Blood when shed, loses its fluidity in few second and becomes jelly like. This phenomenon is called as coagulation. On further keeping the clot retracts to smaller size and gives out clear straw colored fluid called as serum. Serum is plasma without clotting factor. Microscopic features of coagulation first stage granules appear near disintegrating platelets- granules join to form needles- needles join to form threads passing along whole bulk of blood- these threads form a net work in which are entangled R.B.C’s and W.B.C’s. Coagulation is a property of plasma alone not of blood cells. Normal clotting time with lee’s white method is 6 – 17 min.
Morawitz in 1904 described the basic mechanism of blood clotting.
2) Factors involved in clotting mechanism:
I. Fibrinogen: This is factor I. Globulin in nature, mol. Wt. 330,000D. It is distinguished by other plasma proteins by its property of clotting during which fibrinogen converts to fibrin.
II. Prothrombin: It is protein in nature and is present in normal plasma. Molecular weight is 62,7000D, very labile in aqueous solution & in oxalated plasma. Two forms of prothrombin are A & B. Prothrombin A is destroyed by oxygen and is heat labile. B form is removable by Al(OH)3. In human average prothrombin time is 12 sec. It will be longer in deficiency of factor V and factor VII. Prothrombin is manufacture in liver and vit. K is necessary for formation of prothrombin. During clotting it converts into thrombin.
III. Tissue Thromboplastin: It is derived from 2 sources intrinsic and extrinsic. Intrinsic is from plasma and extrinsic from tissue. Intrinsic factor is formed in plasma due to interaction between different plasma proteins such as Hagman factor, plasma thromboplastin antecedent, factor IX, Factor VIII and factor III. Extrinsic thromboplastin is derived from different tissue.
IV. Calcium: Calcium ions help in coagulation. They help in formation of intrinsic and extrinsic thromboplastin and also conversion of prothrombin to thrombin.
V. Proaccelerin or Labile factor: This factor is necessary for conversion of prothrombin to thrombin by intrinsic or extrinsic thromboplastin.
VI. There is no factor VI.
VII. Serum prothrombin conversion accelerator or stable factor: This factor is present in plasma not used up in clotting. It is a protein which remains associated with prothrombin. It accelerates extrinsic and intrinsic thromboplastin formation being activated by extract released from damage tissue. It is formation is retarded after administration of dicoumarin and in deficiency of vit. K.
VIII. Anti-Haemophilic Factor (AHF): This factor helps in formation of intrinsic thromboplastin and it is used in conversion of prothrombin. It is present in plasma and it disappears when clot forms. In haemophilia the defect is not in platelets and release of platelet co- factor 1 or thromboplastin. Deficiency of AHF results in classical haemophilia it is controlled by a recessive gene which is sex linked. Body fails to synthesize this globulin due to the absence of specific enzyme, which is controlled by mutant genes.
IX. Plasma Thromboplastin component (Christmas factor): This factor is responsible for intrinsic thromboplastin formation. Absence of this factor leads to disease which stimulates haemophilia i.e. haemophilia B. It is transmitted through sex linked recessive genes.
X. Stuart factor: This factor has similar properties as factor VII.
XI. Plasma Thromboplastin Antecedent: This factor is activated by activated Hagman factor and leads to formation of thrombin.
XII. Hagman or surface factor: It is a protein which gets activated when it comes in contact with surface. This is in turn activates protein splitting enzyme kallikarin to produce plasma kinins. Resulting affects are increased vascular permeability and dilation of blood vessels.
XIII. Fibrin stabilizing factor: It along with calcium ions, converts soft clot to solid. It also decreases solubility of clot in urea solution. People deficient in this factor present with poor wound healing.
3) Physiology of blood coagulation:
More than 50 substances that effect blood coagulation have been found in the blood and the tissue some that promote the coagulation are called as pro-coagulants and other that inhibit coagulation are called as anticoagulants. Anticoagulants normally predominate in blood and blood does not coagulate but when the blood vessel rupture pro-coagulants become activated and over ride the anticoagulants and then clot develops.
There are 4 phases to haemostasis and thrombosis:
- Constriction of the injured vessel.
- Formation of loose temporary platelet aggregate at the site of injury.
- Formation of fibrin mesh that binds to the platelet aggregate, forming a more stable haemostatic plug or thrombus.
- Partial or complete dissolution of the haemostatic plug or thrombus by plasmin.
A) Activation of platelets:
Platelets bind to collagen at the site of vessel wall injury and are activated by thrombin formed in the coagulation cascade or by ADP released by other activated platelets upon activation platelets change their shape and in the presence of fibrinogen aggregate to form haemostatic plug. In formation of hemostatic plug platelets undergo 3 processes:
- Adhesion to exposed collagen fibers.
