Detailed coagulation functioning and their mechanism was presented as per Indian Pharmacopoeia and some revised articles

1. COAGULATION FACTORS
ACHARYA NAGARJUNA UNIVERSITY
COLLEGE OF PHARMACUETICAL SCIENCES
SEMINAR PRESENTED BY
G.MADAN MOHAN
PHARMACUETICAL ANALYSIS
Under the guidance of
SRUJANI . M.Pharmacy PH.D

2. Introduction ;-
Heme = Blood and Stasis = Halt . It is the process of forming clots in the wall
of damaged blood vessels & preventing blood loss while maintaining blood in a
fluid state with in the vascular system.
coagulation
Vascular wall constriction Platelet plug formation Clotting factors
Smooth muscle constriction
luminal diameter reduction
retarding blood loss
enhancing platelet adherence
Endothelial cells
VIII-vWF synthesis
thromboplastin (III) release
activation of ext pathway
 activation of Factor IX & X
Sub endothelium
collagen  platelet
adherence, activation

3. Coagulation factors

4. ASSAY OF HUMAN COAGULATION FACTOR –VII:-
Human coagulation factors is assayed by its biological activity as a factor VIIa- tissue
factor compose in the activation of factor X in presence of calcium ions and phospholipids
PRINCIPLE:-
The potency of factor VII preparation is estimated by comparing the quantity necessary to
achieve a certain rate of factor Xa formation in a test mixture containing the substance
that take part in the activation of factor X and the quantity of the international standards or
of a reference preparation calibrated in International Units, required to produce the same
rate of factor Xa formation.
The preparation consists of 0.99IU/ ampoules (coded 07/228) containing
1ml aliquots of Factor VIIa concentrate, freeze-dried plasma.
The preparation consists of 0.99IU /ampoules (coded 89/688) containing
1ml aliquots of Factor VIIa concentrate, freeze-dried plasma.
Preparation 2nd international standard factor VIIa concentrate (who international
standard):-
Preparation 1nd international standard factor VIIa concentrate (who international
standard):-

5. chromogenic assay consists of 2 steps:-
Step :-1
Factor VII
Tissue factor + ca++
Factor VIIa
Step:-2
Factor X
Factor VIIa + ca++ Phospholipids and tissue factors
Factor Xa
Chromogenic substrate
Peptide + chromophore
Factor Xa
Both steps comprises of reagents these are obtained from different source
The coagulation factor reagent comprises purified proteins derived from human or bovine sources.
Reagents :-
It includes., Factor X ,Phospholipids & tissue factor thramboplastin etc..,
The second step comprises the quantification of the formed factor X.
it was carried by spectrometric methods
Step :-1
Step:-2

6. ASSAY:-
Reconstitute of the one ampoule of standard and PBE contents with sufficient quantity of
water. It was used with in one hour
Addition of prediluent
To reconstituted preparations to produce solutions containing
between 0.5 to 2.0 IU of factor VII per ml.
Prepare the further dilution of PBE and standard using isotonic non-chelating buffer
containing 0.1% of bovine and human albumin (buffer PH:-7.3 & 8 )
Prepare the dilutions such that the final factor VII concentration is below
0.005 ill per ml.
Prepare a control solution that includes all components except
factor VII.
Prepare all dilutions in plastic tubes and use within 1 hour.

7. Step:-1
Mix dilution of the standard and PBE with appropriate volume of the pre warmed coagulation
factor reagent
Incubate at 37°(in plastic tubes or
micro-titer plates)
Factor X concentration 10 nmol per litre to 350 nmol per litre, preferably 14 nmol per litre to
70 nmol per litre.
Allow the activation of factor X to proceed for a suitable time, usually terminating the reaction
before the factor Xa concentration has reached its maximal level in order to obtain a satisfactory
linear dose-response relationship. The activation time is also chosen to achieve linear production
of factor Xa in time. Appropriate activation times are usually between 2 to 5minutes, but
deviations are permissible if acceptable linearity of the dose-response relationship is thus
obtained.

