Haemostasis8

The injury to the vascular endothelium causes disruption of the endothelial cells and brings the platelets into contact with underlying subendothelial tissue. Platelets get adhere to subendothelial tissue. The platelets are activated and adhere to each other to form a haemostatic platelet plug.

The traces of plasma clotting proteins are exposed to subendothelial tissue and the coagulation mechanism is initiated.

Processes also set in motion to limit the growth of platelet plug and fibrin clot. From the injured endothelial cells a prostaglandin called prostacyclin is released which impedes platelet aggregation. Plasma proteinase inhibitors prevent the activated enzymes of coagulation.

Endothelial cells also release plasminogen activators that activate plasminogen bound to fibrin clot and plasmin is formed with the clot. This plasmin formed dissolves the (fibrinolysis) fibrin strands. These soluble degradation products re-enter the circulation. For number of days fibrin clot formation and fibrinolysis go on as balanced reactions at the injured site.

Meantime proliferation of smooth muscle cells and fibroblasts, deposition of new connective tissue matrix, ingrowth of lining endothelial cells repair the vessel wall. These haemostatic mechanism prevent excessive bleeding after minor tissue injuries of daily life. Platelet plugs are particularly useful to prevent bleeding from capillaries and venules, in erosions of the mucosal injury.126

When there is failure of clot formation or due to excessive fibrinolysis, a trivial injury may cause excessive bleeding (e.g.) Haemophilia.

von Willibrand disease is a genetic disorder due to deficiency of vWF.

Endothelial cells synthesis vWF as dimmers which undergo further complex processing inside the cell within a large intracellular organelle called Weibel - Palade body and vWF multimeres formed and secreted into the plasma. In the plasma vWF is present as multimers. In von Willibrand disease the plasma level vWF multimers level is reduced. Normal plasma vWF is also synthesised by megakaryocytes and normal constituent of platelet granules and secreted along with other granules during activation.

The coagulation factor VIII circulates in the plasma bound to vWF, Plasma level of factor VIII is reduced in von Willebrand disease.

Thrombin promotes clotting of fibrinogen in and around the platelets giving rise to a scaffolding of fibrin to which fused platelet masses adhere.

Shape change and initial phase of aggregation are reversible and loosely aggregated platelets may break away and re-enter into the circulation.

In hereditary - disorder called Glanzmann's disease GP II - III receptors for fibrinogen on platelet surface membrane are not formed. Hence aggregation does not take place and serious bleeding occurs. (GP= Glycoprotein)

α granules contain proteins albumin and IgG - These are plasma proteins taken up by platelets. The plasma concentration decides the platelet concentration of these proteins. The other group of proteins includes Fibrinogen, vWF, and factor V. Their concentration in platelets is high 127than in the plasma. These proteins participate in haemostatic mechanisms.

The α granule proteins which are absent in the plasma until secreted by activated platelets includes thrombospondin, β-thromboglobulin, platelet factor 4 (PF4), platelet derived growth factor (PDGF) and transforming growth factors (TGF).

Thrombospondin - helps aggregation

PF4 - neutralises the anticoagulant heparin and promote blood coagulation at the site of injury.

PF4, PDGF and TGF are chemoattractants and facilitate chemotaxis of white blood cells, smooth muscle cells and fibroblasts, accelerate wound healing. Thus, they contribute during inflammation and repair.

The essential reaction in coagulation of the blood is the conversion of soluble fibrinogen (hydrosol) into an insoluble protein fibrin (hydrogel). The fibrin forms ultra microscopic crystal-like needles. When as they are deposited, create tenuous, inter lacing filaments within the structure of the protein. The fibrinogen itself is composed of long fibre-like molecules. The immobilisation of the molecules in this manner is called coservation by De Jung. If the clot is permitted to stand for an hour or so, the clot shrinks and serum (yellow fluid) is squeezed out.

Normally the blood remains as fluid in the vessels because:

The coagulation time is the time which the blood takes to clot after it has been shed. Normal coagulation time is 5–10 minutes.

The theory of Morawitz is the simplest of all. When blood is shed, thromboplastins are liberated from injured tissues, disintegrated platelets and from plasma itself and the clotting mechanism is initiated. The thromboplastins act on the prothrombin present in the blood and convert it into active thrombin in the presence of calcium ions. Thrombin thus formed acts on the fibrinogens and converts it into fibrin threads in which solid elements of the blood are entangled. Thus the clot is formed. There is almost an universal agreement as to conversion of fibrinogen to fibrin by the action of thrombin.

