Normal haemostasis

Learning outcomes

1. Explain the physiological process of haemostasis involving the blood vessel, platelet and coagulation factors
2. List the coagulation factors that are associated with the intrinsic and extrinsic pathways of coagulation

Introduction

Haemostasis is the process that occurs when there is vascular injury. It is a complex dynamic process that involves interaction between the endothelium that covers the inner wall of the blood vessel, sub endothelial structures, platelets and various coagulation factors that are present in circulation or expressed by cells.

The process of haemostasis is broadly divided into two parts i.e. primary and secondary haemostasis. It has to be emphasised though that the two parts are not mutually exclusive, but are highly interactive and often occur at the same time.

Components of haemostasis

The main players in the initiation, amplification and propagation steps of haemostasis include;

1. Endothelium and pro-coagulant sub-endothelial structures sich as collagen
2. Platelets
3. Tissue factor
4. von Willebrand factor
5. Fibrinogen
6. Soluble coagulation factors (FVII, FXI, FIX, FX, Prothrombin) and co-factors (FV, FVIII)

Endothelium

Endothelial cells (EC) form a natural barrier against inappropriate coagulation occurring within blood vessels. In the normal state, EC express factors such as prostacyclin, tissue-factor pathway inhibitor (TFPI), thrombomodulin (TM) and heparin-like proteoglycans which inhibit the activation of the coagulation cascade.

However, damaged EC such as following a cut, induces the cells to release tissue-factor (TF) and von Willebrand factor that promote coagulation and platelet adhesion to the site of endothelial injury. Collagen which is exposed following EC damage can also attract platelet biding to the vessel wall surface.

Platelets

Platelets are non-nucleated cells produced by cytoplasmic budding of megakaryocytes in the marrow. Megakaryocytes are the largest haematopoietic cells in the bone marrow and do not appear in the peripheral blood.

On the peripheral blood, the platelets appear as small bluish-purple dots with a diameter of 2-3 um.

In the resting form, platelets circulate as biconvex (lens shaped) discoid cells. They can however be activated by various agonists. On activation, platelets become spheroidal and form pseudopodia to facilitate clot formation. Platelets also contain various granules and release intracellular substances such as ADP and serotonin that act as platelet-activating mediators as well as Factor V that mediate coagulation during secondary haemostasis.

Tissue factor (TF)

Tissue factor is a cell surface glycoprotein. It is expressed by activated endothelial cells and acts as the primary initiator of coagulation, responsible for the first step of the ‘extrinsic pathway’. Unlike the other coagulation factors that circulate as nonfunctional precursors, TF is a potent initiator that is fully functional when expressed on cell surfaces. FVII binds at high-affinity to TF, forming a TF-FVIIa complex, that cleaves FX and FIX to its active states.

von Willebrand Factor (vWF)

VWF is a large multmeric glycoprotein that is synthesized by endothelial cells and megakaryocytes. The ultra large multimers are cleaved when they are secreted into the circulation by a proteinase called ADAMTS13 into shorter multimers . Failure of proteolytic cleavage due to deficiency or abnormality of the ADAMTS13 protein causes a condition called thrombotic thrombocytopenia purport (TTP) characterised by excessive platelet adhesion and activation causing thrombocytopenia with microangiopathic haemolytic anaemia.

vWF have important roles in;

1. Initiation of primary haemostasis by binding to subendothelial collage following endothelial injury.
2. Promotes platelet adhesion and activation upon binding to its cognate receptor (GP Ib/IX/V) on platelets.
3. Binds FVIII in circulation and prevents its degradation. FVIII is released from vWF on action of thrombin at the vascular injury site, thereby amplifying coagulation during secondary haemostasis.

Fibrinogen

Fibrinogen is a glycoprotein that is synthesized in the liver. It circulates as monomers that can bind to fibrinogen receptors (GP IIb/IIIa) on platelets and promote its aggregation at sites of vessel injury. Fibrinogen is also the precursor protein to fibrin which forms a meshwork polymer on cleavage of its terminal ends by thrombin. The cleavage of fibrinogen ends creates ‘sticky-ends’ that cause individual fibrin monomers to attach together, forming long intertwined polymers that seal the vessel wall injury.

Other coagulation factors and co-factors

The coagulation factors are a series of inactive proteases (zymogens) that are activated in a step-wise manner. The cascade sequentially amplifies each of the protease’s s activity, thereby generating thrombin and converting fibrinogen to fibrin in large amounts within a short time.

Traditionally, coagulation factors can be ascribed to separate pathways based on their in-vitro characteristics of activation.

