Seminar on:-Screening Models
for Drugs Acting on Blood
Constituents
.
1
Contents


Introduction,
Blood Clotting Mechanism & Clotting Factors,
A. In vitro models1. Blood Coagulation test,
2. Chandler Loop,
3. Platelet Aggregation in Whole Blood,
B.
1.
2.
3.
C.
In Vivo Models –
Foreign surface thrombosis,
Photochemical induced Thrombosis,
Experimental Thrombocytopenia.
Genetically Modified animalsKnock out Mice Method.
References
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Introduction –
 Blood is fluid connective tissue. It circulates


1.
2.
3.
continuously around the body.
It transports Oxygen from lung to tissue &
carbon dioxide from tissue to lung for excretion.
There are three types of blood cells Red Blood Cells,
White Blood Cells,
Thrombocytes/Platelets.
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Clotting (Coaggulation) Mechanism –

When a blood vessel is damaged loss of blood is stopped
& healing occurs in series of overlapping processes in
which platelet play vital part.

Stages in blood Clotting –
1. Vasoconstriction,
2. Platelet Plug Formation,
3. Coagulation (blood clotting).
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Clotting Mechanism
PRIMARY
AGGREGATION
Platelet Aggregation
Clotting
Hemostatic
clot
Fibrin
SECONDARY
COAGULATION
Thrombin
0 min
5 min
10 min
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Clotting Factors –
I
Fibrinogen
II
Prothrombin
III
Tissue factor
IV
Calcium
V
Labile Factor, AC-Globulin
VII
Stable factor, Proconvertin.
VIII
Antihemophilic globulin (AHG)
IX
Christmas Factor, Plasma Thromboplastin
component (PTA)
X
Stuart Prower Factor
XI
Plasma Thromboplastin antecedent (PTA),
XII
Hageman Factor
XIII
Fibrin Stabilizing Factor
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Blood Coagulation Test
PURPOSE AND RATIONALE
•The coagulation cascade consists of a complex network
Of interactions resulting in thrombin-mediated Cleavage
of fibrinogen to fibrin which is one major Component of
a thrombus.
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PROCEDURE
1.Male Sprague Dawley rats weighing
200-220 gm.
2.Anesthised by i.v. of 60mg/kg sod pentobarbital
3.Blood collected to plastic syringe of 0.2 ml 100
mm citrate buffer (pH 4.5)
4.Sample immediately agitated and centrifuged in
plastic tube at 1500g for 10 min.
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EVALUATION
 Mean values of TT, PT and APTT are calculated
in dosage groups and vehicle controls.
 Statistical evaluation is performed by means of
the unpaired Student’s t-test.
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Other Points –
1. Prothrombin Time (PT).
2. Activated Partial Thromboplastin Time (APTT)
3. Thrombin Time (TT).
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Chandler loop
PURPOSE AND RATIONALE
 The Chandler loop technique allows to produce in
vitro thrombi in a moving column of blood
(Chandler 1958).
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PROCEDURE
1. 1 millimeter of non anticoagulant whole blood
drawn into polyvinyl tube.
2. 2 ends of tube are brought together & closed by
outside plastic collar.
3.Tube is placed and rotated at 17 rpm.
4. Blood column become static and moves in
direction of rotation tube.
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EVALUATION
Time to occlusion of the tube by the thrombus
establishes a definite end point in this system.
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Platelet aggregation in whole
blood
PURPOSE AND RATIONALE
 The method uses a whole blood platelet counter
which counts single platelets does not require their
separation from other blood cell types.
 The method has been described by Lumley et al.
(1981).
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PROCEDURE1.Blood taken in polystyrene tube.
2. 9 ml venous blood anticoagulant with 1ml Sod. Citrate for
30-60min.
3.For aggregation study 10 μl test subs. Are added to 480 μl
citrated blood.
4.No. of platelets determined in 10 μl samples before & 10
min after addition of aggregating agent.
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EVALUATION
 IC50 values (50% inhibition of aggregation) are
determined from the dose-response curves (log
concentration test substance versus % inhibition of
aggregation).
 % maximal aggregation and % aggregation are
also calculate.
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Foreign-surface thrombosis
Wire-coil induced thrombosis
PURPOSE AND RATIONALE
 A classical method to produce thrombosis is based
on the insertion of wire coils into the lumen of
blood vessels.
 The model was first described by Stone and Lord
(1951)
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PROCEDURE
 Male Sprague-Dawley rats weighing 260–300 g receive
the test compound or the vehicle (controls) by oral,
intravenous or intraperitoneal administration.
 Through a mid-line incision the caudal caval vein is
exposed and a stainless steel wire coil is inserted into the
lumen of the vein just below the left renal vein.
 The thrombotic material is dissolved in 2 ml alkaline
sodium carbonate solution (2% Na2CO3 in 0.1 N NaOH) in
a boiling water bath for 3 min.
 The protein content is determined in 100 μl aliquots by the
colorimetric method of Lowry.
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EVALUATION
 Thrombosis incidence (= number of animals with
thrombi in dosage groups as compared to vehicle
controls) is assessed.
 Statistical significance is assessed by means of the
unpaired Student’s t-test.
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Photochemical-induced
thrombosis
PURPOSE AND RATIONALE
 In 1977, Rosenblum and Sabban reported that
