This module will look at the process of haemostasis: the formation and removal of a healthy clot. This will then enable us to understand one of the consequences inappropriate activation of haemostatic mechanisms – arterial and venous thrombosis.
The physiology of haemostasis – how blood clots
The ability of blood to clot, for example in response to injury, is vital – without it, even the smallest cut could lead to fatal haemorrhage. But excessive activation of clotting mechanisms, or clots in the wrong places, can be equally disastrous. A thorough understanding of the physiological mechanisms which lead to blood clotting is essential for going on to understand what happens when things go wrong – and how we can intervene pharmacologically when they do.
Over 150 years ago, the German physician, Rudolf Virchow (figure 1) recognised that a change in any one of three factors could lead to formation of a blood clot (thrombogenesis):
- blood flow
- the vessel wall
- constituents of the blood.
This has been modified by modern understanding (figure 2) of the endothelium and its function, of blood flow characteristics (rheology, including loss of laminar flow and development of turbulence), and a full understanding of blood constituents (coagulation factors and platelets).
In health, clotting is most often the result of changes in the vessel wall due to injury. Changes in blood flow (such as in atrial fibrillation) or blood constituents (such as in inherited thrombophilias) become more important when we consider pathological clotting.
The blood vessel wall
The endothelium is a highly dynamic organ with roles in inflammation (mediating the passage of leukocytes into the tissues), in blood pressure control (by the release of nitric oxide and endothelin), and in haemostasis. In terms of haemostasis, endothelial cells express and release a selection of molecules that promote thrombosis, and others that counter thrombosis (table 1). These factors are in balance; in the healthy state, the balance is in favour of suppression of coagulation, to keep blood flowing freely through the vessel. When endothelial cells are activated – by injury or inflammation – the balance is shifted and procoagulant factors are favoured.
There is evidence that cardiovascular risk factors exert at least some of their pro-thrombotic effect by inducing endothelial activation2.
Blood constituents – platelets and the coagulation cascade
In response to injury, platelets and coagulation factors act simultaneously and synergistically to seal the hole and secure haemostasis.
Coagulation factors are a collection of mostly liver-produced proteins, some of which rely on vitamin K for their synthesis. Many are zymogens: inactive precursors of an enzyme that, once activated, themselves act on another zymogen. Several factors have names (such as prothrombin); others are denoted by Roman numerals, such as factor V. The ‘resting or inactive’ form is denoted by the simple factor number, but if activated carries the notation ‘a’, an example being factor Va. Table 2 lists the coagulation factors.
The coagulation cascade
The function of the coagulation cascade is to generate fibrin (see figure 3). This happens via a sequence of highly regulated enzyme/substrate reactions,2 which occur largely on the surface of endothelial cells and platelets. Although traditionally divided into an ‘extrinsic’ pathway (tissue-factor dependent) and an ‘intrinsic’ pathway (contact factor dependent), it is currently thought that the tissue factor pathway is the main mechanism for haemostasis in vivo. The physiological role for the components of the ‘intrinsic’ pathway are less clear – factor XII deficiency, for example, does not seem to be associated with a bleeding phenotype. However, the division into ‘extrinsic’ and ‘intrinsic’ pathways is still useful in the interpretation of coagulation tests: broadly speaking, the prothrombin time (PT) assesses the extrinsic pathway, and the activated partial thromboplastin time (APTT) the intrinsic pathway.
Tissue damage leads to exposure of blood to tissue factor, which complexes with factor VIIa. This complex has the ability to convert small amounts of factor X to factor Xa, and also IX to IXa.
Factor Xa, generated in the initiation phase, can, in turn, generate a small amount of thrombin from prothrombin. This thrombin then acts on factors V and VIII (generating Va and VIIIa), and also and also converts XI to XIa which increases production of IXa from IX. VIIIa and IXa form a complex – the ‘tenase’ complex – which massively increases production of Xa from X. This increased amount of Xa complexes with Va to form the ‘prothrombinase complex’, which produces much more thrombin from prothrombin than Xa could alone. Thus by a sequence of amplification steps, there is an explosive generation of thrombin, which has a central role in production and stabilisation of a clot. The ‘tenase’ and ‘prothrombinase’ complex are assembled on phospholipid surfaces – hence the requirement for activated platelets.
Formation of fibrin clot – the central role of thrombin
Thrombin cleaves fibrinogen to create fibrin monomers, which then spontaneously polymerise before being stably cross-linked by factor XIIIa (see figure 4).
