willebrand factor (vWF) that binds to platelet
receptors and collagen/subendothelial cells
2. PLATELETS
Life span - 9 days (7-10 days)
enucleated cells that are fragments of larger progenitor cells called megakarocytes production primarily regulated by
thrombopoietin and other growth factors
PLATELET PRODUCTION
2. PLATELETS
Normally 2/3 of the platelets released from the bone marrow stay in the circulation; the remaining 1/3 sequestered in a splenic
pool that is freely exchangeable with circulating platelets
Platelet specific granules
1. Dense granules : ADP/ATP, serotonin, calcium
2. Alpha granules:
a. adhesive proteins (von Willebrand factor, fibronectin)
b. coagulant proteins (factor V, fibrinogen)
c. growth factors/selectins (PDGF, P-selectin)
3. Plasma coagulation factors Nomenclature
Plasma coagulation factors have various names but an internationally standardized nomenclature system is using Roman numeral
designations. A lower case a indicates the active factor (e.g. factor IXa)
Roman numerals indicate inactive forms as they exist in the plasma except factors III & IV.
They reflect order of discovery but not the sequence of reaction in the coagulation system
Coagulation Factors
- Factor I Fibrinogen
- Factor II Prothrombin
- Factor III Tissue Thromboplastin
- Factor IV Calcium Ions
- Factor V Labile Factor, Proaccelerin
- Factor VII Stable Factor, Proconvertin
- Factor VIII Antihemophilic Factor
- Factor IX Christmas Factor
- Factor X Stuart-Prower Factor
- Factor XI Plasma Thromboplastin Antecedent
- Factor XII Hageman Factor
- Factor XIII Fibrin Stabilizing Factor
PROENZYMES
Factors XII, XI, IX, VII, X and II (prothrombin) are each activated by proteolytic cleavage to become enzymatically active. The
activated factors are denoted by the subscript "a".
The enzymes of the coagulation cascade all belong to the family of serine proteases exemplified by trypsin.
Vitamin K dependent clotting factors
Factors II, VII, IX and X
COFACTORS
Non enzymatic proteins that is essential for the optimal activity of the enzymes of the clotting cascade Tissue factor, factor VIII and
factor IV are cofactors
EVENTS IN HEMOSTASIS
HEMOSTASIS: 3 phase process
I. Primary Hemostasis
Vascular spasm
Platelet plug formation
I. Secondary Hemostasis
Blood coagulation
Clot organization
I. Tertiary Hemostasis
Fibrinolysis
PRIMARY HEMOSTASIS
I. VASCULAR CONSTRICTION
Reduces the flow of blood from the vessel rupture.
The more a vessel is traumatized, the greater the degree of spasm. This local vascular spasm can last for many minutes or
even hours.
VASCULAR CONSTRICTION FACTORS
The contraction results from nervous reflexes, local myogenic spasm, and local humoral factors from the traumatized tissues and
blood platelets.
The nervous reflexes are initiated by pain that originate from the traumatized vessel or from nearby tissues.
Most of the vasoconstriction results from local myogenic contraction of the blood vessels initiated by direct damage to the
vascular wall.
For the smaller vessels, the platelets are responsible for much of the vasoconstriction by releasing the vasoconstrictor
substance thromboxane A2.
II. FORMATION of PLATELET PLUG
- Platelet adhesion
- Platelet secretion
- Platelet aggregation
FORMATION OF THE PLATELET PLUG
A. Platelet adhesion
It is the binding of platelet to non-platelet surface: sub endothelial collagen
Involves changes from a disc shape to a slightly broader, plate like form to increase surface area
FORMATION OF THE PLATELET PLUG
When platelets come in contact with a damaged vascular surface, such as the collagen fibers in the vascular wall or
damaged endothelial cells, they immediately change their characteristics.
Platelet Changes:
1. Begin to swell
2. Assume irregular forms with pseudopods
3. Their contractile proteins contract forcefully and cause the release of granules that contain multiple active factors;
4. They become sticky so that they stick to the collagen fibers;
5. They secrete large quantities of ADP;
6. Their enzymes form thromboxane A2 (secreted into the blood).
Under electron microscopy
FORMATION OF THE PLATELET PLUG
A. Platelet adhesion
A number of plasma proteins are required for normal platelet adhesion.
Thrombin
Fibronectin
vWF - is the largest component of factor VIII and secreted by platelets and by vascular endothelial cells.
B. Platelet secretion
(platelet release reaction)
It is the release of contents of the granules of platelet:
Primarily ADP stimulates aggregation
Serotonin enhance vasoconstriction
Platelets contain 3 types of secretory granules:
Lysosome containing acid hydrolyses
-granules
o platelet specific proteins (Plt factor 4, - thromboglobulin)
o Platelet derived growth factor
o coagulation proteins found in plasma (fibrinogen & von Willebrands factor)
-granules
o containing ATP, ADP, Calcium & serotonin
C. Platelet aggregation
PLATELET ACTIVATION
SECONDARY HEMOSTASIS
The process of coagulation involves a cascade of reactions i.e. activation of one factor leads to activation of next.