ultraviolet light can produce platelet aggregation
in cerebral microvessels of the mouse after
intravascular administration of sodium
fluorescein.
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PROCEDURE
 Studies are performed in mesenteric arteries of
15–30 μm diameter in anesthetized rats. After
intravenous injection of fluorescein
isothiocyanate-dextran 70 (FITCdextran, Sigma,
10%, 0.3 ml), the FITC-dextran in arterioles is
exposed to ultraviolet light (wavelength of
excitation 490 nm, wavelength of emission 510
nm).
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EVALUATION
 Thrombus formation is quantitated by determining
the time between onset of excitation and
appearance of the first platelet aggregate adhering
to the vessel wall.
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CRITICAL ASSESSMENT
 In contrast to other thrombosis induction methods,
photochemically-induced thrombosis can be Easily
used in smaller animals.
Experimental
thrombocytopenia
PURPOSE AND RATIONALE
 Intravenous administration of collagen,
arachidonic acid, ADP, PAF (platelet
activating factor) or thrombin activates
thrombocytes leading to a maximal
thrombocytopenia within a few minutes.
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PROCEDURE Male guinea pigs (Pirbright White) weighing 300–600 g, or
male NMRI mice (25–36 g), or Chinchilla rabbits of either
sex weighing 2–3 kg are used.
 The marginal vein of the ear of rabbits is cannulated and the
thrombocytopenia-inducing substances collagen or
arachidonic acid are injected slowly.
 The number of platelets and leukocytes is determined within
1 hr after withdrawal in 10 μl samples of whole blood using
a microcellcounter suitable for blood of various animal
species.
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EVALUATION
 Percent inhibition of thrombocytopenia (or
leucocytopenia) is calculated in dosage groups
relative to controls.
 Statistical significance is evaluated by means of the
unpaired Student’s t-test.
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CRITICAL ASSESSMENT
 The method of collagen epinephrine induced
thrombocytopenia is presently widely used to
study the phenotype of mice knocked out for a
specific gene with suspected role in
haemostasis /thrombosis.
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Knock out mice
 PURPOSE AND RATIONALE
Genetically modified animals, in particular
knock-out mice, help to understand the role of
various factors in blood clotting, thrombolysis,
and platelet function. They are useful to verify
the mode of action of new drugs.
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Factor-I
(Fibrinogen)Phenotype-
Born in normal appearance, ~10% die shortly after
birth and another 40% around 1–2 months after birth
due to bleeding, failure of pregnancy, blood samples
fail to clot or support platelet aggregation in vitro
Factor-II
(Prothrombin)Phenotype-
Partial embryonic lethality: 50% between embryonic
day at least 1/4 survive to term, but fatal hemorrhage
few days after birth; factor II important in
maintaining vascular integrity during development as
well as postnatal life.
Factor VPhenotype-
Half of the embryos die at E9–10, possibly as a result
of abnormal yolk sac vasculature, remaining 50%
progress normally to term, but die from massive
hemorrhage within 2 h of birth, more severe in mouse
than in human (due to residual activity in humans).
Factor VIIPhenotype-
Develop normally but suffer fatal perinatal bleeding.
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References –
 H. Gerhard Vogel (Ed.), Drug Discovery and Evaluation
Pharmacological Assays , 2nd Edition Springer Publications,
Page No. 277-309
 Anne Waugh & Allison Grant, Ross & Wilson Anatomy &
Physiology in Health & Illness, 11th Edition, Churchil
Livingstone Publications, Page No. 56, 64.
 Cardinal DC, Flower RJ (1980) The electronic aggregometer: a
novel device for assessing platelet behavior in blood. J
Pharmacol Meth 3:135–158
 Sawyer PN, Pate JW, Weldon CS (1953b) Relations of
abnormaland injury electric potential differences to
intravascular thrombosis Am J Physiol 9:108–112
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 Palmer RL (1984) Laboratory diagnosis of bleeding
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disorders:Basic screening tests. Postgrad. Med 76:137–148
Chandler AB (1958) In vitro thrombotic coagulation of
theblood. A method for producing a thrombus. Lab Invest 7:
110–114
Meng K (1975) Tierexperimentelle Untersuchungen zur
antithrombotischen Wirkung von Acetylsalicylsäure. Therap
Ber 47:69–79
Just M, Tripier D, Seiffge D (1991) Antithrombotic effects of
recombinant hirudin in different animal models. Haemostasis
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Suh TT, Holmback K, Jensen NJ, Daugherty CC, Small K,
Simon DI, Potter SS, Degen JL (1995) Resolution of
spontaneous bleeding events but failure of pregnancy in
fibrinogen-deficient mice. Genes Dev 9:2020–2033
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 Bugge TH, Xiao Q, Kombrinck KW, Flick MJ, Holmback
K(1996) Fatal embryonic bleeding events in mice lacking
tissuefactor, the cell-associated initiator of blood
coagulation.Proc Natl Acad Sci U S A 93:6258–6263
 Robbie LA, Young SP, Bennett B, Booth NA (1997)
Thrombiformed in a Chandler loop mimic human arterial
thrombi in structure and PAI-1 content in distribution. Thromb
Haemost 77:510–515
 Yang TL, Cui J, Taylor JM, Yang A, Gruber SB. Ginsburg D
(2000) Rescue of fatal neonatal hemorrhage in factor V
deficientmice by low level transgene expression. Thromb
Haemost 83:70–77
 www.google.com
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