Haemostasisis a physiological process of blood clotting and dissolution of the clot,following by repair of the injured tissue. It results from interplay ofvascular endothelium, platelets, coagulation factors, anti coagulationmechanisms and fibrinolytic system.
The fluidity of blood in the absence ofinjury is maintained by the balance between pro-coagulant pathway, and themechanisms that inhibit the pro-coagulant pathway. Imbalance between the twomechanisms, for example during clinical illnesses or preoperative period,predisposes a patient to either bleeding or thrombosis. To stop bleeding after injury a complex process is initiated withinseconds.
After vasoconstriction, which reduces blood flow, begins the firstphase of haemostasis –primary haemostasis. The primary haemostasis leads to theformation of initial platelet plug. Activated platelets adhere to the site ofinjured tissue and to each other, plugging the injury. However, this initialplatelet plug is unstable and will be stabilized during coagulation-process, inthe phase of secondary haemostasis. Platelets derived from megakaryocytes are the main player in the primaryhaemostasis. In health they do not adhere to surfaces of vessels or to eachother, but after injury platelets are exposed to subendothelial matrix thatleads to activation and adhesion of platelets into haemostatic plug.
Plateletsplay a central role in haemostasis providing proper flow of sequential eventsafter injuries: platelet adhesion, activation, aggregation, and expression ofprocoagulant activity. Thus, they are involved in cell-based thrombingeneration, which amplifies the blood coagulation cascade, by supplying aprocoagulant surface provided by the phospholipids of the platelet plasma membraneon which the coagulation enzyme complexes can be assembled. Differenttransmembrane receptors are embedded in the platelet membrane, which act incell signalling providing activation and adhesion of thrombocytes: integrins(?IIb?3, ?2?1, ?5?1, ?6?1, ?V?3), leucine-rich repeated (LRR) receptors(Glycoprotein GP Ib/IX/V, Toll-like receptors), G-protein coupled seventransmembrane receptors (PAR-1 and PAR-4 thrombin receptors, P2Y1 and P2Y12ADPreceptors, TP? and TP? TxA2 receptors), proteins belonging to theimmunoglobulin super family (GP VI, Fc?RIIA), C-type lectin receptors(P-selectin), tyrosine kinase receptors (thrombopoietin receptor, Gas-6,ephrins and Eph kinases) and a lot of other types (e.g. CD63, CD36, P-selectinligand 1, TNF receptor) (Figure 2) (Rivera J et al, 2009). It has been alsoknown that some of receptors are involved in other platelet functions such asinflammation, tumor growth and metastasis, or immunological response.
Uponendothelial injury plasma protein called von Willebrand factor (VWF) binds tothe exposed collagen. Platelets move to the site of injury and become activatedthrough the binding to the VWF via glycoprotein (GPIb) and to the collagen viaGPVI and ?2?1receptors. After activation, the GPIIb:IIIa (?IIb?3)-receptorchanges conformation and binds fibrinogen or VWF, initiating plateletaggregation. To support the aggregation and to recruit unactivated circulatingplatelets , thrombocytes release proteins imported for their proper haemostaticfunction such as VWF, fibrinogen, P-selectin, PECAM-1, CD40 ligand (CD154),platelet factor-4, ?-thromboglobulin, thrombospondin, platelet derived growthfactor (PDGF), FV, as well as ADP, thromboxane A2 (TXA2), serotonin, histamine,pyrophosphate, and calcium . Bleeding isthe common cause of platelet disorders. They can be caused by a reduction inthe number of platelets or thrombocytopenia (e.g. Wiskott-Aldrich syndrome,Fechtner syndrome, May-Hegglin anomaly) as well as by platelet function defectsor thrombocytopathies (e.
g. Glanzmann’s thrombasthenia, vonWillebrand’sdisease-platelet type, Bernard-Soulier syndrome. Further, the initial plateletplug must be stabilized via fibrin – clot formation- through secondaryhaemostasis. In 1964 Davie, Ratnoff, and Macfarlane published separatelyarticles in Nature and Science outlining the basic principle of a “waterfall”and a “cascade” of proenzymes activated through proteolytic cleavage that inturn activate downstream enzymes. The waterfall-cascade hypothesis was latermodified as the function of the clotting factors, which were betterinvestigated and defined.