DIFFERENCE BETWEEN EXTRINSIC AND INTRINSIC PATHWAYS
Extrinsic pathway
Explosive in nature once initiated, its speed of occurrence is limited only by the amount of tissue factor released from the
traumatized tissues and by the quantities of Factors X, VII, and V in the blood
With severe tissue trauma, clotting can occur in as little as 15 seconds
Intrinsic pathway
Much slower to proceed, usually requiring 1 to 6 minutes to cause clotting
Calcium ions
Are required for promotion and acceleration of almost all blood clotting reactions
Except: activation of XII and XI (intrinsic mechanism)
Roles played by Thrombin
Positive feedback role of thrombin:
It accelerates the rate of formation of prothrombin activator by activating factors VIII, V & XIII.
Thrombin itself cause further conversion of prothrombin into thrombin. (Amplification Effect)
It also activates Protein C ( anticoagulant).
TERTIARY HEMOSTASIS
Clot retraction- SERUM
FIBROUS ORGANIZATION
(DISSOLUTION OF THE CLOT)
Once a blood clot has formed, it can follow one of two courses:
1) it can become invaded by fibroblasts, which subsequently form connective tissue all
through the clot
2) it can dissolve.
FIBROUS ORGANIZATION
(DISSOLUTION OF THE CLOT)
The usual course for a clot that forms in a small hole of a vessel wall is invasion by fibroblasts, beginning within a few hours after the
clot is formed. This continues to complete organization of the clot into fibrous tissue within about 1 to 2 weeks.
When additional blood coagulates to form a larger clot, such as blood that has leaked into tissues, special substances within the clot
itself usually become activated, and these then function as enzymes to dissolve the clot.
LYSIS OF BLOOD CLOTS:
PLASMIN SYSTEM
When a clot is formed, a large amount of plasminogen is trapped in the clot along with other plasma proteins. This will not become
plasmin or cause lysis of the clot until it is activated.
The injured tissues and vascular endothelium very slowly release a powerful activator called tissue plasminogen activator (t-PA) , so
after the clot has stopped the bleeding, it eventually converts plasminogen to plasmin and removes the clot. (fibrinolysis)
In fact, many small blood vessels in which the blood flow has been blocked by clots are reopened by this mechanism.
Endogenous anticoagulants
Why blood does not clot in circulation?
Physiologic inhibitors of coagulation
Antithrombin III
Activated protein C and protein S
Thrombomodulin
Prostacyclin
1. Antithrombin III (AT-III)
primary function : inactivate Thrombin,
attaches to Thrombin IIa and inactivates it, preventing thrombin from converting fibrinogen to fibrin
binds to activated factors (factors IXa, Xa, XIa and XIIa) rendering them inactive
Antithrombin III (AT-III)
Its action is enhanced and accelerated by the presence of Heparin (either naturally released from basophils or given therapeutically
as an anticoagulant)
Patients with decreased AT-III levels are subject to an increased risk of thromboembolism even in cases of slightly reduced AT-III
levels.
2. Protein C
Produced by liver
Vitamin K dependent
Inactivates factors V and VIII
Stimulates fibrinolysis
Protein C is activated by Thrombin (IIa)
Enhancement of its anticoagulant functions is achieved by Protein S.
3. Protein S
Produced by liver
Vitamin K dependent
Activated by thrombin
Acts as a cofactor to Protein C to enhance its ability to degrade factors V and VIII
Protein C and Protein S
Patients with Protein C and/or Protein S deficiencies have a thrombotic tendency.

Patients also may acquire deficiencies of Protein C and Protein S with liver disease and disseminated intravascular coagulation (DIC).
4. THROMBOMODULIN
Binds with thrombin
Thrombomodulin thrombin complex
Activates PROTEIN C inactivates FACTORS V and VIII
5. PROSTACYCLIN
aka PGI
2

has anti-aggregate action
released from normal endothelial cells in the adjacent vessel
Natural Anticoagulants
Heparin from basophils and mast cells potentiates effects of antithrombin III (together they inhibit IX, X, XI, XII and thrombin)
Antithromboplastin (inhibits tissue factors tissue thromboplastins)
HEPARIN
Mast cells are abundant in the tissues surrounding the capillaries of the lungs and to a lesser extent in the capillaries of the liver.
It is easy to understand why large quantities of heparin might be needed in these areas because the capillaries of the lungs and liver
receive many embolic clots formed in the slowly flowing venous blood; sufficient formation of heparin prevents further growth of
the clots.
