HAEMOSTASIS AND THROMBOSIS

The integrity of the circulation is maintained by blood flowingthrough intact vessels lined by endothelial cells. Injury to thevessel wall exposes collagen and together with tissue injurysets in motion a series of events leading to haemostasis

HAEMOSTASISHaemostasis is a complex process depending on interactionsbetween the vessel wall, platelets and coagulation andfibrinolytic mechanisms.

Vessel wallThe vessel wall is lined by endothelium which, in normalconditions, prevents platelet adhesion and thrombus formation.This property is partly due to its negative charge butalso to:

■thrombomodulin and heparan sulphate expression ■synthesis of prostacyclin (PGI2) and nitric oxide (NO),

which cause vasodilatation and inhibit plateletaggregation

■production of plasminogen activator Injury to vessels causes reflex vasoconstriction, while endothelialdamage results in loss of antithrombotic properties,activation of platelets and coagulation and inhibition of fibrinolysis

PlateletsPlatelet adhesion. When the vessel wall is damaged, theplatelets escaping come into contact with and adhere tocollagen and von Willebrand factor that is bound below theendothelium. This is mediated through glycoprotein Ib (GPIb).

Glycoprotein IIb–IIIa is then exposed, forming a secondbinding site for VWF. Within seconds of adhesion to thevessel wall platelets begin to undergo a shape change, froma disc to a sphere, spread along the subendothelium andrelease the contents of their cytoplasmic granules, i.e. thedense bodies (containing ADP and serotonin) and the α-

granules (containing platelet-derived growth factor, plateletfactor 4, β-thromboglobulin, fibrinogen, VWF, fibronectin,thrombospondin and other factors.(

Platelet release. The release of ADP leads to a conformationalchange in the fibrinogen receptor, the glycoproteinIIb–IIIa complex (GPIIb–IIIa), on the surfaces of adherentplatelets allowing it to bind to fibrinogen

Platelet aggregation. As fibrinogen is a dimerit can form a direct bridge between platelets and so bindsplatelets into activated aggregates (platelet aggregation) andfurther platelet release of ADP occurs. A self-perpetuatingcycle of events is set up leading to formation of a plateletplug at the site of the injury

Coagulation. After platelet aggregation and release ofADP, the exposed platelet membrane phospholipids areavailable for the assembly of coagulation factor enzymecomplexes (tenase and prothrombinase); this platelet phospholipidactivity has been called platelet factor 3 (PF-3). Thepresence of thrombin encourages fusion of platelets, andfibrin formation reinforces the stability of the platelet plug.Central to normal platelet function is platelet prostaglandinsynthesis, which is induced by platelet activation and leadsto the formation of TXA2 in platelets (Fig. 8.34). Thromboxane

)TXA2 (is a powerful vasoconstrictor and also lowers cyclicAMP levels and initiates the platelet release reaction. Prostacyclin

)PGI2 (is synthesized in vascular endothelial cells andopposes the actions of TXA2. It produces vasodilatation andincreases the level of cyclic AMP, preventing platelet aggregationon the normal vessel wall as well as limiting the extentof the initial platelet plug after injury

Coagulation and fibrinolysisCoagulation involves a series of enzymatic reactions leadingto the conversion of soluble plasma fibrinogen to fibrin clot

.Roman numerals are used for most of the factors,but I and II are referred to as fibrinogen and prothrombinrespectively; III, IV and VI are redundant. The active formsare denoted by ‘a’. The coagulation factors are primarilysynthesized in the liver and are either serine protease enzyme

precursors (factors XII, XI, X, IX and thrombin) or cofactors)V and VIII ,(except for fibrinogen, which is degraded to

formfibrin.

Coagulation pathwayThis enzymatic amplification system was traditionally dividedinto ‘extrinsic’ and ‘intrinsic’ pathways. This concept is usefulfor the interpretation of clinical laboratory tests such as theprothrombin time (PT) and activated partial thromboplastintime (APTT) but unrepresentative and an oversimplificationof in vivo coagulation. Coagulation is initiatedby tissue damage. This exposes tissue factor (TF) whichbinds to factor VII. The TF–factor VII complex directly convertsfactor X to active factor Xa and some factor IX to factorIXa. In the presence of factor Xa, tissue factor pathwayinhibitor (TFPI) inhibits further generation of factor Xa andfactor IXa.