- Release of contents.
Platelets adhere to collagen by specific receptors GP Ia-IIa. In this reaction Von- Willebrand Factor is also involved. (Von- Willebrand Factor is a glycoprotein secreted by endothelium with stabilized factor VIII and binds to collagen and sub-endothelium. Platelets bind to Von-Willebrand factor VI a glycoprotein complex). Following are some facts about platelets,
- Platelets circulate in un-stimulated state but during coagulation platelets become activated.
- Thrombin and collagen are two most important platelet activators. ADP is considered to as weak agonist, it causes aggregation of platelet but not granule release.
- Collagen induced activation of platelet phoshoplipase A2 by increased level of cytosolic calcium ions and result in liberation of arachidonic acid from platelet phospholipids, leading to formation of TXA2 which promotes platelet aggregation.
- Anti-platelet drugs like aspirin block platelet cycloxygenase synthesis and inhibit platelet aggregation.
B) 3 types of thrombi:
- White thrombus: it is composed of fibrin and platelets and is relatively poor in erythrocytes. It forms at the site of injured vessel particularly areas where blood flow is rapid.
- Red thrombus: it primarily consists of R.B.C’s and fibrin. It morphologically resembles the clot formed in test tube or in areas where blood flow is retarded or stasis (e.g. veins) with or without vascular injury or it may form at the site of injury or in an abnormal vessel in conjunction with an initiating platelet plug.
- Fibrin deposites: Disseminated fibrin deposits occur in very small blood vessels and capillaries.
4) Clotting pathways:
In an area where there is restricted blood flow or an abnormal vessel wall without injury the initiation of clot formation is by the intrinsic pathway. The initiation of fibrin clot formation in response to injury to tissue is carried out by extrinsic pathway. These both pathways converge in a final common pathway involving conversion of prothrombin to thrombin and thrombin catalyzed cleavage of fibrinogen to fibrin.
Common things in two pathways:
- Both extrinsic and intrinsic pathways lead to formation of fibrin.
- These pathways are not independent.
These pathways involve various proteins such as:
- Zymogens of serine- these are dependent proteases which becomes activated during process of coagulation.
- Trans-glutaminases which stabilize the fibrin clot.
- Regulatory and other proteins.
A) Intrinsic clotting pathway:
Intrinsic pathway involve: Factor XII, factor XI, factor IX, factor VIII, factor X, as well as prekellikerin, high mol. weight kininogen, calcium ions and platelets phospholipids.
Activation of this pathway needs some activation surface. This surface is provided by altered surface of blood surface of blood vessel which is negatively charged. There is no rupture of vessel wall (as in extrinsic pathway) just endothelium surface alteration is there. This is called as ‘contact phase.’
In contact phase, prekallikrein, high molecular weight kininogen, factor XII and factor XI are exposed to negatively charged surface. In vivo collagen provides this site and in vitro glass or kaolin can be used.
- When components of contact phase assemble on the activation surface factor XII is activated to factor XIIa due to proteolysis by kallikrein.
- Once factor XIIa is formed it attacks pre-kallikrein to produce more kallikrein setting up reciprocal reaction activation.
- Activated XIIa factor activates factor XI to XIa and also release bradykinin from high molecular weight kininogen which is vasodilator.
These steps are presented in following flow chart:
Further steps in intrinsic pathway:
- Factor XIa, in the presence of calcium ions activates factor IX to form factor IXa which is a serine protease.
- Activated factor IXa in turn cleaves the Arg-ile bond of factor X to produce Xa which is two chain serine protease.
- Now where is Factor VIII?
Note: Factor IXa, cannot itself activate factor X. It requires the formation of a complex called as ‘Tenase complex’. Factor VIII is the component of this complex. Tenase complex is formed on this surface of activated platelets consisting of calcium ions + IXa + VIIIa+ X + phospholipids. A similar type of complex is formed in next step formed by combination of factor Xa + Va + calcium ions + phospholipids which forms prothrombin activator, the “prothrombinase complex”.
- Minute quantity of thrombin acts as an enzyme for conversion of factor VIII to VIIIa and factor V to Va.
- Reaction containing Gla-containing zymogens requires calcium ions as the Gla residues in the amino terminals regions act as high affinity binding site for Ca++ ions. These reaction are present in factor II, VII, IX and X. (Ca++ is present in the reactions of these factors)
- Factor VIII is not a protease precursor but it is a glycoprotein that acts as a co-factor. This cofactor act as a receptor for IXa & X on the platelet surface. Factor VIII is activated by minute quantity of thrombin which is then inactivated by farther cleavage by same thrombin i.e. factor VIII is activated and deactivated by thrombin.
