Physiology of haemostasis

Abstract

Haemostasis is a complex process that ensures the maintenance of blood flow under normal physiological conditions and prevents major blood loss following vascular injury. The process is tightly regulated to prevent pathological thrombosis. Normal haemostasis relies on the delicate balance of prothrombotic and anticoagulant processes, where five components play a significant role in maintaining the haemostasis, these include: (i) endothelial cells; (ii) platelets which are key to platelet plug formation; (iii) coagulation factors that are essential to formation of insoluble fibrin clot; (iv) coagulation inhibitors; and (v) fibrinolysis. This article will provide an overview of the current concepts of haemostasis, and through this we will explain how antiplatelets and antithrombotic drugs work, as well as provide a basic understanding of how to interpret clotting tests used to measure coagulation disorders.

Learning objectives

After reading this article, you should be able to:
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identify the key components of haemostasis
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describe the principles of how some major inherited bleeding disorders impact haemostasis
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interpret abnormal coagulation assays
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describe how antiplatelets and anticoagulants affect coagulation

Introduction

The haemostatic pathway is a tightly regulated process that ensures the maintenance of blood flow under normal physiological conditions and also facilitates the prevention of significant blood loss following vascular injury. The normal haemostatic response depends on the closely linked interaction between blood vessel wall (endothelial cells) platelets and blood coagulation factors. While an efficient and rapid mechanism for stopping bleeding is essential for survival, it is equally important that this mechanism is tightly controlled, so that pathological thrombosis is prevented. Therefore, normal haemostasis relies on the delicate balance of prothrombotic and anticoagulant processes.

There are five components of haemostasis:

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blood vessels and endothelial cells
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platelets
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coagulation factors
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coagulation inhibitors
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clot dissolution or fibrinolysis.

This article will provide an overview of the current concepts of haemostasis, and through this we will explain how antiplatelets and antithrombotic drugs work, and also provide a basic understanding of how to interpret clotting tests used to measure coagulation disorders.

Haemostasis

Blood vessels and endothelial cells

Following vascular injury, vessel walls vasoconstrict immediately to slow blood flow to the site of injury as well as prevent exsanguination from widespread damage. In addition, collagen and tissue factor (TF) are brought into contact with flowing blood. Exposed collagen triggers the accumulation and activation of platelets at the site of the vessel wall damage resulting in the formation of a platelet plug, while the exposed TF initiates the activation of coagulation factors and generation of thrombin which in turn will lead to formation and stabilization of insoluble fibrin clot.

Platelet structure and function

Platelets are anucleate, extremely small, discoid cells which circulate in abundant numbers in the peripheral blood. They are formed by fragmentation from megakaryocytes within the bone marrow and have a lifespan of 7–10 days. Platelets are essential in the initial formation of a mechanical plug in response to vessel injury and they achieve this through four main functions: (i) activation; (ii) adhesion to the vessel wall; (iii) aggregation; and (iv) secretion. Activated platelets provide a surface for activation and recruitment of more platelets, as well as activation of coagulation factors, which will ultimately lead to fibrin formation.

Platelet membrane: this invaginates into the interior of the cell and forms an extensive canalicular system, which provides a large area of numerous membrane receptors and proteins. Of particular importance in the platelet membrane are phospholipids, which activate coagulation factors such as factor X (FX) and factor II (prothrombin). Surface receptors activate intracellular pathways which lead to a conformational change in platelet structure and shape upon activation. The membrane also contains numerous glycoproteins which serve as binding sites for various other molecules such as von Willebrand factor (vWF) and fibrinogen (adhesion), as well as binding to other platelets (aggregation). Table 1 summarizes the roles and clinical significance of some of the platelet membrane proteins.

Table 1

Role and clinical significance of key platelet membrane proteins

Membrane protein	Role	Clinical significance
GP1a	Binds collagen Activated intracellular pathways leading to thromboxane A2 (TXA2) generation	Aspirin suppresses TXA2 synthesis by inhibiting COX
GP1b	Binds von Willebrand factor	Defective in Bernard Soulier disease – results in bleeding disorders
GPVI	Binds collagen	GP VI Absence results in severe bleeding diathesis
GPIIb/IIIa	Binds fibrinogen and von Willebrand factor Binding site for other platelets in aggregation	Defective in Glanzmann’s thrombaesthenia – results in bleeding disorders
Membrane phospholipid	Activates coagulation factors	Activates factor X→ Xa and factor II → IIa
P2Y12	Activated by ADP, leads to generation of TXA2 and aggregation	P2Y12 Inhibited by clopidogrel and ticagrelor

ADP, adenosine triphospate; COX, cyclooxygenase; GP, glycoprotein; P2Y12, platlelet membrane protein receptor; TXA2, thromboxane A2.

Storage granules: two important intracellular components within platelets are alpha and dense storage granules:

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α granules: contain P-selectin, fibrinogen, fibronectin, factor V, factor VIII, platelet factor IV, platelet-derived growth factor and tumour growth factor-α (TGF-α)
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dense granules: contain adenosine triphosphate (ATP), adenosine diphosphate (ADP), calcium (Ca), serotonin, histamine and epinephrine.

Platelet activation: there are numerous agonists of platelet activation such as ADP, collagen serotonin – all these lead to activation of intracellular pathways. Collagen a potent activator of platelets, is released from vessel endothelium and binds to glycoprotein (GP) 1a and GPVI, which activates the cyclooxygenase (COX) system that will generate thromboxane A2 (TXA2). TXA2 has several haemostatic functions, such as: it causes vasoconstriction, it leads to recruitment and further activation of more platelets via thromboxane surface membrane receptors, and also causes platelet aggregation. Aspirin inhibits TXA2 production by irreversible inhibition of COX enzyme ( Table 2 ).

