1. BLOOD PLATELETS (THROMBOCYTES)
Blood platelets, or thrombocytes, are non-nucleated cytoplasmic fragments derived from megakaryocytes in the bone marrow.
They have a discoid biconvex shape, 2- 4 µm in diameter and µm in thickness.
The normal platelet count is x 103/mm3 of blood. They contain actin and myosin filaments which could contract and shrink the size of the platelets.

2. Platelets (cntd)
Platelets remain in the circulation for 8-10 days. They are removed by the monocyte-macrophage system, particularly in the spleen.
About one third of the platelets in the body are found in the spleen. Splenectomy increases the blood platelet count.

3. Platelets (cntd)
The production of blood platelets (thrombocytopoiesis) is stimulated and controlled by a group of growth factors which include the granulocyte macrophage-colony stimulating factor (GM-CSF), and a group of interleukins (IL-1, 3, 4, 5, 6).
These growth factors are produced by T-lymphocytes as well as other cells.

4. Functions of the platelets
1. Haemostasis:
Platelets are important for haemostasis. This is because:
They aggregate to form platelet plug, which stops bleeding from small wounds.
They release serotonin and thromboxane A2 which constrict local blood vessels.

5. Functions of the platelets (cntd)
They release platelet factor 3 (PL3) which activates blood clotting factors X and II.
They release von Willebrand factor which regulates the circulating level of clotting factor VIII.
They release platelet factor 4 (PL4) which neutralizes heparin.
They release clotting factor V

6. Functions of the platelets (cntd)
2. Clot retraction: After fibrin is formed, platelets get attached to fibrin fibers then contract leading to shrinking of the clot.  
3. Wound healing: Platelets release a polypeptide called “Platelet derived growth factor - PDGF”, which stimulates wound healing. This substance is also released by tissue macrophages and endothelial cells.

7. Abnormalities in platelet number and function
An increase in the number of platelets is called “thrombocytosis” or “thrombocythemia “ It occurs in pregnancy, after hemorrhage, and after surgical operations. The risk of thrombosis is increased.

8. A decrease in the number of platelets is called “thrombocytopenia “
A decrease in the number of platelets is called “thrombocytopenia “. It occurs in cases of aplastic anemia, pernicious anemia, and during menstruation. The platelet haemostatic function is defective. The condition could lead to thrombocytopenic Purpura.
When the number is normal but the platelets are unable to carry out their function properly the condition is called “thrombasthenia”, It is a congenital condition which leads to thrombasthenic purpura

9. Haemostasis
Haemostasis means stoppage of bleeding. It involves all reactions involved in sealing of a wound to prevent loss of blood from the cut vessels.

10. Haemostatic reactions
Injury of a blood vessel initiates the following haemostatic reactions: 1. Vascular reactions.
2. Platelet reactions.
3. Blood clotting (also called blood coagulation).
1. Vascular reactions
Vascular reactions aim at constricting or even obliterating the injured vessels. They include:
(a) Inward rolling of the inner layers of the blood vessel. (b) Elastic retraction of the vascular wall.

11. (c) Vasoconstriction, which is caused by:
i. Reflex sympathetic discharge because of pain.
ii. Local myogenic contraction of the vascular wall in response to trauma. iii. Serotonin (5-hydroxytryptamine; 5-HT) and thromboxane A2 which are released from the platelets.
These reactions are sometimes so powerful so as to constrict a vessel as large as the radial artery and obliterate it completely for several minutes after vascular injury.

12. Haemostasis

13. 2. Platelet reactions
(a) Adhesion: Adhesion is the sticking of the platelets to the exposed sub endothelial collagen fibers. This makes the platelet wall more “sticky”.
(b) -Release:
After adhesion, the platelet membrane allows the release of the following substances:
Serotonin which potentiates vasoconstriction.
Thromboxane A2 which potentiates the vasoconstriction and platelet aggregation.
ADP (adenosine diphosphate) which potentiates platelet aggregation.