1. Extrinsic pathways: FVII
2. Intrinsic pathway also known as the contact activation pathway : FXII, FXI, FIX
3. Common pathway: FX, Prothrombin

The coagulation factors FII (Prothrombin), FVII, FIX and FX are also Ca++ dependant factors.

Primary haemostasis

Initiation of haemostasis occurs upon vascular injury, resulting in activation of endothelial cells, and exposure to sub-endothelial cells that include smooth muscle cells and fibroblasts. Subendothelial collagen is exposed allowing the binding of vWF to the collagen via its A3 collagen-binding domain. vWF undergoes a configurational change in the process, assuming a long linear multimeric chain extending into the blood stream where it may attract passing platelets.

Platelets express vWF receptors (GP Ib/IX/V) that bind to the A1 domaion of vWF. vWF ligand binding to platelet GPIb/IX causes activation of the platelet with consequent conformational change and activation of the platelet fibrinogen receptor (GPIIb/IIIa). The activation of GPIIb/IIIa leads to binding of fibrinogen and cross-linking platelets to form a primary haemostatic platelet plug.

Secondary haemostasis and coagulation activation

The traditional approach to explaining the coagulation cascade is by referring to the intrinsic and extrinsic pathways as described in the Khan Academy video above.. This understanding is important when interpreting results of in-vitro tests of coagulation such as APTT, PT and TT. However, in the physiological setting, components of the intrinsic pathway are not recruited to the cascade by FXIIa.

Rather, the extrinsic pathway plays the primary role in initiating coagulation through activation of FX to FXa by TF-FVIIa, which in turn generates thrombin. Thrombin thereafter activates many of the coagulation factors within the intrinsic pathway, leading to a ‘thrombin burst‘. This would form the basis for the ‘cell-based model’ approach to explain physiological basis of haemostasis, consisting of four phases – initiation, amplification, propagation and termination.

Initiation phase

The initiation phase occurs when tissue-factor (TF) expressed by activated endothelial cells on its surface. FVII, which is found in blood, binds to TF, forming the highly active TF/FVIIa complex. FVII in complex with TF is almost a thousand time more active than FVII alone. TF/FVIIa enzymatically cleaves inactive FX to the active FXa as well as FIX to FIXa.

FXa forms a complex with prothrombin (FII) and the cofactor, FVa, on phospholipid surface in the presence of Ca++. This is also referred to as the ‘prothrombinase complex‘. Within this complex, prothrombin is cleaved to the active form, thrombin.

Similar to the prothrombinase complex, FIXa can also form an ‘intrinsic tenase’ complex with FX, FVIIIa and Ca++ on phospholipid surfaces to cleave inactive FX to active FXa, which in turn can participate in the ‘prothrombinase complex’.

The main result of these activation steps is the generation of thrombin that can contribute to further amplification steps.

Amplification

Thrombin is a key molecule in the coagulation cascade as it has several functions. These include;

1. Cleavage of fibrinogen to fibrin monomers that aggregate to form a fibrin mesh and seal the area of vessel injury.
2. Release and activation of vWF-bound FVIII to FVIIIa
3. Activation of FV to FVa as well as promote its release from endothelial cells and platelet
4. Activation of FXI to FXIa
5. Activation of FXIII to FXIIIa. FXIIIa is a transglutaminase that cross-links fibrin strands by forming is-peptide bonds between glutamine and lysine residues of fibrin, thereby stabilising the clot.
6. Activation of platelets.

The various actions of thrombin amplifies the coagulation cascade and promotes clot formation.

Propagation

Termination

The coagulation process must be regulated and limited to the injury site to prevent thrombotic occlusion of the vessel. Four natural anticoagulants are involved to control the spread of coagulation activation;

1. tissue factor pathway inhibitor (TFPI),
2. protein C (PC),
3. protein S (PS) and
4. antithrombin (AT). 

TFPI is a protein secreted by the endothelium inhibits TF/FVIIa complex and limits coagulation.

Thrombin also counterbalances its pro-coagulant actions with its anticoagulant and fibrinolytic activity. This occurs through its co-factor activity with thrombomodulin (TM) that activates protein C (PC). PC has an inhibitory action on thrombin. The activity of PC is also increased by another cofactor inhibitor, which is PS. Another natural anticoagulant, AT, inhibits the activity of thrombin and other serine proteases such as FIXa, FXa, FXIa and FXIIa.

Thrombin can also promote secretion of tissue plasminogen activator with subsequent conversion of plasminogen to plasmin, that digests the fibrin meshwork.