Abnormalities of hemostasis
CAUSES OF EXCESSIVE BLEEDING
1. Vitamin K Deficiency
2. Hemophilia
3. Thrombocytopenia
Vitamin K deficiency
Vit K is necessary for production of Prothrombin, factor VII, IX , X & protein C.
Thrombocytopenia
Lethal when PLTs<10,000/L
Bleeding occurs when PLTs<50,000/L
Normal:
150-400,000/L
Disseminated intravascular coagulation (DIC)
Widespread coagulation thrombosis in small blood vessels increased fibrinolysis, and
depletion of coagulating factors generalized bleeding
It may result from:
- bacterial infections (endothelial damage)
- disseminated cancers (release of procoagulants)
- complications of pregnancy
- severe catabolic states
HEMOPHILIA
Occurs only in males
Transmitted genetically by X (female) chromosome as a recessive trait.
prolonged bleeding occurs after trauma
Hemophilia A : Factor VIII deficiency
85%
Hemophilia B: Factor IX deficiency
ANTI-COAGULANTS FOR CLINICAL USE
A. Heparin
B. B. Coumarins: (e.g. warfarin) competes with Vit K
C. Aspirins
A. HEPARIN AS THERAPEUTIC ANTICOAGULANT
When it combines with antithrombin III, this increases effectiveness of antithrombin III in removing thrombin
The complex of heparin and antithrombin III removes several other activated coagulation factors in addition to thrombin, further
enhancing the effectiveness of anticoagulation. The others include activated Factors XII, XI, IX, and X.
Heparin
Administered IV
Causes immediate inhibition of blood clotting
Accelerates the action of ATIII to inactivate Thrombin IIa
Monitored using the PTT test
B. Coumadin (Warfarin, Dicoumarol)
Oral anticoagulant
Takes couple days for effects to show
Inhibits production of vitamin K dependent factors (II, VII, IX, X) (Protein C & S)
Monitored using the PT test (since factor VII has the shortest life and becomes deficient first)
C. Aspirin
Administration results in irreversible inhibition of the platelet enzyme cyclooxygenase, which is needed for proper platelet
aggregation (platelet adhesion is unaffected)
Affects last for the lifetime of the platelets at least seven days
Patients undergoing certain platelet function tests should avoid aspirin ingestion for at least seven days
Tests of coagulation
Laboratory Tests to Evaluate Hemostasis
platelet count
bleeding time
clotting time
aPTT
Prothrombin time
thrombin
PLATELET COUNT
examination of blood smear for platelet numbers
BLEEDING TIME
time it takes for a small skin lesion to stop bleeding;
evaluates the function of capillaries in the hemostatic process
normal bleeding Time: 3 - 6 min
CLOTTING TIME
time it takes for blood to clot in a test tube
Clotting time: 6 - 12 min
Activated Partial Thromboplastin Time (APTT)
time it takes for blood plasma to clot after a lipid substance and are added to the plasma sample;
evaluates the pathway
Clotting factors VIII, IX, XI, XII, X, V, II, fibrinogen
norm: 25-35 secs
Most common cause of prolonged aPTT heparin!!!
PROTHROMBIN TIME
Blood removed is immediately oxalated and Ca
++
& tissue factor are added.
The time taken for coagulation is known as prothrombin time.
evaluates the EXTRINSIC pathway
PROTHROMBIN TIME
The normal PT is 11-15 secs
Prolonged PT :
a deficiency in any of factors VII, X, V, prothrombin (factor II), or fibrinogen (factor I)
The extrinsic factors not measured in the PT test are Factors III (Thromboplastin), and IV (Calcium).
The PT is also used to monitor oral anticoagulant therapy such as warfarin.
PROTHROMBIN TIME
Clinical conditions with prolonged PT:
1. vitamin K deficiency (vitamin K is a co-factor in the synthesis of functional factors II (prothrombin), VII, IX and X)
2. liver disease
3. Warfarin therapy (coumadin)
4. DIC
5. excesive heparin
THROMBIN TIME
Primarily measures the level of fibrinogen
Normal: 14-15 sec
Prolonged TT:
Heparin (much more sensitive to heparin than aPTT)
Hypofibrinogenemia
IMMUNITY
An Introduction
Immunity
Constitutes ALL the physiological mechanism which allow the body to recognize materials that is foreign to itself and to neutralize or