14. Platelet factor 3 (PL3): a group of phospholipids which provide a catalytic surface for activation of the clotting factors X and II.
Platelet factor 4 (PL4): a heparin-neutralizing factor.
Clotting factor V.
Von Willebrand factor which is important for initiating the platelet aggregation process.
(c) -Aggregation: Aggregation is the adhesion of platelets to each other.
The aggregated platelets make a “platelet plug” seals the injured vessel

15. 3. Blood clotting (Blood Coagulation)
There are 14 plasma factors plus one platelet factor involved in the process of blood clotting.
Factor
Names I
Fibrinogen II
Prothrombin
III
Tissue thromboplastin (TPL) IV
Calcium

16. V
Labile factor, Proaccelerin
VII
Stable factor, proconvertin
VIII
Antihaemophilic globulin (AHG) IX
Christmas factor X
Stuart-Prower factor XI
Pl. thromboplastin antecedent (PTA)
XII
Hageman factor, contact factor
XIII
Fibrin-stabilizing factor
HMW-K
High-molecular-weight kininogen Ka
Kallilikrein
PL3
Platelet factor 3 (phospholipoprotein)

17. With the exception of factors III (tissue thromboplastin) and IV (Ca2), all the plasma clotting factors are proteins made by the liver.
The platelet factor (PL3) is mentioned above. There is no factor VI. Blood clotting occurs in three steps as follows.

18. (i) Activation of factor x
This step occurs through either of two pathways. The “intrinsic pathway” is a pathway where all the needed clotting factors are found in the blood.
They are found inactive and only need activation .
The “extrinsic pathway” is a pathway where clotting is initiated by a factor not normally found in the blood but provided by tissue extract released by injured tissues.

19. This factor is a protein-phospholipid mixture called “tissue thromboplastin “.
The process of activation of factor X proceeds as shown below.

21. Stages of Blood Coagulation
a = activation
+ = thrombin induces formation of more thrombin

22. The intrinsic mechanism starts with contact activation of factor XII when it gets in contact with the sub endothelial collagen of the injured vessel.
This contact activate factor XII to active factor XII (XIla). Some proteolytic fragments of XIIa act as prekallikrein activator which converts plasma prekallikrein (a β-globulin made by the liver) into active kallikrein.
This step is catalyzed by high molecular weight kininogen (HMWK, an α-globulin made by the liver).

23. kallikrein in the presence of HMWK, catalyzes further activation of factor XII. Factor XII and kallikrein go on auto activating each other.
In vitro, factor XII is activated by contact with electronegative water- wettable surfaces such as that of glass.

24. When factor XIIa is formed, it starts a cascade enzymatic reaction, where factor XIIa activates factor XI to XIa, then factor XIa activates factor IX to IXa.
Factor VIII is found in a complex with von Willebrand factor (a protein made vascular endothelial cells and megakaryocytes) in an inactive form.

25. When it is released from that complex, it becomes activated → VIlla.
Factor IXa releases factor VIII from the complex.
Next, Factors VIlla and IXa combine to make a VIlla/IXa complex. This complex in the presence of Ca2 and PL3 activates factor X to Xa.

26. The extrinsic mechanism starts when tissue thromboplastin (TPL - Factor III) is released in the juice of the injured tissues.
TPL activates factor VII to VIla. Factor VIla in the presence of Ca2, PL3, and TPL activates factor X to Xa.

27. (ii). Activation of prothrombin to thrombin
Active factor X (Xa) converts the inactive prothrornbin (factor II) into the active thrombin (factor IIa).
The reaction is accelerated 100,000 times in the presence of PL3, Ca2, and active factor V (Va).
When thrombin is formed it activates V to Va, then Va activates more prothrombin to thrombin by an autocatalytic mechanism.
Thrombin also helps platelet aggregation, activation of factor VIII and factor XIII.

28. (iii). Formation of fibrin clot
Thrombin acts on fibrinogen (factor I) to form fibrin monomer.
The fibrin monomer makes a soft, unstable clot which is soluble in urea solutions.
The activated fibrin-stabilizing factor (XIIIa) catalyzes the polymerization of the fibrin monomer into a firm, stable clot which is  Insoluble in urea solutions.

29. Remember
Blood clotting is a plasma function. Neither the RBCs nor the WBCs are needed.
The extrinsic pathway for activation of factor X is a rapid one that takes 6-10 seconds. The intrinsic pathway is a slow one that takes 6-10 minutes.
Steps (ii) and (iii) are the same for intrinsic and extrinsic pathways of clotting. They are rapid reactions that take seconds.
Factors I, V, VIII and XIII are consumed during clot formation, but other factors act only as catalysts.

