Haemostasis

Haemostasis is the cessation of blood loss from a damaged vessel. A wound causes vasoconstriction, accompanied
by: adhesion and activation of platelets, formation of fibrin. Platelets first adhere to macromolecules in the
subendothelial regions of the injured blood vessel; they then aggregate to form the primary haemostatic plug. Platelets
stimulate local activation of plasma coagulation factors, leading to generation of a fibrin clot that reinforces the
platelet aggregate. Later, as wound healing occurs, the platelet aggregate and fibrin clot are degraded. Over a century
ago, Rudolph Virchow defined three predisposing factors-'Virchow's triad': injury to the vessel wall, and abnormal
coagulability of the blood.
The process of platelet aggregation and blood coagulation are briefed as follows.
1. Formation of haemostatic plug:
 Platelet activation leads to the formation of a haemostatic plug, which stops the bleeding and is subsequently
reinforced by fibrin.
 Platelet function in response to vascular injury includes four phases: (1) adhesion, (2) secretion, (3)
aggregation, and (4) elaboration of procoagulant activity.
 Release of von Willebrand factor from activated platelets and fibrinogen from the endothelium causes
adhesion of platelets at the site.
 Platelets release granular contents, such as adenosine diphosphate (potent platelet aggregator) and
thromboxane A2 (platelet activator and potent vasoconstrictor) that lead to platelet aggregation.
 Activation of platelets results in a conformational change in the IIb III integrin (IIb/IIIa) receptor, enabling
it to bind fibrinogen, which cross-links adjacent platelets, resulting in aggregation.
 A platelet plug is formed that occludes the blood vessel lesion.
 Simultaneously, the coagulation system cascade is activated, resulting in thrombin generation and a fibrin
clot, which stabilizes the platelet plug
2. Coagulation cascade:
 Activated coagulation factors are generated at the site of bleeding on the activated platelets.
 Two pathways, called the extrinsic pathway and the intrinsic pathway involves in the formation of
prothrombinase.
 Prothrombinase converts prothrombin (a plasma protein formed by the liver) into the enzyme thrombin.
 Thrombin converts soluble fibrinogen (another plasma protein formed by the liver) into insoluble fibrin.
Fibrin forms the threads of the clot.
The Extrinsic Pathway
 It is so named because a tissue protein called tissue factor (TF), also known as thromboplastin (throm-bo¯-
PLAS-tin), leaks into the blood from cells outside (extrinsic) to blood vessels and initiates the formation of
prothrombinase.
 TF is a complex mixture of lipoproteins and phospholipids released from the surfaces of damaged cells. In
the presence of Ca2+, TF begins a sequence of reactions that ultimately activates clotting factor X.
 Once factor X is activated, it combines with factor V in the presence of Ca2 + to form the active enzyme
prothrombinase, completing the extrinsic pathway.
The Intrinsic Pathway
 The intrinsic pathway of blood clotting is more complex than the extrinsic pathway, and it occurs more
slowly, usually requiring several minutes.
 The intrinsic pathway is so named because its activators are either in direct contact with blood or contained
within (intrinsic) to the blood; outside tissue damage is not needed.
 If endothelial cells become roughened or damaged, blood can come in contact with collagen fibers in the
connective tissue around the endothelium of the blood vessel.
 In addition, trauma to endothelial cells causes damage to platelets, resulting in the release of phospholipids
by the platelets.
 Contact with collagen fibers (or with the glass sides of a blood collection tube) activates clotting factor XII
(Figure 19.11b), which begins a sequence of reactions that eventually activates clotting factor X.
 Platelet phospholipids and Ca2+ can also participate in the activation of factor X.
 Once factor X is activated, it combines with factor V to form the active enzyme prothrombinase (just as
occurs in the extrinsic pathway), completing the intrinsic pathway.
The Common Pathway
 The formation of prothrombinase marks the beginning of the common pathway. In the second stage of blood
clotting, prothrombinase and Ca2+ catalyze the conversion of prothrombin to thrombin.
 In the third stage, thrombin, in the presence of Ca2+, converts fibrinogen, which is soluble, to loose fibrin
threads, which are insoluble.
 Thrombin also activates factor XIII (fibrin stabilizing factor), which strengthens and stabilizes the fibrin
threads into a sturdy clot.
 Plasma contains some factor XIII, which is also released by platelets trapped in the clot.
 Thrombin has two positive feedback effects.
 In the first positive feedback loop, which involves factor V, it accelerates the formation of prothrombinase.
Prothrombinase in turn accelerates the production of more thrombin, and so on.
 In the second positive feedback loop, thrombin activates platelets, which reinforces their aggregation and the
release of platelet phospholipids.
