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Year 1 MBChB – Medicine Student Selected Component (SSC)

The Molecular Basis of

Blood Coagulation Disorders

Student : U Bhalraam Supervisor : Dr David Martin

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Abstract

This report includes a detailed overview of the mechanism by which blood

coagulates to form the secondary haemostatic plug from the primary haemostatic

plug via the action of the coagulation cascade. It is structured into several parts. The

first of which describes how the blood maintains its low viscosity by various blood

thinning mechanisms, enabling blood to course, smoothly through our vasculature

without clotting problems. Next, we explore the coagulation cascade and how it plays

a vital role in strengthening the primary clot formed by platelet aggregation. The

essential biochemical modification (gamma-carboxylation) that is essential to forming

the structure of coagulation factors IX, X, VII and II will then be explored. Lastly, we

focus our discussion on the Factor Xa/Va (Prothrombinase) complex and stress its

importance in the coagulation cascade. After reviewing the various methods in which

blood coagulation can be measured quantitatively, we cover various specific

mutations to the coagulation factors and how that affects its structure and hence its

function.

Introduction

Blood is the fluid that courses through our arteries and veins, delivering oxygen,

amongst other substances, to every tissue in the body. (1) It plays an essential role

in the distribution of hormones, enzymes and nutrients in addition to gaseous

exchange. Its buffer properties allow enzymes to work effectively despite pH

changes. (1) It is therefore essential that blood is kept liquid in order to perform its

functions and also be able to coagulate to prevent its loss. The endothelial

vasculature has adapted to actively avoid unwanted coagulation. This keeps blood in

the liquid state, when it performs its functions best.

Virchow’s Triad for thrombogenesis suggests that there are 3 factors that influence

coagulation.(2) However, the ones that hold significant importance are endothelial

damage and hyper-coagulability.(2) Our body regulates hypercoagulability in such a

way that it reacts sufficiently when endothelial damage occurs, but doesn’t trigger

coagulation without any stimulus.(3) A fine balance must be achieved. There are

various processes that control the hypercoagulability of the blood.(3)

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Active anti-coagulative processes of the blood.

The first of the many mechanisms is the action of nitric oxide (NO) released by

healthy endothelial vasculature.(4) It is a vasodilator and an anti-platelet aggregator.

(4,5). Prostacyclins or Prostaglandin I2 are anti-platelet aggregation agents,(6) and

they work synergically with NO to prevent the coagulation of blood. (5) Endothelial

cells also secrete ADP de-phosphatases to break down ADP which stimulate platelet

aggregation (3).

The endothelial vasculature also expresses certain proteoglycans which also spur

anti-coagulant reactions in the blood.(6) Most cells express heparin-sulfate on their

membranes.(6) These proteoglycans have a high affinity for Anti-thrombin III

(ATIII),(6) thus, once bound, it causes a conformational change that activates the

serine protease.(7) This leads to the activated ATIII then inactivates thrombin and

coagulation Factors XIa, Xa and IXa as shown in fig.1.(3)

Fig.1 – Visual representation of the action of anti-thrombin III(3)

Healthy vascular endothelium also expresses thrombomodulin on its surface.(6) This

glycoprotein has a high affinity for thrombin.(6) Once bound, thrombin activates

protein C. (6)With the help from protein S co-factor, activated protein C inactivates

coagulation factors Va and VIIIa. (8)

One of the other anti-coagulant glycoproteins expressed on vascular endothelium is

tissue plasminogen activating factor.(6) This activates plasminogen proteins into

plasmin, (6) which then proceeds to actively break down fibrin strands, thus inhibiting

clot formation.(3)

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Blood coagulation cascade

In haemostasis, there are 2 parts to the formation of the haemostatic plug. There is

the formation of the ‘primary haemostatic plug’ from the processes of platelet

adhesion, degranulation and aggregation. However, fibrin is required to strengthen

this clot, converting it into a ‘stable haemostatic plug’. The reactions that give rise to

the conversion of fibrinogen into a cross-linked fibrin mesh, are governed by

coagulation factors. Table 1 shows the complete list of coagulation factors and their

roles in their active forms. (3)