Thrombin also has a number of other functions (see table 3): positive feedback and amplication of the coagulation cascade, platelet activation, inhibition of fibrinolysis, and activation of factor XIII. The central role of thrombin in coagulation has made it an attractive target for anticoagulant drugs (see module 3 for a full discussion of direct thrombin inhibitors). Thrombin generation has also attracted interest as a laboratory measure of global haemostatic function, which may have future clinical applications.3
Coagulation inhibitors ensure the coagulation pathway does not develop too rapidly or too extensively. The primary regulator is antithrombin, which, in addition to inhibiting thrombin, also suppresses factors VIIa, IXa, Xa and XIa. However, in itself, antithrombin is a relatively weak inhibitor: 90% of its bioactivity is accounted for by its binding to heparin on the surface of the endothelium.
Protein C is a vitamin-K dependent zymogen, or pro-enzyme, or inactive enzyme precursor. The endothelial membrane component, thrombomodulin, binds thrombin, and in this form converts protein C to an active molecule (hence, activated protein C) that inhibits factors Va and VIIa and, to a lesser extent, thrombin. This activation is enhanced by the binding of protein C to the endothelial protein C receptor, and is stabilised by the presence of protein S. Tissue factor pathway inhibitor (TFPI) suppresses the activity of tissue factor in the initiation of the cascade.
Platelets are small, anucleate, but highly complex cells, whose cytoskeleton, cell surface receptors and organelles all play vital roles in achieving haemostasis – roles which are becoming increasingly well understood. Produced by bone marrow megakaryocytes at a rate of around 1011 daily, platelets circulate in the blood in an inactive state. Upon activation – for example by vessel injury – they undergo a number of rapid changes leading to formation of a platelet plug and achievement of haemostasis, in collaboration with the coagulation cascade.
The major components of the platelet membrane are listed in table 4. Each can be bound by a specific ligand, and upon doing so a chain of actions is initiated that results in the adhesion and/or activation of the cell. The consequences of this are:
- shape change, from discoid to spherical, and the extrusion of pseudopodia, thus increasing surface area
- the increased expression of phospholipids and phosphatidylserine, thus providing the optimum surface for the assembly of components of the coagulation cascade
- granule release, resulting in the extrusion of a larger number of biologically active mediators, and the appearance of further adhesion molecules, increasing platelet–platelet adhesion and platelet–subendothelial adhesion.
The major organelles participating directly in thrombosis are dense bodies and alpha granules. These granules are released upon platelet activation, and can be best thought of as promoting further platelet recruitment through positive feedback, while also promoting coagulation locally. The contents of dense bodies include serotonin (5-hydroxytryptamine), adenosine triphosphate (ATP), adenosine diphosphate (ADP) and calcium. Alpha granules contain a huge variety of mediators including:
• components of the coagulation cascade (fibrinogen, von Willebrand factor and factor V)
• molecules involved in fibrinolysis and repair (plasminogen, PAI-1, platelet derived growth factor)
• chemokines for leucocyte recruitment (platelet factor 4, beta-thromboglobulin) and
• promoters of further platelet aggregation (thrombospondin and fibronectin).
Fusion of alpha granules with the platelet membrane also increases membrane concentrations of receptors involved in platelet adhesion, such as P-selectin and GpIIb/IIIa.
As well as granule release, activated platelets generate thromboxane A2 from membrane phospholipids via the enzyme cyclooxygenase. Thromboxane A2 further augments platelet activation. The importance of its role is highlighted by the effectiveness of the cyclooxygenase inhibitor aspirin in reducing platelet function.
Summary – formation of a platelet plug in response to injury1,4,5
Although platelets and the coagulation cascade work together to secure haemostasis, formation of a platelet plug is arguably the most important mechanism behind securing primary haemostasis in conditions of high shear stress, such as in arterial injury. Considering the steps involved provides a useful summary of platelet recruitment and activation:
(i) Initial platelet tethering. Damage to the vessel wall leads to exposure of collagen, which binds von Willebrand factor. The latter then binds platelets via GpIb/IX/V.
(ii) Stable adhesion and activation. Stable adhesion at the site of injury is mediated through receptors including GpVI and GpIIb/IIIa. GpIIb/IIIa also binds fibrinogen and other platelets via fibrinogen, increasing platelet recruitment. Binding of receptors leads via intracellular signalling to platelet activation, and resultant shape change, granule release and thromboxane A2 generation.
(iii) Spreading and aggregation. Positive feedback mechanisms described above lead to further platelet recruitment. Platelet conformational change exposes phosphatidylserine, providing procoagulant surface for optimal function of components of coagulation cascade. Activated platelets become stably cross-linked (aggregation). Activation of coagulation cascade generates thrombin which stabilises the platelet plug.
