Year 1 MBChB Medicine

Student Selected Component (SSC)

The Molecular Basis of
Blood Coagulation Disorders
Student : U Bhalraam
Supervisor : Dr David Martin

Abstract
This report includes a detailed overview of the mechanism of 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. Next we explore
the essential biochemical modification (gamma-carboxylation) that is essential to
forming the structure of coagulation factors IX, X, VII and II. Next 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.
Virchows 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 doesnt trigger
coagulation without any stimulus.(3) A fine balance must be achieved. There are
various processes that control the hypercoagulability of the blood.(3)

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) 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 Antithrombin III
(ATIII).(6) Once bound, it causes a conformational change that activates the serine
protease.(7) 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)Plasmin then actively breaks down fibrin strands, thus, inhibiting clot
formation.(3)

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 peptides 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)

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

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. This complex 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. Thrombin then 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 doesnt 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)

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, 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. FIXa
then activates 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)

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

Gamma-carboxylation of glutamic acid residues in coagulation factors

As mentioned previously, on activation of platelets by ADP, Delta granules degranulate and secrete Ca2+.(13) The presence of calcium ions is essential in
speeding 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) All the glutamic acid residues are
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)
Thus, 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)

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 why; I have
decided to narrow the scope of the study of coagulation disorders in this report to that
of coagulation factors X, V and the Pro-thrombinase complex. (Xa/Va)
I have chosen these coagulation factors as they occupy a pivotal position in
coagulation. Either the intrinsic or the extrinsic pathways activate them. Hence, I felt
that understanding coagulation disorders in the critical portion of the cascade was of
significant importance.
Tests of haemostatic function

There are 3 main tests that help us to evaluate whether the haemostatic process in
the body are proceeding normally. They are thrombin time, activated partial
thromboplastin time, prothrombin time, Russells 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)
Russells viper venom time (RVVT) is a diagnostic test performed, exposing the
blood plasma to Russels 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)

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 disorders that are not directly genetic and have other factors
involved. Vitamin K deficiency is one such example. 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

coagulation

factor

Factor X is a vitamin K-dependent precursor serine protease that, 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. 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)

10

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 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.

11

Factor XKetchikan
Factor X Ketchikan 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 X Ketchikan, 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 X Ketchikan 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) 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 X Kurayoshi is another mutation that leads to Factor X deficiency. Factor X
Kurayoshi 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

12

the second EGF-like domain of factor X that plays a major role in setting the stage for
the 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.
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)
Manifestation of the disease, clinically, 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)

13

Diagnosis of Factor X deficiency

The suspicion of FX deficiency is raised when PT ,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 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 nonfunctioning 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 shouldnt 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)

14

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.
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.

15

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 vander-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. Factor Xa 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 werent 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, doesnt show any cofactor 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

16

parahemophila.(27) This 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 doesnt 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 the

spontaneous

bleeding, tranexamic

acid

should

be

considered.(18)
If the bleeding persists despite the patients 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)

17

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 APCs target sites, it impairs both the
efficiency of FVa degradation via APC as well as FVs 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 enable 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.

18

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