8. Step-2:-
Terminate the step -1 by using the pre warmed reagent containing chromophoric substance.
Quantify the rate of substrate which is linear to that formed Xa factor
Absorbance is measured by using spectrophotometer(monitor the absorbance continuously ,
thus allowing the initial rate of cleavage to be calculated.
terminating the hydrolysis reaction after a suitable interval by lowering the pH by the addition
of a suitable reagent, such as acetic acid (50.0 per cent w/v CzHPz) or a citrate solution (1 mol
per litre) at pH 3. Adjust the hydrolysis time to achieve a linear development of chromophore
with time.
Check the validity of the assay and calculate the potency of
the test preparation by the usual statistical methods.
(or)
Suitable time interval is 3-12min.This gives the linearity of dose-response relationship

9. ASSAY OF HUMAN COAGULATION FACTOR VIII:-
Human coagulation factor VIII is assayed by its biological activity as a cofactor in the
activation of factor X by activated factor IX (factor IXa) in the presence of calcium ions
and phospholipids.
PRINCIPLE:-
The potency of a factor VIII preparation is estimated by comparing the quantity necessary to
achieve a certain rate of factor Xa formation in a test mixture containing the substances that
take part in the activation of factor X, and the quantity of the International Standard, or of a
reference preparation calibrated in International Units, required to produce the same rate of
factor Xa formation.
The preparation consists of 0.98U/ ampoules (coded 07/228) containing 1ml
aliquots of Factor VIII concentrate, freeze-dried plasma.
Preparation 2nd international standard factor VIIa concentrate (who international
standard):-
Human coagulation factor VIll reference preparation is calibrated in International Units
by comparison with the International Standard.

10. chromogenic assay consists of 2 steps:-
Step :-1 Factor X
Activated factor VIII
&
Factor IXa
Phospholipids +calcium ions
Factor Xa
Step :-2
Chromogenic substrate
Factor Xa
Peptide + Chromomphore
Both steps comprises of reagents these are obtained from different source
Reagents. The coagulation factor reagent comprises purified proteins derived from
human or bovine sources. These include factor X, factor IXa, and a factor VIII activator,
Usually thrombin.
Step :-1
Step :-2
The second step comprises the quantification of the formed factor X.
it was carried by spectrometric methods

11. Reconstitute of the one ampoule of standard and PBE contents with sufficient
quantity of water. It was used with in one hour
ASSAY:-
Addition of pre diluent
To reconstituted preparations to produce solutions containing between 0.5 to 2.0
IU of factor VIII per ml.
The prediluent consists of hemophilia A plasma
Von willebrand factor
or
Prepare further dilutions of the reference and test preparations using a non-chelating,
appropriately buffered solution tris(hydroxymethyl)aminomethane or imidazole, containing
1.0 percent of human or bovine albumin.
Prepare the dilutions such that the final factor VIII concentration is below
0.01 IU per ml.
Prepare a control solution that includes all components except factor VIII
Prepare all dilutions in plastic tubes and use immediately

12. Step :-1
Mix dilution of the standard and PBE with appropriate volume of the pre warmed
coagulation factor reagent
Incubate at 37°(in plastic tubes or micro-titer
plates)
Allow the activation of factor X to proceed for a suitable time, terminating the reaction
(step 2) when the factor Xa concentration has reached approximately 50.0 per cent of the
maximal (plateau) level. Appropriate activation times are usually between 2 to 5 minutes
Step :-2
Terminate the step -1 by using the pre warmed reagent containing chromophoric
substance.
Quantify the rate of substrate which is linear to that formed Xa factor
Absorbance is measured by using spectrophotometer monitor the
absorbance continuously , thus allowing the initial rate of cleavage to be
calculated.

13. Suitable time interval is 3-15min.This gives the linearity of dose-response
relationship
Check the validity of the assay and calculate the potency of
the test preparation by the usual statistical methods.
Terminating the hydrolysis reaction after a suitable interval by lowering the pH
by addition of a suitable reagent, such as a 50.0 per cent v/v solution of acetic
acid,
or
A I MpH3 citrate buffer solution. Adjust the hydrolysis time to achieve a linear
development of chromophore over time.

14. ASSAY OF HUMAN COAGULATION FACTOR IX:-
PRINCIPLE
The principle of the assay is to measure the ability of a factor IX preparation to
reduce the prolonged coagulation time of factor IX-deficient plasma .
Kaolin ,silica ,or ellagic acid Phospholipids& contact activator
Potency is assessed by comparing the dose –response curve of the preparation under examination
to that of a reference preparation , calibrated in IU.
Preparation 4th international standard factor IX concentrate (WHO international
standard):-
The preparation consist of 0.86 IU/ampoule, , coded( 09/172), containing
approximately 1 ml aliquots of normal human plasma, freeze dried.
Human coagulation factor IX is concentrate preparation is calibrated in International Units
by comparison with the International Standard.