This theory is the fundamental one and forms the basis for all modern concepts of coagulation.128

In addition, a factor called platelet factor 3 has been described.

Along with several other cells liver also synthesise factor VIII.

Endothelial cells form EPI.

Endothelial cells are also source for von Willebrand factor. Vit. K. is necessary for synthesis of prothrombin, factor VII, Ix and X which are zymozens of procoagulation serine proteases. Protein C and protein S, recently identified coagulation proteins not been given Roman numbers.

Once active thromboplastins are formed, they act on the inactive prothrombin and convert it into active thrombin in the presence of Ca⁺⁺ ions. This reaction is accelerated by factor V and VII.

Factor XIII acts on transmidase in the presence of Ca⁺⁺ ions. Once thrombin is formed it acts enzymatically to convert the soluble fibrinogen to insoluble fibrin threads.

The key event triggering blood coagulation during haemostasis in vivo is the exposure of blood to tissue factor. Tissue factor is a transmembrane apoprotein that acquires coagulant activity when associated with phospholipid of the cell surface membrane. Tissue factor is present in pericytes and fibroblasts in the adventitia of blood vessels; on fibroblasts of loose connective tissues, and on many other cells in different organs. When the blood vessel is damaged, blood readily comes in contact with cells possessing surface membrane tissue factor activity. Tissue factor is the co-factor for factor VII and factor VII gets bound to tissue during clotting. Normal circulating blood contains traces of factor VII a which allows the formation of VII a/tissue factor complex at the site of vessel damage. This complex activates minute amount of X to Xa which in turn by feedback reaction promotes more formation of VII a/tissue factor complexes. Traces of factor VII a may be present in the circulating blood. Factor VII a once formed will persist in the circulation with an intravascular half time of over 2 hours.

Severe hereditary deficiency of factor VII causes serious bleeding. In moderate deficiency there is no abnormal bleeding (5–20% factor VII).

Reactions leading to the formation of thrombin and conversion of fibrinogen to fibrin.

In the intrinsic pathway contact reactions result in the formation of XI a.

In the extrinsic pathway exposure of blood to tissue factor result in the formation of VII a/tissue factor complex, XI a and VII a/tissue complex come together at the step of activation of IX to IX a.

Hereditary deficiency of factor IX gives rise to serious bleeding tendency - haemophilia ‘B’.

Factor VIII deficiency gives rise to haemophilia ‘A’.

Factor VII a in the factor VIIa tissue factor complex and factor IX a in a IX a/factor. VIIIa anionic phospholipid complex activate factor X to Xa. As increasing amount of factor Xa are formed there is increasing inhibition of factor VIIa/Tissue factor catalytic activity.

Factor IX a cannot activate Xa effectively in vitro unless VIIIa, a source for anionic phospholipid and calcium ions are present. Factor Xa/factor VIII a/phospholipid complex activates X on the platelet surface during normal haemostasis. Moreover IXa/VIII a induced activation of X occurs on the surface of vascular endothelial cells.

Factor X a is the sole known physiological activator of prothrombin. Activation of prothrombin to thrombin requires participation of factor Va, phospholipid and calcium ions. As with factor VIII plasma factor V cannot function as co-factor until activated by proteolysis catalysed by thrombin, factor Xa or both. However, Factor V secreted from activated platelets is partially activated by platelet protease.

Assembly of prothrombin activating complex on the surface of activated platelets takes place by the following steps. Factor Va binds to the platelet surface where it serves as the platelet surface binding site for factor Xa. Factor Xa/factor Va/coagulant phospholipid complex is then formed in which phosphatidyl serine transferred from inner surface to outer surface of platelet membrane. Prothrombin binds to its activator on cell surface by a mechanism requiring participation of calcium ions. Gla groups, and Va, factor Xa cleaves two peptide bonds giving rise to meizo thrombin first then with second cleavage to thrombin proper. Thrombin can move off of all surfaces and catalyse conversion of fibrinogen to fibrin in the surrounding plasma and extra cellular fluid. Thrombin also activates factor XIII to XIII a which promotes cross linking of fibrin molecules to each other and to fibronectin, an adhesive protein of 131plasma and extracellular matrix, binding of fibrin to collagen. These reactions stabilise fibrin and protect it from fibrinolysis.

Deficiency of factor XIII leads to severe bleeding disorder. Thrombin cleaves only small fibrinopeptides from fibrinogen, leaving the remainder of the molecule intact and to polymerise to fibrin strands.

The important points to be noted in understanding the mechanism of coagulation.