30. Which pathway is taken in vivo, intrinsic or extrinsic?
This depends on the type of wound.
In clear cut wounds with minimal tissue damage (e.g. surgical wounds), the intrinsic mechanism predominates.
In wounds with excessive tissue damage (crush injuries), the extrinsic mechanism predominates.

31. Clot retraction {serum formation}
lf clotted plasma is left for few minutes it retracts (shrinks), squeezing out a yellowish clear fluid called “serum”.
Serum is the remaining fluid of the plasma after clotting.
It is plasma without factors I (fibrinogen), V, VIII, and XIII.

32. Platelets are essential for clot retraction to occur
Platelets are essential for clot retraction to occur. Clot retraction is induced by platelets. Failure of retraction is an indicator that the number of platelets in the circulating blood is low.
The main function of clot retraction is to reduce the size of any intravascular clot (thrombus) to restore the patency of the blood vessel which was blocked by the thrombus.

33. Fibrinolysis (clot lysis)
Fibrinolysis (i.e. dissolution of the clot) starts shortly after the blood clot is formed.
Tissue plasminogen activator is a polypeptide secreted by the endothelial cells.
It converts the inactive circulating α-globulin “plasminogen” (profibrinolysin) into the active proteolytic enzyme “plasmin” (fibrinolysin,).
Plasmin digests fibrin into smaller soluble molecules called fibrin degradation products (FDPs). These steps proceed as follows:

35. During the formation of the clot, a large amount of plasminogen is adsorbed to fibrin and is entrapped inside the clot.
Plasminogen activator diffuses into the clot and activates plasminogen. Plasmin digests the clot. FDPs inhibit thrombin, thus preventing further blood clotting.

36. The activity of the tissue plasminogen activator (TPA) is controlled by a polypeptide released by the endothelial cells called”TPA inhibitor”.
This inhibitor suppresses the activation of plasminogen to plasmin and maintains the normal coagulability of blood.

37. Plasmin can also digest other plasma clotting factors including factors V. VIII, XII and prothrombin.
The presence of free plasmin in the blood could hinder the normal clotting system of the blood leading to hypocoagulability. Circulating plasmin is inactivated by irreversible binding to α2-antiplasmin. a circulating glycoprotein.

38. The kidney produces a plasminogen activator called “urokinase” which is found in urine. It dissolves any clots that may be formed in renal tubules and block them.

39. Streptokinase is another plasminogen activator produced by streptococci bacteria. It can be used clinically to dissolve recently formed clots.
Human TPA is produced now by recombinant DNA techniques and is available for clinical use.

40. Importance of fibrinolysis: Fibrinolysis dissolves minute clots which are frequently formed in the small vessels. Without fibrinolysis, a large number of small vessels would have been occluded. In addition, after sealing a wound, fibrinolysis slowly clears off blood clots from the tissues to help wound healing, and allow reopening of the blocked vessels.

41. Some important factors in blood clotting platelets
Platelets are important for haemostasis because: (a) They form a platelet plug that seals the cut vessel. (b) They release vasoconstrictor substances (serotonin and thromboxane A2).;
(c) They release phospholipids (Platelet factor 3) which are essential for blood clotting.

42. Calcium ions Calcium ions are essential for blood clotting.
Calcium concentration in plasma is 9 — 11 mg/dL. If it falls below 7 mg/dL, manifest tetany occurs, which could be fatal.
Blood clotting needs a calcium concentration of only 4mg/dL.
Therefore blood clotting cannot be prevented in vivo by lowering plasma calcium level (e.g. by injection of sodium citrate or EDTA), because the patient would die of tetany before clotting is prevented.

43. Vitamin k
Vitamin K is essential for the formation of clotting factors II, VII, IX and X by the liver.
Vitamin K deficiency leads to deficiency of these factors in blood and reduce blood coagulability.
In obstructive jaundice (post-hepatic jaundice), bile salts do not reach the intestine. This reduces the absorption of fat and fat-soluble vitamins. Lack of vitamin K absorption leads to vitamin K deficiency, and blood hypocoagulability. Also in chronic diarrhea, fat absorption is impaired → impaired vitamin K absorption → vitamin K deficiency → blood hypocoagulability.

