Haemostasis

CLINICAL SCIENCES

Haemostasis

Key points

Steven K Austin

C

The four principal elements essential for functional haemostasis are endothelium, platelets, coagulation factors and their regulators, and the process of fibrinolysis

Haemostasis involves an explosive reaction, designed to curtail blood loss, restore vascular integrity, provide a barrier to infection and ultimately preserve life. Haemostatic balance is maintained through four key components e vascular endothelium, platelets, coagulation pathway and fibrinolysis. Any imbalance in this tightly regulated process can result in thrombotic or haemorrhagic conditions with associated morbidity and mortality.

C

Primary haemostasis refers to the role of platelets recognizing injury and interacting with the endothelium and components of the subendothelium to cause an initial arrest of bleeding

C

Secondary haemostasis refers to the coagulation process that leads to fibrin formation

Keywords Anticoagulant pathways; coagulation; endothelium; fibri-

C

Fibrinolysis and natural anticoagulants aim to limit and remodel a thrombus, and ultimately regulate the control of haemostasis to restore and maintain the delicate haemostatic balance

Abstract

nolysis; haemostasis; platelets

Introduction

fibrinolysis by secreting plasminogen activator inhibitor (PAI-1) and reduces surface expression of the anticoagulant, thrombomodulin. Furthermore, stimulated endothelial cell attract leucocytes by synthesizing chemokines and expressing intracellular adhesion molecules (leucocyte integrins). These procoagulant events are themselves regulated, limiting intravascular extension of the thrombus. Proposed mechanisms include the negative charge of intact endothelium (repels platelets), adjacent prostacyclin release (inhibits platelet activation), heparan inhibition of thrombin, thrombomodulin enhancement of thrombin anticoagulant effects, and secretion of tissue plasminogen activator (tPA), which can initiate fibrinolysis. The extent to which each property (metabolic, structural) of the endothelium dictates the fine balance between procoagulant and anticoagulant phenotype varies, and led to the concept of vascular bed-specific haemostasis. Responsible mechanisms include growth factors, cytokines, mechanical forces, circulating lipoproteins, coagulation factors and components of extracellular matrix. Hence the prevalence of pathological thrombosis varies at different vascular sites, and may be associated with different acquired factors or disease states.

Haemostasis is an essential protective mechanism that depends on a balance between procoagulant and anticoagulant processes. Rapid transformation of blood from its fluid state into a localized thrombus at the site of tissue damage is controlled by an intricate interplay of four key components e vascular endothelium, platelets, the coagulation pathway and fibrinolysis; each is discussed in turn.

Endothelium The role of the endothelium is multifaceted. It acts primarily as a physical barrier separating haemostatic blood components from reactive subendothelial structures. It modulates vascular tone and permeability. In addition, endothelial cells produce inhibitors of coagulation and platelet aggregation (Figure 1). Expression of specific proteins (thrombomodulin) and mucopolysaccharides (heparan sulphate, dermatan sulphate) promote an anticoagulant effect by accelerating the action of circulating natural anticoagulants. Platelet aggregation is inhibited by endogenous synthesis of ectoenzymes, which degrade adenosine diphosphate (ADP; a platelet agonist), and production of prostacyclin and nitric oxide. Finally, the endothelium modulates fibrinolysis by producing activators and inhibitors of clot lysis. Tissue damage disrupts the integrity of the endothelial basement membrane, exposing the underlying extracellular matrix and prothrombotic haemostatic factors, including collagen, von Willebrand factor (VWF), fibronectin (promotes platelet adhesion) and tissue factor (TF). Additionally, antithrombotic endothelial properties are lost when stimulated by thrombin, shear stress, oxidants, endotoxin or cytokines interleukin-1, tumour necrosis factor and interferon-g. Activated endothelial cells express TF, which initiates the coagulation pathway, impairs