These steps are presented in following flow chart:
Further steps in intrinsic pathway:
Thrombin formed does 4 things:
- Activates factor VIII to VIIIa.
- Activate factor V to Va.
- Activates Fibrinogen to fibrin.
- Activates Factor XIII to XIIIa.
With conversion of pro-enzymes to active enzymes it acts to convert fibrinogen to fibrin monomer and then to fibrin polymer. It activates factor XIII – XIIIa which helps in cross linkage of fibrin polymer.
These steps are presented in following flow chart:
B) Extrinsic clotting pathway:
Factor Xa is the common factor where both extrinsic and intrinsic pathways converge. Extrinsic pathway involves following factor: Factor VII, factor X, tissue factor and Ca++ ions.
- Activation of the pathway occurs at the site of injury where tissue factor is expressed. At the site of injury tissue factor is expressed which is activated and helps in conversion factor VII to factor VIIa.
- Factor VII (serum prothrombin conversion accelerator) is associated with prithrombin which is present in plasma. It is activated by extract released by tissue damage due to injury.
- Factor VIIa cleaves same arginine- iso-leusine bond of factor X i.e. cleaved by the Tenase complex in extrinsic pathway.
- Factor VII is circulating Gla containing glycoprotein synthesized in liver. It is zymogen. It is also activated by thrombin and factor Xa.
- Activation of factor X (Stuart factor) is the link between extrinsic and intrinsic pathway.
- Another link is complex of factor VIIa + tissue factor in extrinsic pathway. They also activate factor IX to the factor IXa in extrinsic pathway.
C) Final common pathway:
In the final common pathway factor Xa activates prothrombin to thrombin which then convert factor I i.e. fibrinogen to fibrin. Prothrombin like factor X activated by activated platelets on there surface and requires presence of prothrombinase complex. This complex consist of platelet ionic phospholipids, calcium ions, factor Va, factor Xa and prothrombin.
Flow chart showing both intrinsic , extrinsic and final common clotting pathway
5) Inhibitors of coagulation:
These are proteins which inhibit the formation of a thrombus. These constitute a super-family of proteins known as ‘Serpines’. This group functions by combining with proteases and forming alternative high energy substrate that resist complex cleavage. These are,
- Serine protease inhibitors: Anti-thrombin III, heparin co-factor II, and α2 macroglobulin.
- Protein C system: Protein C and protein S.
These inhibitor are consumed up like clotting factors in process of clotting.
- Anti-thrombin III has major anti-thrombin activity contributing about 70% of the capacity of plasma. It also inhibit factor XIIa, XIa, IXa and in particular factor Xa. Inhibition of coagulation factors is greatly enhanced by the presence of heparin. Anti-thombin III is synthesized in hepatocytes and vascular endothelium.
- Heparin cofactor II – It selectively inhibits (Cofactor II) thrombin. Its activity is enhanced by heparin and by other proteoglycans, particularly dermatin sulphate, a major component of vascular wall.
- α2 macroglobulin – It contributes to anticoagulant activity but its physiological significance is not known.
- Protein C- It is vitamin K dependent protein. It inactivates factor Va and VIIIa. It is activated by thrombin and a co-factor present on vascular endothelium termed as thrombomudulin.
- Protein S- It activates binding of protein C to lipid and platelets surface thus enhancing anticoagulant activity.
Note: Cumarin drugs (eg warfarin) which are used as anticoagulant inhibit vitamin K dependent carboxylation of Glu to Gla residues in factor II, VII, IX, X and also in protein C and S. Administration of vitamin K overcomes this inhibition in 12 to 24 hrs.
6) The Palsma Fibrinolyitc system:
This system is used to digest intravascular deposits of fibrin in both large and small blood vessels.
The Plasminogen-Plasmin System- Plasminogen is a glycoprotein of molecular weight of 90,000 which is synthesized in liver. This enzyme not only digests fibrin, but also digests fibrinogen and clotting factors V and VIII.
- Plasmin is a serine protease and any small amount of plasmin that is formed normal physiological conditions is rapidly inactivated by fast acting plasmin inhibitor, α2 anti plasmin. Plasmin that is incorporated in clot, associated with fibrin is protected from α2 to antiplasmin and remains active.