44. Prostaglandins
Thromboxane A2 (TXA2): This is a prostaglandin released from the platelets during the release reaction.
It induces vasoconstriction as well as further platelet aggregation and release → platelet factor 3 which promotes blood clotting.
Aspirin inhibits the formation of TXA2
This reduces the coagulability of blood. Small daily doses of aspirin are useful in reducing the risk of coronary or cerebrovascular thrombosis.

45. Prostacyclin (PGI2): This is a prostaglandin produced by the endothelium and smooth muscles of the vascular wall.
It suppresses platelet aggregation and produces local vasodilatation.
This limits the clotting process and prevents its spread.

46. Factors that prevent spontaneous blood clotting in vivo
The presence of intact, smooth, healthy vascular endothelium.
The presence of a monomolecular layer of mucopolysaccharide on the inner surface of the vascular endothelium which repels the clotting factors and platelets and prevents their activation.
The rapid flow of blood inside the blood vessels. Slow blood circulation increases the risk of contact activation and thrombosis.

47. The clotting factors are present in an inactive form.
The presence of α2-macroglobulin which combines with circulating active clotting factors to make inactive complexes.
Heparin. However, the physiological levels of heparin in blood have insignificant anticoagulant effect.

48. Factors that limit blood clotting when it occurs
Prostacyclin: released from the endothelium and smooth muscles of blood vessels near the clot. It prevents platelet aggregation.
Fibrinolysis
Fibrin and fibrin degradation products (FDPs): They inhibit the action of thrombin. Thrombin is adsorbed to, and inactivated by, fibrin.
This action of fibrin is referred to as ‘antithrombin I”

49. This complex activates protein C, a circulating plasma proenzyme
This complex activates protein C, a circulating plasma proenzyme. Active protein C (protein Ca) is a protease which enhances fibrinolysis.
Also, protein Ca combines with a coenzyme called protein S. The complex inactivates factors Va and VIlla, thus inhibiting thrombin formation.
Activation of protein C: Thrombin makes a complex with a glycoprotein on the endothelial cells surface called “thrombomodulin”.

50. Activation of protein C and its actions to limit the clotting process
Activation of protein C and its actions to limit the clotting process. The process of thrombin inactivation by adhesion to fibrin is called “antithrombin I”. Antithrombin Ill is activated by combining with heparin. Active Antithrombin Ill inactivates clotting factors XIIa, XIa, IXa, Xa, and thrombin.

51. Factors that promote clotting in vivo
Roughening of the endothelial surface of blood vessels (e.g. atherosclerosis).
Slowing of blood flow inside the vessels.
Thrombocythemia (increased number of platelets in blood), e.g. after surgical operations.
Admission of tissue extract into the blood stream (e.g. crush injuries).

52. Anticoagulants
Anticoagulants are substances or methods that prevent blood clotting. They are classified into two categories:
Anticoagulants in vitro:
These are substances or methods which prevent blood clotting outside the body

53. I. Inactivation of Ca2+: This may be done by adding:
1. Sodium, potassium, or ammonium citrate which converts in Ca2+ to non-ionized calcicitrate salts.
This is the most common anticoagulant in vitro.
ii. Sodium or potassium ethylenediamine tetraacetate (EDTA) which convert Ca2+ into non-ionized calcium EDTA.

54. iii. Sodium oxalate which precipitates Ca2+ insoluble calcium oxalate.
iv. A mixture of citrate, dextrose, and phosphate (CD?):
This mixture is used as anticoagulant by blood banks. It has the advantage of preventing the drop in 2, 3-DPG level in the stored blood.

55. V. Sodium fluoride: Fluoride makes a weakly dissociated calcium compound. At the same time it is an enzyme poison. It inhibits the action of the glycolytic enzymes. It is useful in delaying glycolysis if the blood sample is taken for determining the blood glucose level.
2. Defibrination: Removal of fibrin by stirring the blood with feather.

56. 3. Prevention of contact activation: This is done by collecting the blood in a siliconized, or paraffin-lined container. This provides a non-wettable, positively charged surface which prevents contact activation of factor XII and platelets. However, this anticoagulant process is a temporary one that lasts only for about one hour.
4. Heparin: This is used as an anticoagulant in vitro as well as in vivo Rapid cooling of the blood: This inhibits all the enzymatic reactions of clotting.