Platelets The circulating platelet is an anuclear discoid cell produced from megakaryocytes.1 It functions as a vehicle for transporting regulatory factors, prothrombotic proteins, growth factors and other molecules inside platelet granules to the endothelium. The platelet membrane functions as a template for promotion/acceleration of haemostasis and wound healing. Platelets circulate close to the endothelium, facilitating rapid recognition disruption or injury. A reduction in the number of platelets results in a bleeding tendency. The normal platelet count is 150e450  109/ litre; at <80  109/litre, haemostasis may be impaired. The risk of bleeding correlates with the severity of platelet reduction. The process of platelet plug formation is called primary haemostasis, as opposed to the secondary events of the procoagulant system. Primary haemostasis consists of platelet adhesion,

Steven K Austin MB BS(Hons) BMedSci FRACP FRCPA is a Consultant Haematologist, Haemophilia Centre, Guys and St Thomas’ Foundation Trust Haemophilia Centre Director, St George’s Hospital NHS Healthcare Trust, UK. Competing interests: none declared.

MEDICINE --:-

1

Crown Copyright Ó 2017 Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Austin SK, Haemostasis, Medicine (2017), http://dx.doi.org/10.1016/j.mpmed.2017.01.013

CLINICAL SCIENCES

Endothelial haemostatic function In the resting state (upper surface), the endothelium functions as an effective anticoagulant. Its negative surface charge repels platelets, and nitric oxide and prostacyclin inhibit platelet function. Anticoagulant properties are enhanced by surface expression of Negative thrombomodulin and heparan surface charge – sulphate. However, after stimulation by cytokines or tissue damage, the endothelium rapidly becomes prothrombotic (lower surface). Platelet adhesion is promoted by exposure of subendothelial collagen and von Willebrand factor. Meanwhile, tissue generation and clot formation, while of plasminogen activator inhibitor. Anticoagulant properties are also modulated by reduced expression of surface thrombomodulin and heparin sulphate

Endothelial damage

Inhibition of platelet aggregation

Nitric oxide and prostacyclin

–

Thrombomodulin Heparan sulphate

Anticoagulant surface

–

↓Thrombomodulin Plasminogen activator inhibitor

Exposure of Tissue factor collagen and von Willebrand factor Coagulation pathway Platelet adhesion

Fibrin generation

Figure 1

activation and aggregation, a seamless dynamic interaction between the endothelium, plasma proteins and platelets.

second platelet surface glycoprotein, GPIIb-IIIa receptor, undergoes conformational change; this allows synergistic binding between GPIIb-IIIa and fibrinogen, leading to firmer, permanent adhesion. GPIIb-IIIa can also bind with VWF, and other adhesive proteins (fibronectin, vitronectin), which may play a more important role at low shear rates, when they can substitute for the action of VWF. Adhesion is also aided by interaction of collagen with GPIa-IIa.

Adhesion: blood flow is normally laminar, faster at the centre than the edge, creating a shear effect. Shear force is greatest at the endothelium. Platelets are displaced to the mural plasma zone and exposed to maximal vessel wall shearing forces. Under such high shear, platelets contact the endothelium briefly, ‘rolling’ along the endothelial surface. This interaction can occur with both intact (activated) and disrupted endothelium, slowing platelet velocity and encouraging more substantial tethering to the vessel wall. Platelet tethering to exposed thrombogenic surfaces occurs when the platelet surface glycoprotein Iba (GPIba) binds to VWF. VWF is a large, multimeric adhesive protein synthesized by the endothelium and secreted into the plasma and subendothelial matrix. VWF binds to collagen (and possibly other subendothelial components), intact endothelium precludes any interaction between VWF and platelets. In areas of vascular injury exposed to high shear forces, subendothelial VWF is exposed, whereas (more importantly) circulating plasma VWF is immobilized after contact with subendothelial elements. This causes VWF to undergo a conformational change, exposing binding sites for GPIba on the platelet membrane, which ultimately initiates the platelet response to vascular injury. Simply put, VWF acts as a bridge between platelets and subendothelial connective tissue, probably the initial step that tethers platelets to areas of damage. However, this initial attachment is not stable, and a concomitant high dissociation rate allows platelets to continue rolling slowly along the endothelium. Meanwhile, platelet activation begins, and a

