Blood

Blood

I. Definitions:

A. Blood is a liquid consisted of a yellowish fluid called plasma in which red cells, white cells and platelets are suspended.

B. Once the blood has clotted (coagulated); the remaining liquid is called serum. Thus, serum is plasma without clotting factors.

BLOOD COMPOSITION, PROPERTIES, FUNCTIONS

COMPOSITION OF THE BLOOD

 Blood consists of cells suspended in a clear yellowish fluid called the plasma. The cells constitute 40-45 % of the blood volume and include:

(1) Red blood cells or corpuscles (R.B.Cs) or erythrocytes: Normally, there are about 5 million R.B.Cs per mm³. When they are decreased, the condition is called anemia, and when they are increased, the condition is called polycythemia.

(2)White blood cells or corpuscles (W.B.Cs) or leukocytes: Normally, there are 4000-11000 W.B.Cs per mm³. When they are decreased, the condition is called leukopenia, and when they are increased, the condition is called leukocytosis.

(3) Platelets or thrombocytes: Normally, there are about 300000 platelets per mm³.When they are decreased, the condition is called thrombocytopenia, and when they are increased, the condition is called thrombocytosis.

THE PLASMA

The plasma constitutes 55-60 % of the total blood volume, and it consists of water (90%) and dissolved solutes (10%). The latter include:

§  Organic substances :

Plasma proteins (7.1 %), lipids, hormones, enzymes, nutrients and waste products

(2 %).

§  Inorganic substances (0.9%),which include the various electrolytes e.g. Na⁺, K⁺, Cl^-, HCO₃^-, Ca²⁺ and PO₄^3-.

PROPERTIES OF THE BLOOD

(1)The blood colour is red due to hemoglobin.

(2)The pH of arterial blood is 7.4 (that of venous blood is 7.36).

(3)Blood is opaque due to its cellular elements.

(4) The blood specific gravity is about 1060 (that of the cells is about 1090 while that of the plasma is 1025-1030).

(5) The blood viscosity is 5 times that of water due to its cellular elements and the plasma proteins (the plasma viscosity is 2 times that of water).

(6) The osmotic pressure (O.P.) of the plasma is about 5500 mmHg. It is mostly due to crystalloids (electyrolytes, glucose, urea, etc.), since that of the plasma proteins is only about 25 mmHg (which is called the plasma colloid osmotic pressure or oncotic pressure). The total plasma osmolality is 290-300 mOsm/liter, and the plasma proteins contribute by only 0.5% (i.e. less than 2 mOsm/liter).

Solutions that have the same osmolality as that of the plasma are called iso-osmotic or isotonic solutions e.g. a 0.9 % NaCl solution (which is the same concentration of inorganic substances in the plasma).

On the other hand, solutions that have higher osmolalities than the plasma are hyperosmotic or hypertonic while those having lower osmolalities are hypoosmotic or hypotonic. A 5% glucose solution is initially isotonic, but after glucose metabolism in the body the effects of this solution will be similar to those of hypotonic solutions.

THE HEMATOCRIT (H)

This is the percentage of the blood volume that is made up of cells, so it is also called the packed cell volume (PCV). It is calculated as follows:

Blood cell volume x 100

(H) =---------------------------------------------

Total blood volume

Determination of HEMATOCRIT (H)

A blood sample from the subject is placed in a special tube called Wintrobe tube that contains an anticoagulant and is then centrifuged.

The red cells will be packed in the bottom of the tube while the clear plasma remains above and the white cells form a small buffy layer just above the red cell column (figure 2). (H) is then calculated by dividing the blood cell column by the total blood column and multiplying by 100.

Normally, (H) averages 45 % in adults (40-47 % in males and 36-42 % in females) and 60 % in newly born infants (due to polycythemia).

Figure 2 : The HEMATOCRIT (H) in normal persons, and in anemia and polycythemia.

Factors that affect (H)

HEMATOCRIT (H) is affected by changes in the red cell volume relative to the plasma volume. Accordingly, it is changed as follows:

(1) It is increased in polycythemia and cases of hemoconcentration (e.g. when the plasma volume is decreased due to dehydration).