Clot Retraction
 Once a clot is formed, it plugs the ruptured area of the blood vessel and thus stops blood loss.
 Clot retraction is the consolidation or tightening of the fibrin clot.
 The fibrin threads attached to the damaged surfaces of the blood vessel gradually contract as platelets pull
on them.
 As the clot retracts, it pulls the edges of the damaged vessel closer together, decreasing the risk of further
damage.
 During retraction, some serum can escape between the fibrin threads, but the formed elements in blood
cannot.
 Normal retraction depends on an adequate number of platelets in the clot, which release factor XIII and other
factors, thereby strengthening and stabilizing the clot.
 Permanent repair of the blood vessel can then take place. In time, fibroblasts form connective tissue in the
ruptured area, and new endothelial cells repair the vessel lining.
Role of Vitamin K in Clotting
 Normal clotting depends on adequate levels of vitamin K in the body. Although vitamin K is not involved in
actual clot formation, it is required for the synthesis of four clotting factors.
 Normally produced by bacteria that inhabit the large intestine, vitamin K is a fat-soluble vitamin that can be
absorbed through the lining of the intestine and into the blood if absorption of lipids is normal.
 People suffering from disorders that slow absorption of lipids (for example, inadequate release of bile into
the small intestine) often experience uncontrolled bleeding as a consequence of vitamin K deficiency.
COAGULATION DEFECTS
Genetically determined deficiencies of clotting factors are not common.
Examples:
 Classic haemophilia, caused by lack of factor VIII, and
 An even rarer form of haemophilia (haemophilia B or Christmas disease) caused by lack of factor IX (also
called Christmas factor).
Missing factors can be supplied by giving fresh plasma or concentrated preparations of, respectively, factor VIII or
factor IX.
Acquired clotting defects are more common than hereditary ones. The causes include liver disease, vitamin K
deficiency (universal in neonates) and excessive oral anticoagulant therapy, each of which may require treatment
with vitamin K.
ANTICOAGULANTS:
 The ideal anticoagulant drug would prevent pathologic thrombosis and limit reperfusion injury, yet allow a
normal response to vascular injury and limit bleeding.
 Theoretically this could be accomplished by preservation of the TF-VIIa initiation phase of the clotting
mechanism with attenuation of the secondary intrinsic pathway propagation phase of clot development.
 At this time such a drug does not exist; all anticoagulants and fibrinolytic drugs have an increased bleeding
risk as their principle toxicity.
Natural Anticoagulant Mechanisms
 Platelet activation and coagulation normally do not occur within an intact blood vessel.
 Thrombosis is prevented by several regulatory mechanisms that require a normal vascular endothelium.
 Prostacyclin (prostaglandin I2; PGI2), a metabolite of arachidonic acid, is synthesized by endothelial cells
and inhibits platelet aggregation and secretion.
 Antithrombin is a plasma protein that inhibits coagulation factors of the intrinsic and common pathways.
 Heparan sulfate proteoglycans synthesized by endothelial cells stimulate the activity of antithrombin.
 Protein C is a plasma zymogen that is homologous to II, VII, IX, and X; its activity depends on the binding
of Ca2+ to Gla residues within its amino-terminal domain.
 Activated protein C, in combination with its non-enzymatic Gla-containing cofactor (protein S), degrades
cofactors Va and VIIIa and thereby greatly diminishes the rates of activation of prothrombin and factor X
(Esmon, 2003).
 Protein C is activated by thrombin only in the presence of thrombomodulin, an integral membrane protein of
endothelial cells.
 Like antithrombin, protein C appears to exert an anticoagulant effect in the vicinity of intact endothelial cells.
 Tissue factor pathway inhibitor (TFPI) is found in the lipoprotein fraction of plasma.
 When bound to factor Xa, TFPI inhibits factor Xa and the factor VIIa–tissue factor complex.
 By this mechanism, factor Xa may regulate its own production.
Classification of anticoagulants:
1. Parenteral anticoagulants:
a. Heparin,
b. Low-Molecular-Weight Heparin Preparations
i. Enoxaparin (LOVENOX),
ii. Dalteparin (FRAGMIN),
iii. Tinzaparin (INNOHEP, others),
iv. Ardeparin (NORMIFLO),
v. Nadroparin (FRAXIPARINE, others), and
vi. Reviparin (CLIVARINE)
c. Heparinoids
i. Heperan sulfate,
ii. Danaparoid,
iii. Lepirudin,
iv. Bivalirudin
v. Ancrod.
d. Drotrecogin alfa
 2. Oral anticoagulants:
i. Coumarin derivatives:
1. Dicumarol (bishydroxycoumarin)
2. Warfarin sodium
3. Acenocoumarol
4. Phenprocoumon
ii. Indandione derivative:
1. Phenindione
2. Anisindione
iii. Direct thrombin inhibitor
1. Ximelagatran
iv. Serine protease inhibitor
1. dabigatran etexilate
2. Rivaroxaban
PARENTERAL ANTICOAGULANTS:
A. HEPARIN
Heparin was discovered in 1916 by a second-year medical student at Johns Hopkins Hospital. He was
attempting to extract thromboplastic (i.e. coagulant) substances from various tissues during a vacation
project, but found instead a powerful anticoagulant activity. This was named heparin, because it was first
extracted from liver.