The two forms are identical in their action. The total reaction time for blood thromboplastin formation is about 4–9 mts. Tissue thromboplastins formation is rapid, 12–20 seconds. Although thromboplastin formation is spoken of as a single principle, the term refers to an ‘activity’ rather than a precise factor. Thromboplastins in the presence of Ca⁺⁺ activate prothrombin to thrombin.

Antithromboplastins are present in the normal plasma which inhibits thromboplastin formation and inactivate the formed thromboplastin.

Vitamin K deficiency occurs due to lack of vitamin K in food. Destruction of bacterial flora of the intestines by the administration of large amounts of wide spectrum antibiotics. In infants vitamin K deficiency might occur due to lack of bacterial flora in the early infancy.

These factors limit coagulation to the site of injury and prevent the clot from spreading to other areas.

Hereditary deficiency of antithrombin III have increased risk for venous thrombotic disease.

Infants who lack in protein C die shortly after birth of fulminant thrombotic disease.

Other plasma serine proteinase inhibitors are α-2 macroglobulin and heparin co-factor II. These neutralise thrombin. Activated protein C also inactivates the inhibitor of tissue plasminogen activator (TPA) and enables plasmin formation and subsequent fibrinolysis.133

There are several mechanisms by which fibrinolysis is confined to the fibrin surface only. Plasminogen activator inhibitors are PAI-1 and PAI-2, PAI 1 is present in the plasma and secreted by the vascular endothelium. In inflammation its secretion is increased by cytokines. PAI I is also present in the platelet granules. Platelet activation gives rise to more release of PAI-1. PAI-1 inhibits both TPA and UPA. PAI-2 is measurable in plasma only in third trimester of pregnancy and responsible for decreased fibrinolytic activation of pregnancy. Placental macrophages secrete PAI 2. PAI 2 inhibits both TPA and UPA. The plasminogen system not only causes fibrinolysis but also plays a role in cell movement and ovulation.

Plasmin catalysed proteolysis on or around cell surfaces in tissues plays an important role in the inflammatory response in tissue remodelling and in the mechanisms whereby tumor cells invade tissue.

Streptrokinase is given to dissolve clot in myocardial infarction.

Fibrinogen degradation products are called fragments ‘x’ and ‘y’ which are large and ‘D’ and ‘E’ which are smaller degradation products. These can compete with fibrinogen for binding to the GII and III a fibrinogen receptor on platelets. Therefore, they can impede the haemostatic plug formation and lengthen the bleeding time.

It is produced mainly in the mast cells of the liver and to a small extent in the lung tissues and in basophils. It is present in the tissues of liver, muscle, intestinal wall, heart, spleen, thymus. Since it is present in negligible quantity in the blood 0.009 mg/100 ml. Or absent, it cannot be responsible for maintaining the normal fluidity of blood. Since it is present in the tissue mostly it is useful as a local anticoagulant. Its concentration is increased in peptonic shock and anaphylactic shock. Heparin prevents clotting both in vivo and vitro. It is an antithrombin; it prevents formation of thrombin and it inhibits the action of thrombin. Heparin binds with antithrombin III as a co-factor and alters its chemical configuration making it a more powerful antithrombin. The unit (Iu) of heparin is defined as the quantity of heparin which will prevent clotting of 1cc of cat's blood for 24 hours, when kept in cold.

Heparin is used to prevent coagulation, (thrombosis) in the blood vessel during vascular surgery, animal experiments and transfusion.

Chemically heparin is a dextrorotatory polysaccharide made up of hexose amine and hexuronic acid containing sulphuric acid groups. Heparin owes its anticoagulant 134action to its strong electronegative charge due to sulphuric acid groups. Electropositive substances such as toluidine blue and protamine neutralise the negative charge of heparin and antagonise its anticoagulant action.

EDTA-Ethylenediamine Tetra acetic acid or its sodium salt is a powerful chelating agent. Anticoagulant in vitro.

Mechanical contact with rough surface. Gentle shaking promotes clotting whereas vigorous shaking retards clotting because it breaks the network as it is formed.

Snake Venom - Bothrops venom.

Calcium ions, thromboplastins, thrombin, Vit. K and other coagulation factors.

All these prevent the formation of prothrombin activator in time by the intrinsic mechanisms.135

Tests to screen for the adequacy of blood coagulation are the prothrombin time and the activated partial thromboplastin time (APTT).

In disseminated intravascular clotting there is wide spread thrombosis in different blood vessels. If vital vessels are involved it may be fatal.