15. ASSAY :-
Re constitute the ampoule of PBE and reference standard preparation and use immediately.
Determine the heparin and neutralize the heparin by addition of the protamine sulphate
Pre dilution of PBE & SP
With Factor IX deficient plasma
To produce solutions containing 0.5-2.0 IU/ml. at least 3 dilution were prepared using buffer
(imidazole buffer pH 7.3) contains 1%w/v of human albumin. use these solutions immediately
Use an apparatus suitable for measurement of coagulation times
or
Carry out the assay with incubation tubes maintained in a water-bath at 37°.
In each test tube add 0.1ml of factor IX deficient plasma and add 0.1ml one of the dilution
PBE & standard preparation

16. Add to each tube 0.1 ml of a suitable activated partial thromboplastin Time (APTT) reagent
containing phospholipids and contact activator and incubate the mixture for a recommended
time at 37°.
Using a timer, measure the coagulation time, i.e. the interval between the moment of
the addition of the calcium chloride and the first indication of the formation of fibrin.
The volumes given above may be adapted to the APTT reagent and apparatus used.
Calculate the potency using the usual statistical methods.
Add 0.1ml of a 0.37 %w/v of cacl2 heated to 37°.

17. ASSAY OF HUMAN COAGULATION FACTOR X:-
PRINCIPLE
Preparation 4th international standard factor X concentrate (WHO international
standard):-
Human coagulation factor X is assayed following specific activation to form factor Xa.
Factor Xa is estimated by comparing its activity in cleaving a specific chromogenic
peptide substrate with the same activity of the International Standard or of a reference
preparation calibrated in International Units.
The preparation consist of 0.89 IU/ampoule, , coded( 09/172), containing approximately 1 ml
aliquots of normal human plasma, freeze dried.
Chromogenic assay consists of 2 steps:-
Snake venom-dependent activation of factor X,
Step:-1
Step:-2
Enzymatic cleavage of chromogenic factor of Xa

18. Under appropriate assay conditions, there is a linear relation between factor Xa activity and
the cleavage of the chromogenic substrate
Reagents
Russell's viper venom specific factor
X activator (RVV).
Factor Xa chromogenic substrate Dilution buffer.
It is a protein derived from Russell's viper
It specifically activates factor X
Reconstitute according to the manufacturer's
instructions
Stored at 4° & use with in a month
N-a-benzyl oxy carbonyl D-
arginyl-L-glycyl-L-arginine-
4-nitroanilide dihydrochloride.
Example:-
0.37% w/v of tris
(hydroxymethyl)
aminomethane
+
Nacl 1.8%w/v
+
0.21%w/v imidazole
+
hexadimethrine
bromide
In 1%w/v of human
plasma
Adjust Ph8.4 with Hcl

19. METHOD:-
Test solution:- Dilute the PBE with dilution buffer to obtain a solution containing 0.18 IU of
factor X per mI. Prepare at least 3 further dilutions with same solvent.
Reference solution:- Dilute the reference preparation with dilution buffer to obtain a solution
containing 0.18 ill of factor X per ml. Prepare at least 3 further dilutions with same solvent.
Warm all solutions to 37° in a water-bath shortly before the test.
The working of the solutions carried by using test tubes or micro titer plates
Micro titer plates maintained at 37° add 12.5 ul of each dilution of the test solution or
the reference solution to each of a series of wells.
To each well add 25 ul of R Wand incubate for exactly 90 seconds. To each well add
150 ul of factor Xa chromogenic substrate, diluted 1 in 6 in dilution buffer
Read the rate of change of absorbance at 405nm continuously a period of 3min and
obtain the mean rate of change of absorbance.
If continuous monitoring is not possible, read the absorbance at 405 nrn at suitable
consecutive intervals, for instance 40 seconds
Plot a graph absorbance against time a linear line is formed

20. REFERENCES :-
INDIAN PHARMACOPIEA 2010 Page no:-244-248
IU standard preparation assay of coagulation factor VII
NCBI JOURNAL WHO Standards 1nd & 4th
IU standard preparation assay of coagulation factor X
NCBI JOURNAL WHO Standards 4th .

21. THANK YOU EVERY ONE