- Plasminogen activators: Plasminogen activators are present in tissue (TPA), in plasma and in urine (urokinase). Tissue plasminogen activator (TPA) is present in endothelium of veins, capillaries, pulmonary arteries and microsomal factors of cells. TPA is released into blood in ischemia, vaso-active drugs and exercise. TPA released is rapidly inactivated inhibitor by complexing. It has half life of 5 minutes.
- Tissue Plasminogen Activators (Altiplase, t-PA): It is a serine protease that is released into the circulation from vascular endothelium under condition of injury of blood vessels, stress and is inactive until it binds to the fibrin. t-PA cleaves plasmingen within clot to form plasmin which in turn digest the fibrin to form soluble degeneration products and thus dissolves clot.
- Urokinase secreted by secretory endothelium (urinary tubules), is involved in dissolving the clot that has been formed in such tubules.
Investigation done in bleeding disorders:
Following investigation are done in bleeding disorders:
- Bleeding time: Time taken for a standardized skin puncture to stop bleeding is calculated. Depending upon method used it varies from 2-9 minutes. Abnormality is found when there is defect in platelet number or function.
- Platelet count: It is obtained on anti-coagulated blood using electronic particle counter. Range is 150 to 450 × 103/mm3. Decreased platelet count is associated with bleeding disorders.
- Prothrombin Time (PT): It measures the adequacy of extrinsic and common coagulation pathway. In this test the tissue factor is replaced by exogenously added source of tissue thromboplastin eg. Brain extract. Calcium ion are provided from outside. Increased PT is present in deficiency of factor V, VII, X, prothombin and fibrinogen. It is calculated in second.
- Partial Thromboplastin Time (PTT): It checks the integrity of intrinsic and common clotting pathway. It is calculated in seconds. Intrinsic pathway requires a contact phase that is provided by the surface of the blood vessels on which surface endothelium is altered. In- vitro it is provided by Kaolin. Cephalin is substitute for platelet phospholipids. Plasma clotting time is checked. Increased PTT indicates deficiency of factor I, II, V, VIII, IX, XI, XII. Any of these factors may be deficient when PTT is increased.
Table showing various hematological tests and clinical conditions associated with them
7) Bleeding disorders:
Hemophilia A: It is one of the most common hereditary bleeding disorder. It is caused by reduction in factor VIII. Factor VIII works as a cofactor for the activation of the factor X. inherited as X-linked recessive trait and occurs in males and homozygous females. Severity of the disease depends upon level of factor VIII. Those with less than 1 percent of the activity develop severe disease, those with 2-5 percent of activity develop moderate disease and those with 6-50 percent of level have mild disease.
Patient with hemophilia-A have typically following findings:
- Bleeding time = Normal,
- Platelet count= Normal,
- PTT= Prolonged.
Hemophilia B: It is clinically indistinguishable from hemophilia-A. Spectrum of mutation found in hemophilia is similar to hemophilia-A, but DNA inversion is different. It is also inherited as an X-linked recessive trait and may occur asymptomatically or with associated hemorrhage. It also has PTT prolonged. Identification can only be done by factor IX assay.
Von-Willebrand disease: In plasma there is a complex made up of factor VIII-vWF. Endothelial cells are major source of plasma vWF. Factor VIII is synthesized separately but they come together circulate in plasma as a unit. Intrinsic Pathway for activation of factor X requires factor VIII pro-coagulant protein. Factor VIII is a combination of factor VIII and vWF. vWF is a large protein and factor VIII is small. 99% of the complexes formed by the vWF. It has got 1b-IX receptor that helps it to bind with collagen and platelets and favors platelet aggregation. Most important function of vWF is to facilitate the adhesion of platelets to sub-endothelial collagen. vWF can be estimated by Ristocetin aggregation Test.
Von-Willebrand disease is one of the most common inherited disorder of bleeding. It is more common than hemophilia-A. It is mainly transmitted as autosomal dominant disorder but rarely autosomal recessive.
Now, vWF helps in formation of platelet plug, so lab finding is prolonged bleeding time in the presence of normal platelet count. More than 20 variants of vWF have been found and have been divided into 2 groups-
Type I and Type III vWF disease: These two types are associated with reduced level of vWF, and Type I=autosomal dominant, Type III=autosomal recessive. Type I is present in almost 70 percent of cases and less severe. Type III is less common and is associated with extremely low level of vWF, so it is severe.
Type II vWF: this type is associated with qualitative defect in vWF. It is autosomal dominant. Present in 25 percent cases that has several subtypes out of which 2A is most common. In this case there is defective multimere assembly. Because vWF stabilizes factor VIII by binding so its loss leads to secondary decrease in factor VIII.
Clinical protocol usually followed in diagnosis of bleeding disorders
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