eliminate them
Function of Immunity
1. Protection against microbes, viruses, bacteria, and other uni-cellular and multi-cellular organisms.
2. Elimination of worn-out or damaged body cells.
3. Immune surveillance (cancer)
4. Involved in the process of aging.
5. Major obstacle to successful transplantation of organs.
Classification of Immunity
A. Non-specific Defense Mechanism
- Phagocytes
- Lysosomes
- Basic polypeptides
- Properdin
- Interferons
B. Specific Immune Mechanism
- Humoral Immunity (antibodies)
- Cell-mediated Immunity (sensitized lymphocytes)
Types of immunity
ACQUIRED IMMUNITY
INNATE IMMUNITY
Innate immunity
Results from general processes:
Phagocytosis of bacteria by WBC and tissue macrophages
Destruction of swallowed organisms by the acid secretion of stomach and digestive enzymes
Resistance of the skin to invasion of organisms
Presence of certain chemical compounds in the blood: lysozymes- attacks bacteria and cause dissolution
Basic polypeptides react with certain types of gram (+) bacteria
Complement complex- a complex of proteins activated to destroy bacteria
Natural killer lymphocytes- recognize and destroy foreign cells, tumors and infected cells
Adaptive/acquired immunity
Results from processes directed at a specific organism
The body is able to develop powerful specific immunity against invading agents such as bacteria, viruses, toxins and foreign
tissues
Caused by a special immune system that forms antibodies and/or activated lymphocytes that destroy the specific invading
organism or toxin
Requires weeks or months to develop
Adaptive immunity
Definition of Terms
ANTIGEN
Any substance capable of provoking an immune response of any type in an immunologically-competent individiual.
ANTIBODIES
Plasma proteins synthesized in immune response which are capable of combining with the antigens
CELLULAR SPECIFICITY
Maximal reactivity of the immune cells to the specific antigen, and lessening reactivity to decreasingly-related compounds
Definition of Terms
THE IMMUNE SYSTEM
Used synonymously with the term lymphoid system; the system that is responsible for ALL types of immune response
THE T-CELL DIVISION
The system responsible for the expression of cellular immunity; requires the presence of a thymus gland
THE B-CELL DIVISION
System responsible for the expression of humoral immunity
Schematic Ontogeny of the Lymphoid System
Differences
B-cells
Secretes antibodies
Confer specific immune resistance against bacteria
T-cells
Major carriers of specific immunity against fungi, viruses, parasites and few bacteria, which to survive must live inside cells
Destruction of cancer cells
Rejection of solid tissue transplant
Antibodies
Composed of polypeptide chains, with small amount of amino acids occupying the first positions in the chains = for
SPECIFICITY.
AKA gamma globulins or immunoglobulins
Functions of Antibodies
Neutralization of antigens
Antigen-antibody precipitation
Agglutination of bacteria
Lysis of cellular structure
Opsonization
Classes of Ig
1. IgG - most abundant, can cross the placenta
i. has antibacterial, antiviral, antitoxic activities in vivo
2. IgA - second most abundant
ii. Chief Ig in exocrine secretions
3. IgM - strong cytolytic and complement-fixing; exceed IgG
iii. found predominantly in intravascular pool, larger molecular size; does not tend to cross placenta
4. IgE - has homocytotropic property
iv. it mediates certain allergic responses
4. IgD - present in very small amounts; function not yet known
Classes of immunoglobulins
Complement
Set of proteins in the blood serum
Inactive enzymes that are activated in a definite sequence to catalyze an intricately-linked series of reactions
END-RESULT: cytolysis
Activation of Complement System
Properdin
Also inactive enzymes in the blood serum
When activated, can initiate the complement series in addition to the usual antibody method of activating it.
Interferon
A protein compound that plays a part in producing immunity
Synthesized in the body after viruses have invaded the body cells
Acts on the other body cells to defend them against viruses
Also, retards the growth of cancer cells
Acquired Immunity
Is that which an individual develops by exposure to a foreign substance or organism from some outside source.
A person is usually immune from second attacks of the same disease.
Acquired Immunity
1. Active Immunity
- Produced in response to natural or artificial stimulation of the antibody-producing mechanisms
2. Passive Immunity
- Produced from antibody derived from another individual either by natural transfer or by injection; only temporary, lasting
only for a few weeks to a few months
Clinical Correlation
A. Defects in Development of Immune System
1. Failure of lymphocytes precursors to migrate to thymus, liver and spleen absence of cellular and humoral
immunity
2. Congenital absence of thymus absence of cellular immunity but humoral immunity is normal
3. Agammaglubulinemia B-cells fail to develop, susceptible to bacterial infections
Clinical Correlation
B. Autoimmunity
o lost of immune tolerance
o self antigens recognized as foreign, antibodies attack own cells
o Myasthenia Gravis
o SLE
Clinical significance
Immunization
Used to produce acquired immunity against specific diseases
1. injecting dead organisms no longer capable of causing disease but with intact antigen
Vaccines against Diptheria and pertussis
2. injecting toxins whose toxic nature already destroyed but with intact antigen
Vaccine against tetanus
3. injecting live organisms that have been attenuated
Vaccine against polio, measles, chickenpox