Seminar on:-Screening Models for Drugs Acting on

Blood Constituents

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Contents Introduction, Blood Clotting Mechanism & Clotting

Factors,A. In vitro models-1. Blood Coagulation test,2. Chandler Loop,3. Platelet Aggregation in Whole Blood,

B. In Vivo Models –4. Foreign surface thrombosis,5. Photochemical induced Thrombosis,6. Experimental Thrombocytopenia.C. Genetically Modified animals- Knock out Mice Method. References 2

Introduction –

Blood is fluid connective tissue. It circulates 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 -

1. Red Blood Cells,

2. White Blood Cells,

3. 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

Thrombin

AGGREGATION

Fibrin

Hemostaticclot

ClottingPlatelet Aggregation

0 min 10 min5 min

SECONDARY

PRIMARY

COAGULATION

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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 Factor6

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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.

Blood Coagulation Test

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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 loopPURPOSE AND RATIONALEThe Chandler loop technique allows to produce in

vitro thrombi in a moving column of blood (Chandler 1958).

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PROCEDURE1. 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 bloodPURPOSE AND RATIONALEThe 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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PROCEDURE-1.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 thrombosisWire-coil induced thrombosis

PURPOSE AND RATIONALEA 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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PROCEDUREMale 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 thrombosisPURPOSE 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 V-Phenotype-

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 VII-Phenotype-

Develop normally but suffer fatal perinatal bleeding.

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 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 21(Suppl 1):80–87

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

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