 Heparin is a glycosaminoglycan found in the secretory granules of mast cells.
 About 10 to 15 glycosaminoglycan chains, each containing 200 to 300 monosaccharide units, are
attached to a core protein and yield a proteoglycan with a molecular mass of 750,000 to 1,000,000
Daltons.
Mechanism of action:
 Heparin catalyses the inhibition of several coagulation proteases, both in vivo and in vitro.
 It inhibits by activating antithrombin III.
 Antithrombin III is a glycosylated, single-chain polypeptide composed of 432 amino acid residues.
 It inhibits activated coagulation factors of the intrinsic and common pathways, including thrombin,
Xa, IXa and has relatively little activity against factor VIIa.
 Antithrombin is a "suicide substrate" for these proteases; inhibition occurs when the protease attacks
a specific Arg-Ser peptide bond in the reactive site of antithrombin and becomes trapped as a stable
1:1 complex.
 Heparin modifies this interaction by binding, via a unique pentasaccharide sequence, to antithrombin
III, changing its conformation and increasing its affinity for serine proteases.
 Antithrombin III deficiency is very rare but can cause thrombophilia and resistance to heparin
therapy.
Pharmacokinetics:
 Heparin is not absorbed from the gut because of its charge and high molecular weight, and it is
therefore given intravenously or subcutaneously.
 Heparin has an immediate onset of action when given intravenously.
 In contrast, there is considerable variation in the bioavailability of heparin given subcutaneously,
and the onset of action is delayed 1 to 2 hours.
 The half-life of heparin in plasma depends on the dose administered. When doses of 100, 400, or
800 units/kg of heparin are injected intravenously, the half-lives of the anticoagulant activities are
approximately 1, 2.5, and 5 hours, respectively.
 Heparin appears to be cleared and degraded primarily by the reticuloendothelial system; a small
amount of undegraded heparin also appears in the urine.
 The half-life of heparin may be shortened in patients with pulmonary embolism and prolonged in
patients with hepatic cirrhosis or end-stage renal disease.
 The elimination half-life is approximately 40-90 min.
  Heparin does not cross blood-brain barrier or placenta (it is the anticoagulant of choice during
pregnancy).
 It is metabolized in liver by heparinase and fragments are excreted in urine.
 Heparin released from mast cells is degraded by tissue macrophages-it is not a physiologically
circulating anticoagulant.
 After i.v. injection of doses < 100 U/kg, the T-half averages 1 hr.
 Beyond this, dose-dependent inactivation is seen and t1/2is prolonged to 1-4 hrs.
 The t-half is longer in cirrhotic and kidney failure patients, and shorter in patients with pulmonary
embolism.
Drug-drug interactions:
 Heparin should not be mixed with penicillin, tetracyclines, hydrocortisone or NA in the same syringe
or infusion bottle.
 Heparinized blood is not suitable for blood counts (alters the shape of RBCs and WBCs), fragility
testing and compliment fixation tests.
Adverse Effects
 Bleeding due to overdose is the most serious complication of heparin therapy. Haematuria is
generally the first sign.
 Thrombocytopenia is another common problem. Generally it is mild and transient; occurs due to
aggregation of platelets. Occasionally serious thromboembolic events result. ln some patients
antibodies are formed to the heparin-platelet complex and marked depletion of platelets occurs
heparin should be discontinued.
 Osteoporosis may develop on long-term use of relatively high doses.
 Hypersensitivity reactions are rare-urticaria, rigor, fever and anaphylaxis. Patients with allergic
diathesis are more liable.
Contraindications
 Bleeding disorders, heparin induced thrombocytopenia.
 Severe hypertensiory (risk of cerebral haemorrhage), threatened abortion, piles, g.i. ulcers (risk of
aggravated bleeding).
 Subacute bacterial endocarditis (risk of embolism), large malignancies (risk of bleeding in the central
necrosed area of the tumour), tuberculosis (risk of hemopfysis).
 Ocular and neurosurgery, lumbar puncture.
 Chronic alcoholics, cirrhosis, renal failure.
 Aspirin and other antiplatelet drugs should be used very cautiously during heparin therapy.