(2) It is decreased in anemia and cases of hemodilution (e.g. when the plasma volume is increased due to hydration).

(3) Its value in small blood vessels is less than its value in large vessels due to plasma skimming (refer to circulation) and is also greater in venous blood than in arterial blood due to chloride shift (refer to respiration).

Uses of HEMATOCRIT (H)

The determination of (H) is used in

(a) Estimation of the blood volume

(b) Diagnosis of anemia and calculation of some blood indices

 (c) Measurement of the renal blood flow (refer to kidney).

II. Functions of blood:

1-Respiration: transport of oxygen from the lungs to the tissues and of CO2 from the tissues to the lungs.

2-Nutrition: transport of absorbed food materials.

3-Excretion: transport of metabolic waste to the kidneys, lungs, skin, and intestines for removal.

4-Maintenance of normal acid -base balance in the body.

5-Regulation of water balance through the effects of blood on the exchange of water between the circulating fluid and the tissue fluid.

6-Regulation of body temperature by the distribution of body heat.

7-Defense against infection by the white blood cells (lymphocytes) and circulating antibodies.

8-Transport of hormones and regulation of metabolism.

9-Transport of metabolites.

10-Coagulation.

III. Composition of plasma: It consists of:

A. Water: about 90 %.

B. Solids: about 10 %. They include:

Organic matters: proteins, lipids (plasma lipoproteins), carbohydrate (glucose and other blood sugars) , non-protein nitrogenous compounds (amino acids, urea, uric acid, creatinine, etc), hormones, enzymes, ketone bodies and other organic compounds .

Inorganic matters: include plasma electrolytes, Na⁺, K⁺, Ca⁺⁺,Cl^- and carbon dioxide (CO₂),

IV. Plasma proteins:

The concentration of total proteins in human plasma is approximately 6-9 g / dl and they comprise the major part of solids in plasma. There are many protein types, some of them are simple proteins and others are glycoproteins.

A. Types of plasma proteins:

1-Pre-albumin: 25 mg/dl: It is responsible for transport of T3, T4 and retinal.

2-Albumin: 4.5 g/dl:

a) It is the most abundant plasma protein of M.W. 68,000.

b) Its functions are maintenance of plasma osmotic pressure and it acts as transport carrier for calcium, bilirubin, fatty acids and aldosterone in blood.

3. Globulins: 2.7 g/dl: Their M.W. ranges 90, 000-1000, 000. They are further subclassified into:

a) a-1-Globulins: prothrombin (for blood clotting), Retinol binding globulin (for retinol transport), transcortin (for cortisol transport), vitamin D-binding globulin (for vitamin D transport), Ct,-antitrypsin, Ct,-acid glycoprotein and a1-fetoprotein.

b) a-2-Globulins: They include: ceruloplasmin (for copper transport), haptoglobin (for plasma hemoglobin binding), a₂-macroglobulin (it has anti protease and transport functions) and thyroxin-binding globulin (for T3 and T4 transport).

c)b-Globulins: They include: plasminogen (for fibrinolysis), transferrin (for iron transport),

C-reactive proteins, b₂­-microglobulin.

d) Gamma globulins: These are antibodies which are: IgG, IgA, IgM, IgE and IgD.

B. Functions of plasma proteins:

1. Maintenance of plasma osmotic pressure, mainly by albumin. Hypoproteinemia leads to edema.

2. Transport functions: many plasma proteins act as carrier proteins of: lipids, hormones (e.g. thyroxin, cortisol), metals. (e.g. calcium, copper and iron) and excretory products (e.g. bilirubin).

3. Defense reactions by immunoglobulins.

4. Coagulation and fibrinolysis.

5. Buffering of H⁺ ions.

6. Special functions, including protease inhibitors e.g.a1-antitrypsin and, a2-macroglobulin.

C. Organs for synthesis of plasma proteins:

1. Liver: All plasma proteins -except gamma globulins-are synthesized in the liver.

2. Lymphocytes: Gamma globulins (antibodies) are synthesized by plasma cells in lymphoreticular system.

D. Albumin / globulin ratio (A/G ratio): It is about 1.6/1. This ratio is inverted in:

1- Liver diseases (due to decreased albumin synthesis)

2- Kidney diseases (due to loss of more albumin than globulins as albumin has smaller molecular weight).