57. Anticoagulants in vivo
(1). Heparin:
taken as injection.
(2). Vitamin k antagonists:
These are derivatives of coumarin which are taken orally. E.G. dicumarol and warfarin.  

58. Heparin
Heparin is a naturally occurring anticoagulant found in the granules of the circulating basophil leukocytes and mast cells which are found in most tissues, particularly the lungs, from which it extracted commercially.

59. Its clinical importance is due to its use as an anticoagulant drug.
Heparin is used as an anticoagulant in vivo and in vitro.
The anticoagulant effect appears as soon as heparin reaches the blood.
Heparin is administered by injections intravenously or subcutaneously, but never intramuscularly because of the frequent formation of hematomas at the site of injection.

60. Abnormalities of haemostasis
hemophilia Haemophilia is a hereditary X-linked, recessive disease characterized by blood hypocoagulability due to lack of certain clotting factors. A small wound may result in excessive, serious bleeding. The disease is transmitted by females to males, but females do not suffer from it.
So, females are the carriers but the males are the sufferers of the disease.
The disease is due to lack of certain genes on the X-chromosome which express the synthesis of specific clotting factors. Because it is a recessive trait, the presence of specific gene on one chromosome is enough to express the synthesis of the specific clotting factor of that gene. This is why women do not usually suffer from the disease

61. Females carrying the abnormal X-chromosome transmit the disease to half their sons, while half their daughters will be carriers.
A hemophilic father will have normal sons but carrier daughters.
If a haemophilic man marries a haemophilic carrier, which is a very rare coincidence, it is possible in this case to have haemophilic daughters.

63. Types of haemophilia Hemophilia A: Due to lack of factor VIII.
Haemophilia B (Christmas disease): Due to lack of factor IX.
Parahaemophilia: Due to lack of factor V

64. Treatment of hemophilia:
Hemophilia can not be cured, but it can be temporarily corrected by transfusion of purified preparations of the defective factor.
In this case, normal coagulability is restored for several days after each treatment.

65. Purpura
Purpura is a disease characterized by easy bruisability and spontaneous small hemorrhages (petichae).
It is due to lack of platelet activity.
The haemostatic platelet reaction is defective
The skin of the patient shows many small purplish blotches, caused by subcutaneous small hemorrhages giving the disease the name “purpura “.
Blood clots are soft and poorly retracted.

66. There are two types of purpura:
Thrombocytopenic purpura: It is due to a decrease in the number of platelets in the blood.
Thrombasthenic purpura: It is due to abnormally weak platelets.
Platelet number could be normal

67. Tests of haemostasis bleeding time
This is the time taken to stop bleeding from small wounds without clotting. It tests the efficiency of the vascular and platelet haemostatic reactions.
A sharp, pointed scalpel is used to pierce the lobe of the ear. The oozing blood is wiped off by a filter paper every few seconds. The end point is when bleeding stops.
The normal bleeding time is 1-3 minutes. It is prolonged in purpura, but is normal in haemophilia.

68. Clotting time
This is the time taken by a blood sample to clot in vitro. It is a test of the activity of the blood factors involved in the intrinsic clotting mechanism.
A blood sample is taken in a chemically clean glass test tube.

69. The tube is then tilted back and forth every 30 seconds in a water bath at 37oC.
The end point is when the blood clots. The normal clotting time is 6-10 minutes. Clotting time is markedly prolonged in hemophilia (1-12 hrs). It is also prolonged in vitamin K deficiency, in thrombocytopenia and in thrombasthenia.

70. Prothrombin time
This is the time taken by blood to clot by the extrinsic mechanism. It is a test of the efficiency of factors I, II, III, V, VII, X.
(‘NB. The normal times mentioned above for different le1s vary according to different materials and techniques used So. normal. standard times must be determined hi each laboratory)

71. A blood sample is collected on sodium oxalate
A blood sample is collected on sodium oxalate. Calcium chloride and tissue extract are added to the oxalated blood, and then time is measured until clotting occurs. The normal prothrombin time is seconds.  
Prothrombin time is prolonged in vitamin K deficiency, thrombocytopenia, and in Parahaemophilia. It is normal in hemophilia.

72. THE END