All coagulation factors, except Fibrinogen, are either enzyme precursors, or

cofactors.(3) All the enzymes are serine proteases except for factor XIII.(9) Serine

proteases are proteins whereby their ability to hydrolyse peptide bonds are

dependant on the serine residue in the peptide’s active centre. (10) Figure 4 shows

the activation of Factor X, a serine protease. (3)

Fig. 4 – Activation of a serine protease: Factor X (3)

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Blood Coagulation Cascade: Initiation

When endothelial cells are damaged, the Tissue Factor (TF) on the basolateral

membrane of these cells are exposed to the vessel lumen.(3) These tissue factors

are usually expressed in the inactive form, and thus, have to be subsequently

activated by disulphide isomerase following vascular injury.(3) TF forms a complex

with activated factor VII, which in turn is known as extrinsic factor Xase.(3) 1 to 2 per

cent of the total VII population lies in the activated state so the initiation complex can

be formed.(3) TF:VIIa complex then proceeds to activate coagulation factors IX and X

into their active forms, IXa and Xa respectively. Factor Xa then activates prothrombin

into thrombin, which subsequently activates fibrinogen into fibrin monomers.(3)

However, this is insufficient to initiate fibrin cross-linking and polymerisation due to

the absence of activated factor XIII.(3)

Blood coagulation cascade: Amplification

If the levels of tissue factor following injury is low and doesn’t warrant a coagulation

response, tissue factor pathway inhibitor (TFPI) stops the initiation pathway.(3) TFPI

is a multivalent protease inhibitor with 3 Kunitz-type domains. They bind to TF, VIIa

and Xa.(11) This forms a quaternary complex TF/VIIa/TFPI/Xa.(9) It inactivates

further activation of Xa via the extrinsic pathway. (9)

Table 1: Outlining the various coagulation factors and their role in their active form (6)

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The thrombin generated from the extrinsic pathway then goes on to activate 5 other

coagulation factors. They are FXI, FVIII, FV, FXIII and FI.(3) The activation of these

coagulation factors are sufficient to kick-start the intrinsic pathway for coagulation.(3)

The coagulation cascade is split into 2 main pathways: the extrinsic and the intrinsic

pathway. The extrinsic pathway is also known as the initiation pathway, as previously

discussed. The intrinsic pathway is governed by a different set of reactions that lead

to the activation of factor X and the eventual breakdown of fibrinogen and formation

of the fibrin mesh.(3)

In the event where collagen fibres are exposed, FXII can be activated into FXIIa.

FXIIa then activates FXI into XIa. (12) It is important to note that the activation of FXII

is not essential in the intrinsic pathway as preformed thrombin from the extrinsic

pathway activates FXI as well. (12) FXIa then proceeds to activate FIX to FIXa, which

eventually results in the activation of FX to FXa. FVIIIa, which thrombin has already

activated, catalyses this process. (12) By the similar mechanism as described above,

FXa activates prothrombin into thrombin, which in turn breaks down fibrinogen into

insoluble fibrin monomers.(11) FXIII is activated by thrombin into FXIIIa which aids in

the crosslinking of the fibrin monomers to form a stable fibrin mesh on the primary

haemostatic plug(3)

A diagram outlining the processes above can be seen in figure 5. (12)

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(12)

Figure 5: Outlining the extrinsic, intrinsic and common pathways of the coagulation cascade (12)

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Gamma-carboxylation of glutamic acid residues in coagulation factors

As mentioned previously, on activation of platelets by ADP, Delta granules de-

granulate and secrete Ca2+.(13) It is precisely the presence of these Ca2+ ions that

speed up the coagulation cascade. (3)

Factors II, VII, IX, X and Protein C and Protein S all have glutamic acid residues that

undergo Vitamin K dependant gamma-carboxylation.(3) Vitamin K gets converted into

Vitamin K epoxide via the enzyme: epoxide reductase. Gamma carboxylation is

necessary in creating negative charges in the coagulation factors, protein C and

protein S. (3)

All glutamic acid residues present in the gamma-carboxyglutamic acid-rich(GLA)

domains are potential carboxylation sites.(14) Additionally, they are also modified to

GLA in coagulation factors by the abovementioned mechanism. GLA domains are

responsible for the high-affinity binding of Ca2+ ions.(14)

Ca2+ ions introduce conformational changes in the GLA domains and are essential for

the proper folding of the domain.(15) A common structural feature of functional GLA

domains is the clustering of N-terminal hydrophobic residues into a hydrophobic

patch that mediates interaction with the cell surface membrane. (15)

Therefore, gamma carboxylation allows for the coagulation factors to be bound down

to a particular phospholipid surface, enabling faster interactions between coagulation

factors, speeding up the process of coagulation.