E. Methods of measurement and separation of plasma proteins:

1. Direct chemical measurement e.g. Biuret method for detecting the presence of peptide bonds. This method measures the total concentration of proteins.

2. Measurement of biological activity e.g. enzymatic activity, coagulation properties.

3. Immunological methods using antigen-antibody reactions as in radial immunodifusion and radioimmuno assays.

4. Physical measurements

e.g. nephelometry, where scattered light by protein particles is measured.

5. Measurement after separation by techniques such as electrophoresis, isoelectric focusing, chromatography, ultracentrifugation, precipitation (by salts or alcohol) and dialysis.

THE PLASMA PROTEINS

The plasma proteins include mainly albumin, globulins and fibrinogen, in addition to small amounts of other proteins (e.g. certain hormones, prothrombin and most of the other clotting factors). Their average amount is 7 gm % (6-8 gm %) divided as follows:

(1)Albumin: This is the most abundant plasma protein. Its amount averages 4 gm % (3.5-5 gill %), and its molecular weight (m.w.) is 69000.

(2)Globulins (alpha 1 & 2, beta I & 2 and gamma globulins): These average 2.7 gm % (2.3 -3.5 gm %), and their (m.w.) range from 90000 to 156000.

(3)Fibrinogen: This is the least abundant plasma protein. Its amount averages 0.3 gm % and its (m.w.) are 340000.

METHODS OF SEPARATION OF THE PLASMA PROTEINS

(1)Plasma ultracentrifugation:

This technique leads to sedimentation of the various plasma proteins at different rates (so they can be separated).

(2) Chemical separation:

This method is based on precipitation of the various plasma proteins, and it can be carried out by two methods:

a-Precipitation by salts :

The plasma proteins can be precipitated by addition of different concentrations of certain salts. For example, albumin can be precipitated by full saturation of the plasma with ammonium sulphate while half saturation with this salt precipitates globulins.

b-Fractional precipitation :

This is precipitation of the various plasma proteins at a low temperature by varying the plasma pH and addition of certain salts and alcohol.

(3) Electric separation (electrophoresis):     This is separation of the plasma proteins by passing a constant electric current in the plasma. It is the most accurate method, and is based on the following principles:

a-Proteins are amphoteric substances (i.e. they can ionize as acids or as bases) since they contain both carboxyl (COOH).and amino (NH2) groups.

b-Each protein is neutral at a specific pH called its isoelectric point (lEF), but it ionizes as a base in solutions that are acid relative to its IEP, and ionizes as an acid in solutions that are alkaline relative to its IEP.

The IEPs of the various plasma proteins are around pH 5, and since the blood pH (7.4) is alkaline relative to these IEPs, the plasma proteins ionize as acids (called proteinic acids). These acids dissociate into H⁺ and free proteinate-anions (which combine with Na⁺ forming Na proteinate).

Accordingly, on passing an electric current in the plasma, the proteins migrate towards the anode by varying rates (depending on their molecular weights), where they can be separated on a paper strip (so this technique is called paper electrophoresis).

SITE OF SYNTHESIS OF THE PLASMA PROTEINS

All plasma proteins are synthesized in the liver, except gamma globulin which is synthesized in lymphoid tissues by the plasma cells.

THE ALBUMIN / GLOBULIN RATIO (A/G)

Normally, this ratio is 1.2 when electrophoresis is used for separation of these proteins, and 1.6 -1.7 when separated chemically. The determination of the A/O ratio is important clinically since it is altered by disease as shown in the following examples:

(1)  It is decreased in

(a) Advanced liver disease (due to decreased synthesis of albumin), so it is frequently used as a liver function test.

(b) Severe infections (due to increased synthesis of gamma globulin).

(2)It is increased when the globulin fraction is decreased e.g. in congenital a gamma globulinemia.

