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Abstract
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)
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)
Table 1: Outlining the various coagulation factors and their role in their active form (6)
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)
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)
Figure 5: Outlining the extrinsic, intrinsic and common pathways of the coagulation cascade
(12)
(12)
As mentioned previously, on activation of platelets by ADP, Delta granules degranulate 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.
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 Prothrombinase 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, 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
Structural
and
functional
analysis
of
Factor
coagulation
factor
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.
11
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)
12
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)
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)
13
14
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, 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 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
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 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.
18
References:
1.
2.
3.
4.
5.
6.
7.
8.
Van Walderveen MC, Berry LR, Atkinson HM, Chan AKC. Covalent
antithrombin-heparin effect on thrombin-thrombomodulin and activated protein
C reaction with factor V/Va. Thromb Haemost. 2010 May;103(5):9109.
9.
Johari V, Loke C. Brief overview of the coagulation cascade. Dis Mon. 2012
Aug;58(8):4213.
10.
11.
12.
13.
14.
Price PA, Fraser JD, Metz-Virca G. Molecular cloning of matrix Gla protein:
implications for substrate recognition by the vitamin K-dependent gammacarboxylase. PNAS. 1987 Dec;84(23):83359.
15.
Freedman SJ, Blostein MD, Baleja JD, Jacobs M, Furie BC, Furie B.
Identification of the phospholipid binding site in the vitamin K-dependent blood
coagulation protein factor IX. J Biol Chem. 1996 Jul 5;271(27):1622736.
16.
Triplett DA. Use of the dilute Russell viper venom time (dRVVT): its
importance and pitfalls. J Autoimmun. 2000 Sep;15(2):1738.
19
17.
McMullen BA, Fujikawa K, Kisiel W, Sasagawa T, Howald WN, Kwa EY, et al.
Complete amino acid sequence of the light chain of human blood coagulation
factor X: evidence for identification of residue 63 as beta-hydroxyaspartic acid.
Biochemistry. 1983 Jun 7;22(12):287584.
18.
Bolton-Maggs PHB, Perry DJ, Chalmers EA, Parapia LA, Wilde JT, Williams
MD, et al. The rare coagulation disorders--review with guidelines for
management from the United Kingdom Haemophilia Centre Doctors'
Organisation. Haemophilia. Blackwell Science Ltd; 2004 Sep;10(5):593628.
19.
20.
21.
22.
23.
Messier TL, Wong CY, Bovill EG, Long GL, Church WR. Factor X Stockton: A
mild bleeding diathesis associated with an active site mutation in factor X.
Blood Coagul Fibrinolysis. 1996 Jan;7(1):514.
24.
25.
Gosselin RC, Dager WE, King JH, Janatpour K, Mahackian K, Larkin EC, et al.
Effect of direct thrombin inhibitors, bivalirudin, lepirudin, and argatroban, on
prothrombin time and INR values. Am J Clin Pathol. American Society for
Clinical Pathology; 2004 Apr;121(4):5939.
26.
27.
28.
Richards AM, Tonolo G, McIntyre GD, Leckie BJ, Robertson JI. Radioimmunoassay for plasma alpha human atrial natriuretic peptide: a comparison
of direct and pre-extracted methods. J Hypertens. 1987 Apr;5(2):22736.
29.
Kane WH, Davie EW. Blood coagulation factors V and VIII: structural and
functional similarities and their relationship to hemorrhagic and thrombotic
disorders. Blood. 1988 Mar;71(3):53955.
30.
20
31.
Duga S, Asselta R, Tenchini ML. Coagulation factor V. Int J Biochem Cell Biol.
2004 Aug;36(8):13939.
32.
33.
21