Secondary haemostasis is the cascade of coagulationserine proteases that results in cleavage of fibrinogen by thrombin to fibrin(Figure 3). It leads to stabilization of the instable primary platelet plug atthe site of an injury and formation of a blood clot. Remarkable, that theprocess of fibrin generation occurs coincident to the process of plateletaggregation.Two different models of coagulation cascade have been accepted: thetraditional classification into extrinsic and intrinsic pathway, both of whichconverge on factor X activation, and the modern model of coagulation pathway.
The modern model describes coagulation pathway with following phases:initiation, amplification and propagation. Tissue factor (TF) is atransmembrane glycoprotein and the cofactor for the serine protease factorVIIa. Its contact with blood leads to binding and activation of FVII in thepresence of calcium. This TF-VIIa-Ca2+-complex considered as the initiator ofcoagulation cascade activates factors IX and X. The coagulation factor Xaconverts prothrombin (factor II) to thrombin. This small amount of thrombin(trace level) is sufficient and crucial for activation of factor XI, which thenactivates factor IX, and factors V and VIII. Upon activation, factors FXI, FVand FVIII promote the amplification of the coagulation pathway. In thepropagation phase of coagulation cascade, conversion of prothrombin to thrombinby prothrombinase complex (FVa-FXa-Ca2+) takes place.
Then thrombin cleavessoluble fibrinogen into fibrin monomers, which are insoluble. The fibrinmonomers polymerize producing a stable clot. Factor XIIIa (plasmatransglutaminase) activated by thrombin cross-links glutamine and lysine residuesbetween fibrin molecules completing the process of secondary haemostasis. Notonly coagulation but also mechanisms which inhibit pro-coagulant pathway play asignificant role in providing haemostasis. Anticoagulant mechanisms have aregulatory and control function in maintaining haemostasis. Thus, thesemechanisms promote blood fluidity in the absence of injury, localize theformation of clot at the site of injury, as well as perform degradation ofblood clot after injury. The balance between procoagulant system andanticoagulant system is critical for proper haemostasis and the avoidance ofpathological bleeding or thrombosis. The main action of anticoagulantmechanisms is to reduce production and activity of thrombin.
Antithrombin (AT),also known as AT III is an inhibitor of the coagulation serine proteases suchas thrombin, factor IXa, Xa, XIa and XIIa. Heparin enhances the enzymaticactivity of antithrombin. Furthermore, heparin cofactor II, ?2 macroglobulinand ?1-antitrypsin are also inhibitors of thrombin. Another physiologicalanticoagulant mechanism is represented by the protein C pathway. Protein C is avitamin K-dependent serine protease which is activated by thrombin andregulates activity of coagulation factors Va and VIIIa. Once activated bythrombin, it forms activated protein C (APC) and inhibits activated factors Vand VIII. Protein S and phospholipids act in this pathway as cofactors.
ProteinZ-dependent protease inhibitor (ZPI) is a serine protease inhibitor, whichinactivates FXa in the presence of protein Z (PZ) (vitamin K-dependentglycoprotein) as cofactor, phospholipids and Ca2+-ions. ZPI also inhibitsfactors IXa and XIa, in protein Z independent pathway. The primary inhibitor of the initiation of blood coagulation process isthe tissue factor (TF) pathway inhibitor. TFPI acts as a high-affinityinhibitor of two coagulation proteases, such as TF-factor VIIa (TF-FVIIa) andfactor Xa (FXa). There are two isoforms of TFPI known: TFPI? and TFPI?. Thesetwo isoforms differ in several characteristics: in affinity for factor V/Va(FV/FVa)and protein S (PS), in expression in plateletsand endothelial cells, in mechanism for association with cell surfaces,and in ability to influence early steps of blood coagulation through distinct mechanismsof inhibition of TF-FVIIa activity or inhibition of prothrombinase.
Protein S is needed ascofactor for optimal inhibition of factor Xa by TFPI?, but is not required for FXa-dependent TF-FVIIa inhibition. As mentioned above, fibrin plays anessential role in secondary haemostasis as the primary product of thecoagulation cascade. Degradation of fibrin is termed fibrinolysis. Thefibrinolytic pathway is a complex physiological pathway controlled by action ofa series of cofactors, inhibitors, receptors. Dysregulation of this pathway isassociated with different pathologies (e.
g. coagulopathies, disseminatedintravascular coagulation (DIC) or congenital bleeding disorders). Degradationof fibrin is performed by serine protease plasmin, which is present in blood asa proezyme plasminogen and need to be activated by tissue plasminogen activator(tPA) and urokinase.