Following inhibition by TFPI the amount of factorXa produced is insufficient to maintain coagulation. Furtherfactor Xa, to allow haemostasis to progress to completion,can only be generated by the alternative factor IX/factorVIII pathway. However, enough thrombin exists at thispoint to activate factor VIII (and factor V) and together withfactor IXa (generated by TF-factor VIIa) further activationof factor X can proceed. The presence of activated factorV dramatically enhances the conversion of prothrombinto thrombin by factor Xa. Without the amplification and consolidatingaction of factor VIII/factor IX, bleeding will ensueas generation of factor Xa is insufficient to sustainhaemostasis.

Thrombin hydrolyses the peptide bonds of fibrinogen,releasing fibrinopeptides A and B, and allowing polymerizationbetween fibrinogen molecules to form fibrin. At the sametime, thrombin, in the presence of calcium ions, activatesfactor XIII, which stabilizes the fibrin clot by cross-linkingadjacent fibrin molecules Factor VIII consists of a molecule with coagulant activity

)VIII:C (associated with von Willebrand factor. Factor VIIIincreases the activity of factor IXa by ~200 000 fold. VWFfunctions to prevent premature factor VIII:C breakdown and locate it to areas of vascular injury. VIII:C has a molecularweight of about 350 000.

Von Willebrand Factor (VWF) is a glycoprotein witha molecular weight of about 200 000 which readily formsmultimers in the circulation with molecular weights of up to

20 × 106 .It is synthesized by endothelial cells and megakaryocytesand stored in platelet granules as well as theendothelial cells. The high-molecular-weight multimericforms of VWF are the most biologically active

Physiological limitation of coagulationWithout a physiological system to limit blood coagulationdangerous thrombosis could ensue. The natural anticoagulantmechanism regulates and localizes thrombosis to thesite of injury.

Antithrombin. Antithrombin (AT), a member of the serine proteaseinhibitor (serpin) superfamily, is a potent inhibitor ofcoagulation. It inactivates the serine proteases by formingstable complexes with them, and its action is greatly potentiatedby heparin.Activated protein C. This is generated from its vitamin K dependentprecursor, Protein C, by thrombin; thrombin activationof protein C is greatly enhanced when thrombin isbound to thrombomodulin on endothelial cells.Activated protein C inactivates factor V and factor VIII, reducingfurther thrombin generation.Protein S. This is a cofactor for protein C, which acts byenhancing binding of activated protein C to the phospholipidsurface. It circulates bound to C4b binding protein but some

30–40% remains unbound and active (free protein S).

Other inhibitors. Other natural inhibitors of coagulationinclude α2-macroglobulin, α1-antitrypsin and α2-antiplasmin

FibrinolysisFibrinolysis is a normal haemostatic response that helps torestore vessel patency after vascular damage. The principalcomponent is the enzyme plasmin, which is generated fromits inactive precursor plasminogen. This is achievedprincipally via tissue plasminogen activator (t-PA) releasedfrom endothelial cells. Some plasminogen activation mayalso be promoted by urokinase, produced in the kidneys.Other plasminogen activators (factor XII and prekallikrein) areof minor physiological importance

Plasmin is a serine protease, which breaks down fibrinogenand fibrin into fragments X, Y, D and E, collectivelyknown as fibrin (and fibrinogen) degradation products (FDPs).D-dimer is produced when cross-linked fibrin is degraded.

Its presence in the plasma indicates that the coagulationmechanism has been activated.

The fibrinolytic system is activated by the presence offibrin. Plasminogen is specifically adsorbed to fibrin andfibrinogen by lysine-binding sites. However, little plasminogenactivation occurs in the absence of polymerized fibrin,

as fibrin also has a specific binding site for plasminogenactivators, whereas fibrinogen does not.

t-PA is inactivated by plasminogen activator inhibitor-1)PAI-1 .(Activated protein C inactivates PAI-1 and therefore

induces fibrinolysis. Inactivators of plasmin, such as α2-antiplasmin .and thrombin-activatable fibrinolysisinhibitor (TAFI), also contribute to the regulation of

fibrinolysis .