MEDICINE --:-

Activation: platelets become activated as they adhere stepwise to endothelial cells (or a variety of other surfaces). This also results in endothelial cell activation and the expression/secretion of chemokines. Activation results in a marked structural change, the platelets becoming spherical with protuberant pseudopodia. Platelet granules centralize secondary to activation of the cytoskeletal contractile apparatus, and secretion follows. Subsequent contraction of these microfibrils may cause clot retraction and promote platelet plug formation. Exposure of negatively charged platelet phospholipids and receptors for specific plasma clotting factors, particularly activated factor V derived from platelet agranules, provide a procoagulant surface for assembly of the enzymeeco-factor complexes of the coagulation pathway. Platelet activation is caused by binding of various agonists (e.g. thrombin, thromboxane A2, ADP, collagen, arachidonic acid) to specific receptors.2 Signal transduction is mediated by G proteins and intracellular cyclic adenosine monophosphate (cAMP); increased cAMP concentrations inhibit platelet adhesion, aggregation and release. Following platelet activation, adenylate cyclase activity is reduced, decreasing cAMP concentrations and increasing mobilization of calcium. Many calcium-

2

Crown Copyright Ó 2017 Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Austin SK, Haemostasis, Medicine (2017), http://dx.doi.org/10.1016/j.mpmed.2017.01.013

CLINICAL SCIENCES

concept of the coagulation cascade was based on the concept of distinct intrinsic and extrinsic coagulation pathways. This model remains useful in vitro for diagnostic purposes as it has led to the development of plasma-based laboratory tests (screening tests) to identify deficiencies in the extrinsic (prothrombin time (PT)), intrinsic (activated partial thromboplastin time (APTT)) and common (thrombin time (TT)) pathways (Figure 2). In vivo, the coagulation pathways are integrated3 most importantly because TF-factor VIIa complex (TF-VIIa) activates both factor IX and factor X (Figure 3). TF, a glycoprotein constitutively expressed on the surface of fibroblasts and expressed on damaged or stimulated cells (monocytes, macrophages, endothelial cells) is crucial to the initiation of the procoagulant system. Following endothelial perturbation, circulating factor VII or activated factor VII (VIIa) binds to exposed TF, resulting in assembly of the potent TF-VIIa complex that activates limited amounts of membrane-bound factors IXeIXa and X eXa. This is called the initiation phase. The initial factor Xa produced by this mechanism combines with factor Va and generates sufficient thrombin to induce local platelet aggregation and activation of the critical co-factors V and VIII (the propagation phase). However, it is insufficient to sustain haemostasis because of rapid factor Xa-dependent inactivation of TF-VIIa by TF pathway inhibitor. Instead, marked amplification is achieved via the action of factors IXa and VIIIa, which form the tenase complex (activates factor XeXa). Absence of these factors in haemophilia syndromes can produce marked haemorrhagic states. Factor XIa, a contact factor protein of the intrinsic

dependent reactions follow, including phosphorylation of myosin light chain (initiating the contractile reaction of the release mechanism), secretion of platelet granule contents (fibrinogen, VWF, thrombospondin, factor V and others) and liberation of arachidonic acid. These events are modulated by several regulatory substances produced by the platelets themselves (cAMP) and also endothelial cells (ADPase, thrombomodulin, nitrous oxide). Aggregation: as platelets become activated, spreading out on the subendothelium, additional platelets arrive and adhere to the adherent activated platelets. This process is mediated through fibrinogen bound to GPIIb-IIIa on the activated platelets, which acts as a bridge-like structure catching passer-by platelets. These new recruits in turn become activated, releasing granules and promoting the process further. A microthrombus of aggregated platelets is rapidly formed. Interaction of other proteins (fibronectin, thrombospondin) with glycoprotein receptors assists in the aggregation process, possibly stabilizing the platelet aggregate.