Figure 6: Summary of Vitamin K dependant gamma carboxylation.(3)

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Scope of the study of coagulation disorders

As seen above, the coagulation process is extremely complex and there are many

processes that can fail and give rise to coagulation disorders. This is the scope of this

study has been narrowed down to that of coagulation factors X, V and the Pro-

thrombinase complex. (Xa/Va)

These coagulation factors have been specifically selected as they occupy a pivotal

position in coagulation. Either the intrinsic or extrinsic pathways activate them.

Hence, the understanding coagulation disorders in the critical portion of the cascade

is of significant importance.

Tests of haemostatic function

There are several diagnostic tests that enable us to evaluate whether the

haemostatic process in the body are proceeding normally. They are thrombin time,

activated partial thromboplastin time, prothrombin time, Russell’s viper venom time,

chromogenic assays and the specific levels of a coagulation cascade. (11,12,16)

Thrombin time (TT) is the time taken for blood plasma to coagulate after thrombin is

added. This tests for the fibrinogen concentration and if anti coagulants are present

(10)

Prothrombin time (PT) evaluates the extrinsic pathway in the coagulation cascade.

(10) It detects abnormalities that result in deficiencies or other forms of inhibition in

coagulation factors II, VII, IX and X.(3)

Activated partial thromboplastin time (aPTT) is an evaluation test that involves partial

thromboplastin (a contact activator) and calcium are added to the blood plasma to

test the intrinsic pathway of the coagulation cascade(10)

Russell’s viper venom time (RVVT) is a diagnostic test performed, exposing the

blood plasma to Russel’s viper to induce hemostasis and thrombus formation.(16)

The active agent in the venom directly activates factor X. A standardised dRVVT

solution us used in the majority of cases, which gives the average clotting time to be

between 23 and 27 seconds.(16) This test is usually used in determining if there are

any problems with the common pathway of coagulation.(16)

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These tests are, for the most part, the basis of which coagulation disorders are

diagnosed.

Causes of blood coagulation disorders

There are 2 main categories of blood coagulation disorders-Inherited and Acquired.

Acquired disorders are not directly genetic and have other factors involved, a prime

example being Vitamin K deficiency. The lack of Vitamin K can prevent normal

gamma-carboxylation of the glutamic acid residues on coagulation factors II, VII, IX

and X, resulting in little or no activation of coagulation factors of the coagulation

cascade.

Inherited disorders, on the other hand, have a much higher genetic correlation. This

is usually due to mutations in the genome which give rise to defective coagulation

factors. These defects affect the normal coagulation of the blood resulting in

prolonged coagulation times.

Structural and functional analysis of Factor X coagulation factor

Factor X is a vitamin K-dependent precursor serine protease which, once activated,

forms thrombin from prothrombin in the presence of factor Va, Ca2+ and a

phospholipid membrane during coagulation.(17)

The factor X gene contains 8 exons and located on chromosome 13q34. (18,19) 105

mutations, as of 2012, on FX have been identified. (19,20)

Factor X is mainly synthesized in the liver.(19) The peptide consists of several

segments, in which the first 40 amino acid residues are named pre-propeptide.(19)

They contain hydrophobic signal sequences that target the protein for secretion.(19)

This pre-propeptide is then removed by 2 subsequent clevages. These propeptides

are important to direct intracellular posttranslational modifications.(19) It also

contains a heavy chain, (Mr 42,000), and a light chain, (Mr 17,000). A single

disulphide bond (between residues Cys89/Cys124(19)) holds these chains together.

The N-terminal of the light chain holds the 11 gamma-carboxylated glutamic acid

residues crucial for phospholipid and Ca2+ adhesion.(17)

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The peptide also contains 2 epidermal growth factor (EGF) domains.(21) The first of

which contains an aspartic acid residue that is essential in the Ca2+ interaction.(19)

This is visually represented in figure 7 below.