The transition from inactive platelet to active can be seen under the electron microscope in figure 5. As an introduction to pharmacological intervention in platelet function, Figure 66 shows a stylised view of platelet activation and the targets of various antiplatelet agents.
Once a thrombus has done its job and reduced blood loss, and the damage is repaired, it must be removed. This is performed largely by the enzyme plasmin in the process of fibrinolysis, sometimes expressed as thrombolysis. Plasmin itself is derived from the zymogen plasminogen by the action of tissue plasminogen activator (tPA), which is an endothelial product.
However, tPA has a regulator, plasminogen activator inhibitor, of which there are several types (hence PAI-1). Plasminogen activator inhibitor can be released from platelets, endothelial cells and other cells. Since tPA and PAI-1 are believed to react in a stoichiometry of 1:1, the balance between the two is crucial for the process of fibrinolysis.
When plasmin breaks down cross-linked fibrin, it generates quite specific protein fragments that are easily identified in the plasma. These fragments are called d-dimers, and high levels are considered proof of active fibrinolysis, and, therefore, of a high general burden of thrombus within the body. This process is summarised in figure 7.
The dynamics of haemostasis
An old view of haemostasis considered it to be a ‘stop–start’ model, where various factors would initiate the process, which would proceed and eventually stop with the formation of a clot. In the light of new research, this view has been superseded by the dynamic hypothesis.
In the dynamic model, the coagulation system is permanently active, but at a low level, and is held in check by inhibitors. The platelet pool is at rest and very few are activated. Upon stimulation, coagulation activity increases, escapes from inhibitor regulation, and thrombus formation follows. However, the inhibitors soon catch up and eventually coagulation activity slows down, which prevents the process from expanding too rapidly. Quite possibly in parallel, platelets are activated (by thrombin for example), degranulate, and so promote the coagulation pathway. The platelet shape change favours adhesion and aggregation, and binding to fibrin results in thrombus formation.
This model predicts that there are always background levels of active factors generating a small amount of clot, but that this is degraded by low levels of plasmin generated from the fibrinolysis pathway. Indeed, the small amounts of plasma d-dimers present in healthy blood support this hypothesis.
Thus, the balance between a semi-active coagulation system, regulation by inhibitors, and fibrinolysis is crucial in thrombosis. The attraction and strength of this model is that it explains many of the causes of clinical thrombosis, predicts outcomes, and provides opportunities to intervene.
Unwanted clots – the pathology of thrombosis
We have seen how blood clots to stop bleeding in response to injury. This is haemostasis – its physiological role. In health, it is tightly regulated to restrict clots to where they are needed. If this regulation fails, or is overwhelmed, clots can form excessively or in the wrong places. When an unwanted clot blocks an artery or vein it is called a thrombosis. Although both platelets and the clotting cascade are involved in the development of all clots, the main driver in thromboses of arteries and veins is thought to be different, because of the different shear stresses involved:
- in arterial thrombosis, platelets take a lead role;
- in venous thrombosis, coagulation factors seem to be more important.
This has crucial implications for management and prevention.
Atherosclerosis is a chronic lipid-driven inflammatory disease of the arterial wall, which is promoted by the well-known cardiovascular risk factors (diabetes, smoking, hypertension and dyslipidaemia). It leads to the formation of plaques of lipids and other material at several key anatomical points, especially the coronary arteries, carotid arteries, cerebral circulation, and the aorta, iliac and femoral arteries. These plaques lead to gradual stenosis of these arteries (see figure 8) which restricts blood flow and can lead to tissue hypoxia at times of high oxygen demand. In such conditions, the tissues are forced to respire anaerobically, leading to the accumulation of lactic acid and the development of pain symptoms (see figure 9) – the syndromes of angina and intermittent claudication.
Atherosclerotic plaques also promote thrombus formation, which, when it occurs acutely and occludes the vessel, leads to acute ischaemia in the supplied organ – myocardial infarction or stroke (see figure 10). The thrombogenicity of atherosclerotic plaques is thought to be multifactorial: plaque rupture exposes sub-endothelial collagen and tissue factor, but there is also evidence that the endothelium overlying plaques loses its anticoagulant function and becomes pro-thrombotic.7 Under the conditions of high shear stress in arteries, platelet tethering via von Willebrand factor is thought to be the predominant initiating mechanism for thrombus formation, leading to the formation of a platelet-rich thrombus.7 However, activation of the clotting cascade and generation of cross-linked fibrin is also important.