Investigation of bleeding disordersAlthough the precise diagnosis of a bleeding disorder willdepend on laboratory tests, much information is obtainedfrom the history and physical examination:

■Is there a generalized haemostatic defect?Supportive evidence for this includes bleeding frommultiple sites, spontaneous bleeding, and excessivebleeding after injury.

■Is the defect inherited or acquired? A family history ofa bleeding disorder should be sought. Severe inheriteddefects usually become apparent in infancy, while mildinherited defects may only come to attention later in life,for example with excessive bleeding after surgery,childbirth, dental extractions or trauma. Some defectsare revealed by routine coagulation screens which areperformed before surgical procedures.

■Is the bleeding suggestive of a vascular/plateletdefect or a coagulation defect

Vascular/platelet bleeding is characterized by easy bruisingand spontaneous bleeding from small vessels. There isoften bleeding into the skin. The term purpura includes bothpetechiae, which are small skin haemorrhages varying frompinpoint size to a few millimetres in diameter and which donot blanch on pressure, and ecchymoses, which are largerareas of bleeding into the skin. Bleeding also occurs frommucous membranes especially the nose and mouth. Coagulationdisorders are typically associated with bleeding afterinjury or surgery, and in more severe forms, haemarthrosesand muscle haematomas. There is often a short delaybetween the precipitating event and overt haemorrhage orhaematoma formation

Laboratory investigations ■Blood count and film show the number and

morphology of platelets and any blood disorder such asleukaemia or lymphoma. The normal range for theplatelet count is 150–400 × 109/L.

■Bleeding time measures platelet plug formation in vivo.It is determined by applying a sphygmomanometer cuffto the arm and inflating it to 40 mmHg. Two 1 mm deep,

1 cm long incisions are made in the forearm with atemplate. Each wound is blotted every 30 seconds andthe time taken for bleeding to stop is recorded, normallybetween 3 and 10 minutes. Prolonged bleeding timesare found in patients with platelet function defects, andthere is a progressive prolongation with platelet countsless than 100 × 109/L. The bleeding time should not beperformed at low platelet counts.

■Coagulation tests are performed using blood collectedinto citrate, which neutralizes calcium ions and preventsclotting

The prothrombin time (PT) is measuredby adding tissue factor (thromboplastin) and calcium to thepatient’s plasma. The normal PT is 12–16 seconds and maybe expressed as the international normalized ratio, INR The PT measures VII, X, V, prothrombin and fibrinogen

)classic ‘extrinsic’ pathway (and is prolonged with abnormalities of these factors. It may also be abnormal in liverdisease, or if the patient is on warfarin-

The activated partial thromboplastin time (APTT) is alsosometimes known as the PTT with kaolin (PTTK). It is performedby adding a surface activator (such as kaolin), phospholipid

)to mimic platelet membrane (and calcium to thepatient’s plasma. The normal APTT is 30–50 seconds anddepends on the exact methodology. The APTT measures XII,XI, IX, VIII, X, V, prothrombin and fibrinogen (classic ‘intrinsic’pathway) and is prolonged with deficiencies of one or moreof these factors. It is not dependent on factor VII.

The thrombin time (TT) is performed by adding thrombinto the patient’s plasma. The normal TT is 12–14 seconds,

and it is prolonged with fibrinogen deficiency, qualitativedefects of fibrinogen (dysfibrinogenaemia) or inhibitors suchas heparin or FDPs

Correction tests can be used to differentiate prolongedtimes in the PT, APTT and TT due to various coagulationfactor deficiencies and inhibitors of coagulation. ProlongedPT, APTT or TT due to coagulation factor deficiencies canbe corrected by addition of normal plasma to the patient’splasma. Failure to correct after addition of normal plasma issuggestive of the presence of an inhibitor of coagulation.Factor assays are used to confirm coagulation defects,especially where a single inherited disorder is suspected.

Special tests of coagulation will often be required toconfirm the precise haemostatic defect. Such tests includeestimation of fibrinogen and FDPs, platelet function testssuch as platelet aggregation and platelet granule contents.