Figure 7: The 4 domains of the Factor X peptide chain

During coagulation, factor X is converted into factor Xa. In the activation of factor X, a

specific arginine-isoleucine bond is cleaved proximal to the N-terminal of the heavy

chain, creating an activation peptide (Mr 14,000) and Factor Xa. (17) This can be

visually presented as in figure 4. The heavy chain contains the catalytic domain that

is structurally homologous to serine proteases. (20).

Upon activation, factor Xa binds to its cofactor factor Va forming the prothrombinase

complex, which then activates prothrombin in the presence of factor V, Ca2+ and

phospholipid membrane.(22)

Factor X deficiency

Factor X deficiency is an extremely uncommon autosomal recessive bleeding

disorder, affecting 1 in every 1,000,000 people. It is the most severe form amoungst

bleeding disorders.(19)

Although it is rare, in populations with a high incidence of consanguineous

marriages, they can be more frequent(22)

Factor X deficiency, if not acquired, is an inherited bleeding disorder caused by an

abnormality of coagulation factor X.(20) As the body produces less factor X than the

body requires, the clotting process eventually comes to a stop.(20)

The severity of the disease is directly proportional to the amount of serum Factor X

or Xa in the blood.(18)

We shall now discuss examples of factor X deficiencies.

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Factor XKetchikan

Factor XKetchikan is an example of factor X deficiency. It is caused by a point mutation

on the 14th residue on the Factor X light chain switching out Gla for Gly. (21)This

prevents gamma carboxylation of that residue, affecting the ability to bind to Ca2+ at

that location. This is seen in the figure 8 below. The model on the right shows the

presence of gamma carboxylation and binding with a calcium ion. However, on the

right, in Factor XKetchikan, there is an absence of this interaction which gives rise to an

abnormal interaction with calcium ions, resulting in an abnormal Factor X. Phenotypic

analysis would show both a decrease in antigenic and functional levels to less than

1%.(21)

Fig. 8 : Models of Factor X light chains

(Normal on the left, Factor XKetchikan on the right)

Factor XStockton

Factor X Stockton is a unique blood coagulation factor X variant. This leads to the

people affected, having prolonged prothrombin times and activated partial

thromboplastin times(23), thus showing very low level of both factor X activity.(23)

The cause of this deficiency is said to be from a single G to A substitution in one

allele of factor X resulting in an amino acid substitution of Asn to Asp at residue

282.(23) This mutation alters the active site of Factor Xa preventing it from activating

prothrombin to thrombin.

Factor XKurayoshi

Factor XKurayoshi is another mutation that leads to Factor X deficiency. Factor XKurayoshi

is due to a C to A mutation in exon 6.(24) This results in the amino acid located in

residue 139 to be changed from Arg to Ser.(24) This mutation is located in the

second EGF-like domain of factor X that plays a major role in setting the stage for the

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interactions between factor X and factors Va and VIIIa.(24) Thus, this mutation will

interfere in the interactions with Va and hence the formation of the prothrombinase

complex.(24)

Acquired Factor X deficiency

Patients who do not have a significant history of persistent haemorrhaging, but still

produce prolonged PT and aPTT should bring into suspicion the presence of

inhibitors or Vitamin K deficiency.(18) The inhibitors could be in the form of direct

thrombin inhibitors, bivalrudin, lepirudin or argatroban.(25)

Vitamin K deficiency can cause bleeding in infants in the first hours to months of life.

This is termed as the Hemorrhagic Disease of the Newborn (HDN). It is diagnosed

when an infant has a prolonged prothrombin time but is cured on vitamin K

administration. This is usually due to limited Vitamin K transfer across the placenta in

the foetus.

Clinical manifestations of Factor X deficiency

Patients affected with factor X deficiency tend to be the most severely affected

amongst all the rare bleeding disorders.(19)

A clinical manifestation of the disease can occur at any age, however, it is more

common to be seen in neonates in the case of umbilical stump bleeding and infancy.

(18,19) The most common symptom is epistaxis in all degrees of severity.