Coagulation The coagulation process is composed of a series of serine protease enzymes and their co-factors that interact on a phospholipid surface (platelet membrane, damaged endothelium) to form a stable fibrin clot.3 This process consists of a recurrent theme involving the rapid assembly of co-factors, enzymes and substrates to result in a maximally efficient rapid molecular reaction. The traditional

Classic waterfall hypothesis of coagulation showing the intrinsic, extrinsic and common pathways and the associated coagulation screening tests

PK

Intrinsic pathway

Extrinsic pathway

HMWK

FXII

FXII

K HMWK

FXIIa APTT

PT

FXIa

FXI HMWK

FIX Ca

FVIIa

FIXa FVIIIaCa

TF

FX

Ca

FX

Common pathway

Fibrinogen

FXa FVa

Ca

FIIa

FII

PT, APTT

TT FXIII

Cross -linked

FXIIIa

APTT, activated partial thromboplastin time; F, factor; HMWK, high-molecular-weight kininogen; K, kalikrein; PK, prekalikrein; PT, prothrombin time; TT, thrombin time. Figure 2

MEDICINE --:-

3

Crown Copyright Ó 2017 Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Austin SK, Haemostasis, Medicine (2017), http://dx.doi.org/10.1016/j.mpmed.2017.01.013

CLINICAL SCIENCES

Revised hypothesis of blood coagulation X Tissue factor (TF) has an integral role in the initiation of coagulation, through activation of factor VII. Activated factor X (Xa) is generated by factor VIIa. TF complex. Because of factor Xa-dependent inactivation of VIIa. TF by TF pathway inhibitor, factors IX and XI are tenase complex to ensure thrombin generation. The central role of thrombin is shown. Coagulation is limited by the action of the anticoagulant system Prothrombin

VII + Tissue factor

IX

XI

Tissue factor pathway inhibitor VIIa.TF

Xa + Va

XIa

Activated protein C Protein S V Thrombomodulin

IXa + VIIIa VIII XI

Phospholipid + Ca2+

Vitamin K-dependent serine proteases Co-factors Anticoagulant system

XIII Thrombin XIIIa

Antithrombin Fibrinogen

Fibrin

Cross-linked

Figure 3

pathway, may be required to produce additional factor IXa if insufficient quantities are generated by TF-VIIa, or if fibrinolysis is particularly active. The remaining components of the intrinsic system are important in vitro, but do not appear to have an important haemostatic role. An abundance of Xa results in the formation of prothrombinase complex (factor Xaefactor Va), which rapidly converts prothrombin to thrombin (thrombin burst). Thrombin hydrolyses the arginineeglycine bonds of fibrinogen to form fibrin monomers, and activates factor XIII, which cross-links the fibrin and improves tensile strength. Thrombin also has a positive feedback role, promoting activation of factor XI and the cofactors V and VIII, thereby influencing the haemostatic plug formation. Ultimately, the formation of the fibrin strands represents the second phase of haemostasis. Modern anticoagulants are now in therapeutic use that target specific coagulation factors such as factor Xa and thrombin. These offer promise as a reliable and predictable means of anticoagulation that may simplify therapy in this area.

selectively inhibits thrombin in the presence of heparin or dermatan sulphate. Another natural anticoagulant is protein Zdependent protease inhibitor, which inhibits factor Xa in a reaction that is enhanced by the co-factor protein Z. Factor Xa is also inhibited by TF pathway inhibitor (TFPI) which complexes with FXa and can subsequently inhibit the activity of FVIIa-TF complex. Cleverly, thrombin also functions as an anticoagulant by binding to surface-bound thrombomodulin. The thrombin ethrombomodulin complex is a potent activator of protein C, which (in the presence of protein S) can inactivate co-factors Va and VIIIa and enhance fibrinolysis. Factor V Leiden is a common mutation of factor V that is resistant to protein C inactivation and hence is associated with increased thrombotic risk. Patients with deficiencies of protein C or S also have a predisposition to venous thromboembolic disease. Interfering in the function of anticoagulants such as antithrombin and TFPI offers a potential therapeutic advance to reset the haemostatic balance in patients with coagulation factor deficiencies (haemophilia) and are currently in advanced stages of clinical development.