SOURCE OF THE PLASMA PROTEINS

This can be investigated in animals by a procedure called plasmapheresis which is carried out as follows: A small amount of blood is withdrawn from the animal, anticoagulated and centrifuged. The plasma is discarded while the cells are reinjected into the animal in an isotonic saline solution. This process is repeated till the protein reserves in the body are exhausted and the plasma protein level. drops to 4 gm %. The animal is then given different types of food, and the rate of synthesis of different plasma proteins and their blood levels are measured. The results of this procedure showed that plasma proteins are primarily formed from food proteins, but they can be formed from tissue proteins if the protein content in food was low.

(A)       Food proteins:

These are the most effective for synthesis of plasma proteins when

(1) Their structure is similar to that of the plasma proteins

(2) They are of a high biological value (i.e. rich in the essential amino acids). Certain proteins favour albumin formation (e.g. those from muscle) while others favour globulin formation (e.g. plant proteins).

(B) Tissue proteins:

These are either fixed tissue proteins that cannot be converted into plasma proteins, or reserve tissue proteins. The latter can be converted into plasma proteins, and are two types:

·       Dispensable reserve proteins:

These are mobilized from the tissues to the liver during starvation where they are used for energy production and plasma protein formation.

·       Labile reserve proteins:

These are structurally similar to the plasma proteins. They are mobilized from the tissues to the bloodstream directly if the amount of the plasma proteins is suddenly decreased e.g. in case of hemorrhage (in the latter case, fibrinogen, globulins, then albumin are then gradually regenerated in that order).

FUNCTIONS OF THE PLASMA PROTEINS

(1)        Hemostatic function:

Fibrinogen is essential for blood coagulation.

(2) They share in production of blood viscosity, especially fibrinogen and globulins (due to their large m.w.) which cause peripheral resistance to blood flow. This helps maintenance of the arterial blood pressure, particularly the diastolic pressure (refer to circulation).

(3) Their osmotic pressure (about 25 mmHg) is essential for reabsorption of fluids from the tissue spaces at the capillary venous ends which maintains the blood volume. 70-80 % of the osmotic pressure is produced by albumin (because of its greater amount and smaller m.w.).

(4) Buffer action: They provide about 15 % of the buffering power of the blood e.g. a strong acid such as lactic acid can be buffered as follows: lactic acid + Na proteinate -> Na lactate + proteinic acid (a weak acid).

(5) Defence (immunity): Gamma globulins are antibodies that attack bacteria, so they are called immunoslobulins

(6)Conservation function: The plasma proteins combine loosely with many substances such as hormones (e.g. thyroxine and cortisol) and minerals (e.g. iron and copper). This serves as a reservoir for these substances and also prevents their rapid loss in the urine.

(7) Control of capillary permeability:

The plasma proteins close the pores in the capillary walls, thus limiting their permeability. This favours development of edema in cases of hypoproteinemia.

(8) Carriage of CO₂:

CO₂ combines with the amino groups of the plasma proteins and is carried as a carbamino compound (NHCOOH).

(9)Nutritional function:

The plasma proteins can be utilized for nutrition of the tissues in cases of prolonged starvation.

(10)Specific functions:

 Certain plasma proteins exert specific functions e.g. the various hormones, the clotting factors and angiotensinogen. Globulins and fibrinogen increase the erythrocyte sedimentation rate by favouring formation of rouleaux shapes of R.B.c.s.

The following; table shows the mainfunctions of various plasma proteins

PLASMA PROTEIN	MAIN FUNCTIONS
Albumin	conservation function and osmotic pressure
Globulins	conservation function and defence (immunity)
Fibrinogen	blood coagulation and blood viscosity

V. Plasma enzymes:

A. Introduction:

1. Enzymes present in plasma are either functional or nonfunctional.

a) The functional enzymes are those, which perform a physiologic function in, blood e.g. lipoprotein lipase and enzymes of fibrinolysis and coagulation.

b) The non-functional plasma enzymes are those, which perform no known physiologic function e.g. lipase and amylase.

2. If a disease causes cell damage of an organ, which produces non-functional enzymes, the plasma levels of its enzymes are elevated and can be used in clinical diagnosis.