Haemathroses, severe post-operative haemorrhage and central nervous system

haemorrhage are also reported as symptoms of severe Factor X. Severe FX

deficiency is when Factor X activity (FX:C) <1 IU/dL.(18)

Moderately affected patients, (FX:C is 1-5 IU/dL), only bleed after haemostatic

challenges such as trauma or surgery. (18,19,26)

Mild FX deficiency, FX:C is 6-10 IU/dL, are rarely diagnosed and only identified on

routine screening. They experience easy bruising or menohagia.(18)

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Diagnosis of Factor X deficiency

The suspicion of FX deficiency is raised when PT and aPTT are unusually slow. The

diagnosis is confirmed by taking plasma FX levels. (18,19)

There are 5 assays for measuring plasma FX levels. PT or aPTT based assay alone

is insufficient for the diagnosis of FX deficiency.(18) They would have to be done

along with a chromogenic assay, RVVT assay and an immunological assay.(18)

The deficiency is then further classified by further assays. A reduction in FX antigen

levels and FX coagulant activity indicates a type I deficiency. This is usually caused

by a problem in protein synthesis. A reduction of FX coagulant activity, but an

increased or normal FX antigen level indicates a type II deficiency due to non-

functioning FX.(19)

It is important to note that Vitamin K deficiency should always be considered before

investigating into rarer causes, such as a coagulation factor deficiency.(19) A

therapeutic trial of vitamin K is advised before serious treatment options are to be

considered.(18)

Treatment of Factor X deficiencies

Tranexamic acid is particularly useful for epistaxis. 10mL of a 5% solution should be

used as a mouthwash every 8 hours.(18)

Fibrin glue could be used in spurring local haemostasis.(18)

Prothrombin complex concentrates are usually 3 factor concentrates, containing FII,

FIX and FX. An infusion would raise serum FX levels and hence curbing the problem

of deficiency.(18) It is important to note that this shouldn’t be given with Transexamic

acid due to the risk of thrombosis.(18)

Virally inactivated fresh-frozen plasma, (FFP) should be used to treat this condition.

A loading dose of 20mL/kg followed by 3-5mL/kg doses twice daily. There are at

least 2 inactivated FFP products available in the UK. They are Methylene blue

treated single unit FFP and a commercial product.(18) The commercial product

comprises of a pooled plasma that is virally inactivated using a ‘solvent detergent’

technique.(18)

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Structural analysis of Factor V/Va coagulation factor

Factor V is present in human plasma at a concentration of 20nM as a singular large

pro-cofactor.(27) 80% of the co-factor is found in this form while 20% is present in

alpha-granules in platelets.(27)

The gene that encodes for factor V is 80kb long and can be found on chromosome

1q21-25 and contains 24 introns. (28)The messenger RNA that is produced from

transcription is 6.8kb long. The liver is the primary source of factor V in plasma,

whereas platelets serve as a secondary source.(28)

The final Factor V is then secreted after post translational modifications as a 2196

amino acid protein.(27) This protein is made up of several domains. Triplicated A

domains, a B domain and duplicated C domains.(29)

The B domain of the human factor V is released as 2 heavily glycosylated and

fragments upon activation to factor Va.

As previously discussed, activated protein C is involved in the deactivation of factor

Va into inactivated factor Va (Vai). This is done by the cleavage of the A2 domain as

2 fragments. Thrombin can also inactivate Factor Va by cleaving the Arg643 residue

on the heavy chain. Figure 9 A, B and C show diagrams depicting these three states.

Figure 9A – showing the domains of factor V.

Figure 9B – showing the domains of factor Va and the cleaved B domain.

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Figure 9C – showing the domains for inactivated Va (Vai) via APC mechanism and

the cleaved A2 domain.

Factor Va is a very efficient co-factor increasing the rate of thrombin formation by

300,000 fold compared to the rate of reaction catalysed by Factor Xa alone.(27)

Functional analysis of factor V/Va in the prothrombinase complex

The fundamental contribution of factor Va to the function of prothrombinase complex

is the retention of factor Xa on the membrane surface.(27)

The domains crucial to this function are in the light chain of factor V. Ionic and van-

der-Walls’ forces of interaction facilitate the binding of the light chain to the

phospholipid surface (27)

The conversion can only take place when factor Xa is immobile, as it has a poor

affinity for membranes compared to factor Va. Thus, the formation of the complex

helps to improve the rate of alpha-thrombin production. The amount of thrombin

produced in 1 minute via the prothrombinase complex would have taken 6 months if

it weren’t for factor Va cofactor.(27)

Factor Va has another important function in the coagulation process. It is also a

cofactor for the APC/Protein S complex in the inactivation of factor VIII. Factor V can

only catalyse this reaction when protein S is present. It increases the rate by 2 fold.