Anticoagulant pathways: a delicate balance between procoagulant and anticoagulant mechanisms is needed to limit thrombosis to the site of injury. Naturally occurring anticoagulants include antithrombins (serpins), which inhibit serine proteases of the coagulation system, and the protein C system, which neutralizes activated coagulation co-factors. At least seven serpins have been identified, but antithrombin and heparin co-factor II are thought to be the major anticoagulants. Antithrombin inhibits factors IXa, Xa and thrombin; this is greatly potentiated by heparin, whereas antithrombin deficiency is associated with a striking risk of venous thrombosis. Heparin co-factor IIa

MEDICINE --:-

Fibrinolysis Clot dissolution, or fibrinolysis, functions as a clot-limiting mechanism and process of repair, and consists of a series of tightly regulated enzymatic steps. Plasminogen, a key player, is cleaved to the active serine protease, plasmin, by tPA. tPA activity is enhanced when bound to the surface of fibrin. Plasmin hydrolyses arginine and lysine bonds in its major substrates, fibrinogen, fibrin and factors V, VIII and XIII. Cleavage of fibrin and fibrinogen generates fragments X and Y, which inhibit thrombin and fibrin polymerization, respectively. Excessive

4

Crown Copyright Ó 2017 Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Austin SK, Haemostasis, Medicine (2017), http://dx.doi.org/10.1016/j.mpmed.2017.01.013

CLINICAL SCIENCES

2 Longstaff C, Kolev K. Basic mechanisms and regulation of fibrinolysis. J Thromb Haemost 2015; 13(suppl 1): S98e105. 3 Hoffman M, Monroe 3rd DM. A cell-based model of hemostasis. Thromb Haemost 2001; 85: 958e65.

fibrinolysis, resulting in fibrinogen consumption and haemorrhage, is prevented by inhibition of tPA by PAI-1, and of plasmin by a2-antiplasmin. Thrombolysis is based upon activating fibrinolysis. Analogues of tPA, other recombinant-based plasminogen activators (e.g. alteplase, reteplase, tenecteplase), streptokinase and urokinase are administered to clear blood clots (e.g. after myocardial infarction or stroke). A

FURTHER READING Gomez K, McVey JH. Normal haemostasis. In: Hoffbrand AV, Higgs DR, Keeling DM, Metha AB, eds. Postgraduate haematology. 7th edn. Blackwell, 2016. Chapter 36. Yau JW, Treoh H, Verma S. Endothelial control of thrombosis. BMC Cardiovasc Disord 2015; 15: 130.

KEY REFERENCES 1 Michelson AD. Platelets. 2nd edn. 2013. Academic Press, 2013.

TEST YOURSELF To test your knowledge based on the article you have just read, please complete the questions below. The answers can be found at the end of the issue or online here. Investigations  Platelet count 22  109/litre (150e400)  International normalized ratio 1.0 (<1.4)

Question 1 A 24-year-old man presented to the emergency department with epistaxis. He had had two previous episodes in the past year. He was noted to have several large bruises from playing rugby. Clinical examination was otherwise normal.

Which of the following is the most important mechanism by which platelets promote clotting? A. Metabolically active nucleus producing high levels of clotting factors B. Development of pseudopodia on contact with damaged endothelium C. Ability to remain in the midstream of blood flow D. Aggregation between platelets that is mediated by thrombin E. Secretion of granules rich in collagen

Investigations  Platelet count 210  109/litre (150e400)  Bleeding time 15 minutes (3e8)  International normalized ratio 1.0 (<1.4)  Factor VIII level 25 IU/dl (50e150)  VWF: Activity 5 IU/dl (50e150) What is the most important defect causing this problem? A. Impaired binding of coagulation factors to the surface of platelets B. Defective platelet-to-platelet binding C. Anchoring of platelets to the subendothelium D. Initiation of the tissue factorefactor VIIa complex formation by exposure to tissue factor E. Failure of cleavage of plasminogen to plasmin

Question 3 A 25-year-old woman was admitted with a deep vein thrombosis following a long journey by air. She was 32 weeks’ pregnant. She was treated with heparin until delivery. Through which target does heparin exert its effect? A. Plasminogen B. Protein C C. Tissue factor pathway inhibitor D. Antithrombin E. Thrombomodulin

Question 2 A 35-year-old woman presented with a rash on her legs. She was otherwise well. On clinical examination, there was purpura in the skin of the lower legs but no other abnormality.

MEDICINE --:-

5

Crown Copyright Ó 2017 Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Austin SK, Haemostasis, Medicine (2017), http://dx.doi.org/10.1016/j.mpmed.2017.01.013