Table 2

Summary of antiplatelet drugs, mechanism of action and their limitations

Adapted from Kottke-Marchat K, Davis B. Anticoagulant, antiplatelet and thrombolytic drugs, Chapter 41 in Laboratory Hematology Practice, Wiley–Blackwell 2012.

Drug	Target	Binding	Route	Half-life
Aspirin	Acetylates Ser529 of COX-1	Irreversible	PO	15–20 minutes
Ticlopidine	ADP-P2Y ₁₂	Irreversible	PO	20–50hours after a single dose
Clopidogrel	ADP P2Y ₁₂	Irreversible	PO	7–8 hours
Prasugrel	ADP P2Y ₁₂	Irreversible	PO	7 hours
Cangrelor	ADP P2Y ₁₂	Reversible	IV	3–5 minutes
Ticagrelor	ADP P2Y ₁₂	Reversible	PO	7 hours (ticagrelor) 9 hours (active metabolite)
Abciximab	GP IIb/IIIa	Irreversible	IV	10–30 minutes
Tirofiban	GP IIb/IIIa	Reversible	IV	1.5–3 hours
Eptifibatide	GP IIb/IIIa	Reversible	IV	2–3 hours

IV, intravenous; PO, oral administration.

Platelet adhesion: upon activation, platelets undergo considerable conformational change in order to maximize surface area for adhesion to other surfaces. vWF is essential in promoting platelet adhesion in high shear conditions. vWF is released from the vascular endothelium where it is continually secreted and is stored within platelet granules and in Weibel–Palade bodies within endothelial cells. vWF is a large multimeric molecule that is essential in platelet adhesion, aggregation, and also act as a carrier for coagulation FVIII. Dysfunction or deficiency of vWF results in a bleeding diathesis. Shear forces, stress, exercise, adrenaline and desmopressin/DDAVP stimulates the release of vWF and thus will raise its plasma levels. DDAVP has therapeutic benefit in functional platelet disorders and in von Willebrand disease.

Platelet aggregation is achieved by platelets cross-linking at GPIIb/IIIa receptors on the platelet membrane. GPIIb/IIIa is the most abundant glycoprotein, and upon activation it undergoes conformational change allowing it to bind to fibrinogen, thus forming platelet-fibrinogen bridges. ADP, TXA1 and thrombin all activate GPIIb/IIIa and are potent enhancers of platelet aggregation.

Platelet secretion: once activated, platelets release procoagulant substances that are responsible for a ‘secondary wave’ of aggregation after the initial activation of platelets. The two most significant substances responsible for this positive feedback are ADP (released from dense granules) and TXA2 generated from the COX pathway. Other substances released from granules include serotonin, fibrinogen, fibronectin and growth factors such as PDGF. Impaired release of these mediators results in qualitative defects of platelet function which can be both congenital or acquired (e.g. drug induced). Antiplatelet therapies target various pathways of platelet activation and aggregation – their target, route of administration and their half-lives are described in Table 2 .

After activation, aggregation and secretion, the membrane phospholipid becomes exposed resulting in activation of the clotting cascade. Platelet phospholipids are essential for activation of FX and FII (thrombin) and the formation of the tenase (factors Xa-VIIIa- IXa) and prothombinase complexes (factors Xa-Va-IIa).

Coagulation pathway

The coagulation cascade involves the marked amplification of procoagulant proteins from relatively few initiation substances by the sequential activation of enzyme precursors (zymogens) to active enzymes. These are usually serine protease enzymes. The result is the rapid and marked generation of thrombin, which converts soluble fibrinogen into the insoluble fibrin. Fibrin enmeshes platelet aggregates and converts the unstable platelet plug into a stable fibrin clot.

Traditionally, the coagulation pathway was classified into extrinsic, intrinsic and common pathways. This classical model still remains useful in interpreting in vitro coagulation screening tests (i.e. prothrombin time [PT], activated partial thromboplastin time [APTT]); however, the classical model does not incorporate the central role that cell surfaces play in coagulation. Further, this system does not explain why some patients with coagulation factor deficiencies have bleeding tendencies (e.g. why individuals with factor IX or factor VIII deficiency have severe bleeding even though their extrinsic and common pathways are normal which should be sufficient for haemostasis), and more importantly, it does not predict which patients are at risk of bleeding or thrombosis.

The cell-based model of haemostasis

The ‘cell-based’ model of haemostasis has replaced the classical pathway and it is now the most widely accepted model of in vivo coagulation. The cell-based model proposes that the coagulation process takes place on different cell surfaces and, occurs not as a cascade but in three overlapping stages which include initiation, amplification and propagation.

Initiation: the primary event of in vivo coagulation is the exposure of tissue factor (TF) which will lead to activation of FVII in flowing blood after vascular injury. The TF:FVIIa complex catalyses the activation of FIX and FX. The activated FX which escapes the cell surface environment is rapidly inhibited by tissue factor pathway inhibitor (TFPI) and antithrombin (AT), whereas that which remains on the TF bearing cell will activate a tiny amount of thrombin from prothrombin. This initial thrombin is essential for the activation of more platelets, as well as activation of FVIII and FV, thus setting the scene for the large-scale thrombin generation. The small initial thrombin generated will also activate FXI in a positive feedback manner, leading to amplification.

Amplification: platelets provide the surface on which the amplification and propagation phases take place. During the amplification phase the procoagulant signal shifts from TF-bearing cells to the surface of platelets as these become activated, while in the propagation phase, a large burst of thrombin is generated on the surface of activated platelets ( Figure 1 ).