The central role of platelets in initiating arterial thrombus formation explains the essential role of antiplatelet agents (aspirin, clopidogrel, dipyridamole) in prevention of cardiovascular disease. Antiplatelet agents are also essential for the treatment of acute thrombotic events, but anticoagulants and fibrinolytics are also used. Antiplatelet and anticoagulant therapy will be discussed in more detail in modules 2 and 3.
Thrombi which form in the venous system do so under low shear. Thus von Willebrand factor and platelet tethering seem to be less important, and activation of the coagulation system takes a leading role to produce a fibrin-rich clot.8
Alterations in blood flow (venous stasis, turbulence) and in the coagulability of blood seem to be particularly important in disposing to venous thromboembolism (VTE). This is reflected in acknowledged risk factors for its development. For example, surgery can lead to immobility, venous stasis and an inflammatory response, which includes an increase in factor VIII levels. High levels of oestrogen (as in pregnancy, the oral contraceptive pill and hormone replacement therapy), lead to a prothrobotic state by a rise in clotting factors and a fall in natural anticoagulants. A number of inherited defects of the natural anticoagulant system can predispose to thrombosis, such as deficiencies of protein C, protein S, or antithrombin, or factor V Leiden (an inherited reduction in sensitivity of factor Va to deactivation by protein C). A current area of much active research is the risk of venous thrombosis associated with malignancy. Malignancy predisposes to thrombosis by a number of mechanisms, including expression of tissue factor by tumour cells, pro-thrombotic mucin production, and release of microparticles containing tissue factor into the circulation.9
The majority of deep vein thromboses occur in the deep veins of the lower limb. It is thought that venous stasis leads to endothelial hypoxia – and hence activation and adoption of a pro-coagulant state as described above – leading to local activation of the coagulation system.10 Many deep vein thromboses (DVTs) are thought to form in the valve pockets and then to grow by elongation, spiralling up the vein. However, the tip of the thrombus can break off (embolise) and be driven up the venous circulation.
Such an embolus will eventually become lodged in the pulmonary circulation, causing a pulmonary embolus (PE). If large, or if added to by local growth or additional emboli, PEs can cause serious haemodynamic complications, such as pulmonary hypertension, right-sided heart disease, and death.
Just as arterial thrombosis is treated and prevented with antiplatelets, VTE is treated and prevented with anticoagulants. Heparin and warfarin were the first anticoagulants to be developed and are remarkably successful in preventing the development of a VTE, be it after a major risk event such as orthopaedic surgery, or in preventing a second VTE. Unfortunately, their use requires close laboratory monitoring and considerable expertise in dosing. Heparin has largely been superseded by a ‘cleaner’ variant, low molecular weight heparin (LMWH). More recently, direct oral anticoagulants (DOACs – previously known as NOACS11) have been developed which offer the prospect of equivalent, or better, efficacy and safety without the need for monitoring. These will be discussed in module 3.
A note about atrial fibrillation
Embolic stroke is one of the most devastating consequences of atrial fibrillation. The mechanism of thrombus formation in atrial fibrillation is thought to be very similar to that in VTE: stasis of blood, with possible contributions from endothelial dysfunction and/or a hypercoagulable state.12 This is further supported by the fact that anticoagulants are more effective at stroke prevention in AF than antiplatelet agents, as will be discussed further in module 3.
The Royal College of Physicians, in a position statement13 said that there is significant evidence to support the view that hospital-acquired VTE can be prevented through a combination of two simple, safe and effective steps:
- a risk assessment of patients for their VTE and bleeding risk, to identify those at risk of VTE and those for whom preventative treatment is appropriate; and
- administering preventative treatment for those identified as being at risk of VTE, in the form of pharmacological prophylaxis and/or mechanical prophylaxis.
Current NICE guidance14 on reducing the risk of VTE says VTE prevention is a cost-effective measure for national health boards to implement. NICE has calculated that compliance with their guidance to prevent hospital-acquired VTE saves money, over and above the cost of managing VTE once it has developed.
Following the publication of this guideline, NICE placed VTE prevention within its list of top 10 cost-effective guidelines. NICE estimated that effective VTE prevention would cost the NHS in the UK an additional £21.9 million nationally, but this figure is more than offset by the anticipated reductions in DVT and PE. In 1993, the Office for Healthcare Economics estimated that the annual cost of treating patients who developed post-surgical DVT and PE alone was in the region of £204.7 to £222.8 million in the UK.
These figures clearly demonstrate that compliance with best practice in VTE prevention (that is, risk assessment of patients for VTE on admission and the administration of appropriate prophylaxis) makes financial sense for the NHS at a time when there are significant pressures to manage costs. VTE prevention is a simple, effective, and cost-efficient measure to save lives.
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