B. Types of enzymes of clinical importance:

1. Transaminases (ALT and AST):

These enzymes are present in most tissues, but especially in cardiac muscle and liver.

a) ALT activity: is widely used as a test for diagnosis of hepatocellular damage e.g. acute viral hepatitis.

 b) AST activity:

1) It is also used for diagnosis of hepatocellular damage.

2) It is increased in myocardial infarction. It gets its maximum level after 2 days of attack.

2. Alkaline Phosphatase:

a) It shows its maximum activity in the range of pH 9.0-10.5.

b) Liver, bone, placenta and intestine are important sources of plasma alkaline Phosphatase:

1) Physiological increase: of alkaline Phosphatase, occurs in growing children (bone) and in pregnancy (placenta).

2) Pathological increase: occurs in rickets and hyperparathyroidism (bone) and in obstructive jaundice (liver).

3. Acid Phosphatase: It shows its maximum activity in the range of pH 4-5. The prostate contains high concentrations of acid Phosphatase, and its measurement is used mainly for the diagnosis of prostatic carcinoma.

4. Lactate Dehydrogenase (LD): It is present in most tissues especially liver, heart and muscles. Its activity is increased in hepatitis, myocardial infarction and muscle diseases.

   In myocardial infarction, LD gets its maximum level after 5 days and returns to normal after 5-7 days of attack.

5. Amylase: It is produced by pancreas and parotid glands. Its activity increases in acute pancreatitis and parotitis.

6. Lipase: It is produced by pancreas. Its activity increases in acute pancreatitis and pancreatic carcinoma.

7. Creatine kinase (CK): Also known as creatine phosphokinase (CPK). It is increased in myocardial infarction and in myopathies, in myocardial infarction; it gets its maximum level after 24 hours, and returns to normal level within 2-3 days.

Note: Enzymes for diagnosis of myocardial infarction:

§  First 24 hours: CPK

§  2-3 Days: AST

§  5-7 Days: LDL

8. Cholinesterase (ChE):

a) There are 2 types of the enzyme:

§  Plasma cholinesterase: known as pseudocholinosterase.

§  Tissue cholinoesterase: known as true cholinoesterase.

b) Succinyl choline apnea: Some patients during anesthesia and after administration of succinyl dicholine as muscle relaxant develop prolonged apnea, often lasting for several hours. The plasma of these patients is usually deficient in pseudocholinesterase enzyme essential for hydrolysis of succinyl dicholine.

9. Gamma-glutamyl transferase (GGT):

Also known as gamma glutamyl transpeptidase. It is found in a number of tissues especially kidney and liver. Its activity increases in cholestasis (i.e. impairment of bile flow) and in 70-80% of chronic alcoholics.

VI.Hemostasis and blood coagulation:

A. Hemostasis is the cessation of bleeding that follows injury of blood vessels.

B. Mechanisms responsible for cessation of bleeding: When blood vessel is injured, bleeding stops by the following mechanisms: 1. Constriction of the injured vessel to diminish blood flow.

2. Formation of a loose and temporary platelet plug (white thrombus): At the site of injury: collagens of blood vessels will be exposedà Platelets bind to the collagen (and activated by thrombin or ADP) à Platelets change shape (and in presence of fibrinogen) à Platelets aggregation à Formation of platelets plug àStop bleeding. This mechanism is measured by bleeding time.

3-Formation of fibrin mesh or clot (coagulation); that contains the platelet plug (white thrombus) and / or red cells (red thrombus) forming a more stable thrombus.

4- Partial or complete dissolution of the clot by plasmin (fibrinolysis).

In normal hemostasis, there is a dynamic steady state in which thrombi are constantly being formed (by coagulation) and dissolved (by fibrinolysis).

C. Mechanism of blood coagulation: Two pathways lead to fibrin clot formation, intrinsic and extrinsic pathways:

§  Intrinsic pathway: It occurs in areas without a tissue injury due to either restricted blood flow or in response to abnormal vessel wall. Theoretically, this pathway may be divided into 3 stages:

1, Generation of active factor X (Xa):

a) When blood vessel is disrupted, its collagen will be exposed, Collagen acts as a negatively charged activating surface which activates prekallikrein into kallikerein. Kallikerein activates factor XII into active factor XII (XlIa). The active factor XIIa attacks:

1) Perkallikerein to generate more kallikerein, setting up a reciprocal activation, High molecular kininogen (HMK) participates as non enzymatic accelerator of this reaction.