The interesting thing is that factor Va, without the B domain, doesn’t show any co-

factor effect of the rate of inactivation of factor VIII with our without protein S. (27,30)

Factor V deficiency

Factor V deficiencies are a very uncommon form of bleeding disorder with an

incidence of 1 in 1,000,000.(27) These deficiencies can be further classified into type

I and type II. Type I factor V disorder are when there is low amounts or

immeasurable levels of FV antigen. (27)Type II is when there are normal or slightly

reduced FV antigen levels. Severe type I factor V deficiency is also known as

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parahemophila,(27) which is usually an autosomal recessive disorder.(27) Null

alleles arise due to nonsense or frame shift mutations. FVNewBrunswick is the only

genetic defect associated with type II deficiency. This mutation doesn’t impair

synthesis but severely affects the stability of the mutant Factor Va.(31)

Diagnosis of Factor V deficiency

This disease is characterised by an increase in both the PT and aPTT. However, a

normal TT is observed.(18) Diagnosis of FV is confirmed by performing a

prothrombin time-based FV assay followed by an immunological assessment of FV

antigen levels.

Treatment of Factor V deficiency

Due to the absence of FV concentrate readily available in the industry, treatment is

15-20mL/kg of fresh-frozen plasma (FFP).(18) As with for the treatment of FX

deficiency, a virally inactivated preparation is to be used.(19) There are at least 2

inactivated FFP products available in the UK. They are Methylene blue treated single

unit FFP and a commercial product.(18) The commercial product comprises of a

pooled plasma that is virally inactivated using a ‘solvent detergent’ technique.(18)

In order to treat spontaneous bleeding, tranexamic acid should be considered.(18)

If the bleeding persists despite the patient’s FFP prescription, or if FV levels fall

below 15 U/dL, a further dose should be considered.(18) A regular reading of the

patients FV levels should be taken to ensure it is within the normal range of 71-125

U/dL.(18)

Acquired Factor V deficiency

Sometimes, the body can produce antibodies against FV, inhibiting them as they are

unable to bind to specific binding sites on enzymes.(18)

Low level inhibition can be overcome by large amounts of FFP.(32) However, in

surgical situations, intravenous immunoglobulins are probably effective in eradicating

the inhibitor.(33)

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Factor VLeiden

Factor V in thrombophilia has been heavily focussed upon in recent years. The

Arg506Gln mutation (FVLeiden) is the most common defect in patients with

thromboembolic disease. (31) Having this mutation gives rise to a peculiar condition

called APC-resistance. Affecting one of APC’s target sites, it impairs both the

efficiency of FVa degradation via APC as well as FV’s function as a cofactor in the

inactivation of FVIIIa. (31) The risk of venous thrombosis increases by 500-700 % in

heterozygotes and a whopping 8000% in homozygotes.(31)

The allele frequency of FVLeiden is between 0.01 and 0.15. (31) The reason for this is

due to the competitive advantage given to heterozygote women who have a reduced

bleeding tendency after delivery(31)

There are 2 additional FV allele variants that give rise to APC resistance and they

are FV Arg306Thr (FVCambridge) and FV Arg306Gly (FVHongKong) Both of these

mutations result in a complete loss of FVa pro-coagulant activity.(27)

APC resistance is also seen with the HR2 haplotype. (His1299Arg mutation) This is

also known as R2.(27) This mutation is a collection of more than 10 linked genetic

variants and present with low levels of FV antigen.(27)

Conclusion

Blood coagulation is an underestimated, yet highly essential life mechanism in living

animals. Its complexity also results in numerous mutations that could lead to fatal

consequences. Understanding how the blood coagulates, and the mechanisms in

which the blood is kept liquid, is imperative to acquiring key knowledge of the

pathology behind blood coagulation disorders. Deep molecular study enables us to

understand unique mutations that give rise to coagulation factor deficiency, and

perhaps in the future, this knowledge can be applied to help make life better for

individuals who are disadvantaged genetically in a more efficient way.

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