2) High molecular weight kininogen to generate bradykinin.

 3) Factor XI, in the presence of HMK as a cofactor, activating it into active factor XIa.

b) Factor XIa in the presence of Ca 2+ ions activates factor IX to serine protease active factor IX (IXa),

c) Active factor IXa activates factor X into serine protease active factor X (Xa). This activation is accelerated about 500 folds in the presence of pnospholipids, Ca²⁺ and factor VIlla, (phospholipids, Ca^{2 +}, Factor VIlla and IXa are called tenase complex).

1) This reaction occurs on the platelet surface. 2) Phospholipids are derived from activated platelets. 3) Factor VIII is activated into VIlla by a minute amount of thrombin. It acts as a receptor for factors IXa and X.

4) Formation of factor Xa occurs at the site where the intrinsic and extrinsic pathways start a final common pathway of blood coagulation.

2. Conversion of prothrombin into thrombin:

a) In the final common pathway, factor Xa activates prothrombin (factor II) to thrombin (factor lla). The activation of prothrombin occurs on the surface of activated platelets and requires phospholipids (from platelets) , factors Xa and Va.

b) Factor V is activated by a minute amount of thrombin into factor Va, which then binds with specific receptors on the platelet membrane, and form complex with factor Xa and prothrombin This complex in presence of Ca²⁺ and phospholipids activates prothrombin to thrombin.

3. Conversion of fibrinogen to fibrin:

a)     Fibrinogen (factor 1) is a soluble plasma glycoprotein that consists of 3 non identical pairs of polypeptide chains covalently linked by disulfide bonds.

 b) Thrombin, a serine protease causes conversion of fibrinogen to fibrin by releasing of fibrinopeptide portions (the black areas) of fibrinogen, converting it into fibrin monomer. Fibrin monomers aggregate spontaneously to form fibrin gel, which in the presence of active factor XIIla, thrombin (lIa) and Ca²⁺, is converted to insoluble fibrin clot. This clot traps platelets, red cells and other components to form white or red thrombi. Factor XIII is activated into active factor XIII (XlIla) by thrombin.

Extrinsic pathway

It occurs at the site of tissue injury with the release of tissue factor that acts as a cofactor for active factor VII (VIla).

1.  Factor VII is activated into active factor VII (VIla) by a minute amount of thrombin. Active factor VII acts as serine protease and together with tissue factor, they activate factor X into active factor X (Xa).

2.  Factor Xa then proceeds in the final common pathway as in the intrinsic pathway.

D. Important notes on blood coagulation:

1. Coagulation factors involve factor I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII as well as prekalikerein, and high M.W. Kininogen. The names and numbers of factors are listed in the following table:

Factor	Common name	Comment
(1) I	Fibrinogen	These factors are usually Prothrombin referred by their common names.
(2) II	Prothrombin
(3) III	Platelets phospholipids	These are usually not referred to as coagulation factors.
(4) IV	Calcium
(5) V	Proaccelerin , labial factor, accelerator (AC-) globulin
(7) VII	Procon vertin, serum prothrombin conversion accelerator   (SPCA) cothromboplastin
(8) VIII	Antihemophilic factor A, antihemophilic globulin   (AHG)
(9) IX	Antihemophilic factor B, Christmas factor , plasma thromboplastin component (PTC)
(10) X	Stuart-Power factor
(11) XI	Plasma thromboplastin   antecedent
(12) XII	Hegman factor
(13) XIII	Fibrin stabilizing factor   (FSF), fibrinoligase.

2. Coagulation mechanism proceeds in a sequential enzyme amplification process, which has been called the cascade reaction. The concentration of factor XII in plasma is approximately 3 mg / ml, while that of fibrinogen is 3000 mg / ml, with intermediate clotting factors increasing in concentration as one proceeds down the cascade(=series).

3. Serine protease factors:

a) These are factors II (prothrombin), VII, IX, X, XI, XII and perkallikerein.

b) Serine proteases are those factors (enzymes), which possess serine residues at their active center, and acting by splitting polypeptides.

4. Vitamin K dependent factors:

a) These are factors II (prothrombin), VII, IX and X.

 b) They are synthesized in liver as precursors containing 10-12 glutamic acid residues. These residues are then carboxylated in a reaction requiring vitamin K as a coenzyme to form g-carboxyglutamate residues which have a high affinity for calcium binding.

5. Role of thrombin in blood coagulation: It is responsible for activation of the following factors: I (fibrinogen), V, VII, VIII and XIII.

6. Role of ionized calcium (Ca²⁺) in blood coagulation: It is important for activation of the following factors: II (prothrombin), IX, X and XIII.

7. Role of platelets in blood coagulation:

a) Formation of platelet plug (white thrombus): discussed before.

b) Platelets provide phospholipids on its membrane surface. Platelet phospholipids are also known as platelet factor 3.

c) Platelets have membrane receptors for factor Va which in turn bind factor Xa.

E. Inhibitors of coagulation:

In normal hemostasis, the concentration of active thrombin must be carefully controlled to

prevent spontaneous clots. The natural inhibitors of coagulation provide mechanism to limit clotting to the location of tissue injury. The major inhibitors of coagulation include:

1. Antithrombin III: It is the main coagulation inhibitor of plasma:

a) It inhibits thrombin, factors IXa, Xa, XIa, and XIlla.

b) Its mechanism of action is enhanced by heparin which binds to specific site on antithrombin III, inducing conformational changes and promoting its binding to thrombin and other factors.

Action of antithrombin III.

Addition of heparin makes it easier for thrombin to interact with antithrombin (positive allosteric effect).

2. Heparin co-factor II: Its activity is enhanced by heparin. It inhibits thrombin.

3.a2-Macroglobulin: It is one of plasma proteins. It inhibits thrombin and kallikrein.

4. Protein C and protein S: protein C is vitamin K dependent protein, which inhibits factors Va and VIlla. Protein S acts as a cofactor for activation of protein C.

F. Hemophilia:

1. These are a group of inherited diseases in which one of clotting factors is deficient. Patient suffering from hemophilia shows frequent bleeding even from minor traumas. Tests that measure whole clotting time are all prolonged.

2. Types:

a) Hemophilia A: is the most common type due to deficiency of factor VIII. The disease is a X-chromosome linked disease. It affects only males.

b) Hemophilia B is also present, due to deficiency of factor IX. Hemophilia C Von Wilbrand disease.

3. Treatment of hemophilia is by repeated blood or plasma transfusion. Factor VIII prepared from pooled donor's plasma may also be given. Nowadays, there are trials to produce sufficient factor VIII by recombinant DNA technology.

G. Fibrinolysis:

1. This is the dissolution of clotted blood after their formation by a blood enzyme called plasmin.

2. Plasmin is present in plasma in an inactive form, which is called plasminogen.

3. Plasminogen is activated by a number of activators, which are derived from tissue, plasma or kidney as urokinase and streptokinase enzymes. These factors are used in treatment of recent blood clots as in myocardial infarction.

H. Anticoagulants:

These are substances that interfere with blood coagulation, either in vivo or in vitro, by removal of any factor of coagulation mechanism. They include:

1. Citrate (that binds ionized calcium) and oxalate (that precipitates calcium as calcium oxalate).

2. Defibrination of blood: Removal of fibrin by stirring by a glass rod.

3. Heparin: See the following table.

4. Dicumarol: See the following table.

5. EDTA.

	Heparin	Dicumarol
Origin	Animal (mast cells)	Plant.
Structure	Proteoglycan	Similar to vitamin K.
Mode of action	Inhibits thrombin.	Antagonizes vitamin K.
Onset of action	Rapid.	Late.
Duration	Remains for a short time	Remains for a long time
Antidote	Protamin sulphate	Vitamin K
Site of action	In vivo & vitro	In vivo only

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