0050_A CONCEPT OF ANTI-THROMBOTIC THERAPY BASED ON THE PHARMACOLOGICAL EFFECTS - 02.pdf

8/22/2019 0050_A CONCEPT OF ANTI-THROMBOTIC THERAPY BASED ON THE PHARMACOLOGICAL EFFECTS - 02.pdf

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Reprint from

Atherosclerosis:Proceedings of the Second International Symposium

Edited byRichard J. Jones

Springer-Verlag New York. Heidelberg. Berlin 1970.Printed in Germany Not for sale)

A CONCEPT OF ANTI THROMBOTIC THERAPYBASED ON THE PHARMACOLOGICAL EFFECTSOF DRUGS PREVENTING PLATELET

AND RED CELL AGGREGATIONH. I. BICHEI~

References ro the literature appearing in the text are available in a comprehensive bibliographywhich may be obtained ar cost from the Chicago Heart Association, 22 West Madison Street,

Chicago, Illinois 60603, U.S.A.


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A CONCEPT OF ANTI-THROMBOTIC THERAPYBASED ON THE PHARMACOLOGICAL EFFECTS

OF DRUGS PREVENTING PLATELETAND RED CELL AGGREGATION

H.I. BICHER


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ANTI-ADHESIVE DRUGSIn previous reports E153, i55, t56, 158] we described the properties of a

group of chemically nonrelated substances, such as Phenylbutazone, Chloro-promazine, local anesthetics, etc. to prevent blood cell aggregation of the typethat may lead to thrombosis, namely red cell and platelet aggregation. Theseproperties were present both in vivo and in vitro and seemed to be mediatedby some type of membrane action El ~8]. They were termed Anti-AdhesiveDrugs .Several ketolactone derivatives ~14~1 ] exhibit these properties. Based on lowtoxicity and potency of action we selected 2-methyl-2-tert-butyl-beta-ketolactone(2-4 BMK) for further experimentation. This drug prevents in vitro platelet toglass adhesiveness and ADP-induced platelet aggregation at a concentrationof I mg/cc (as tested using modifications of the methods of Wright Et~79],Swank [t446]) and red cell aggregation at t00 ~/cc (tested with a modifiedThorsen method).

The effective doseso in vivo to prevent both platelet and red cell aggregationis 200 mg]kg. At this dose, the compound markedly improved the peripheralcirculatory disturbances induced by intravascular red cell aggregation (sludge),as shown by vital microscopy in the cats omentum.

PREVENTION BY ANTI-ADHESIVE DRUGS OF THROMBOSISAND ANOXIC TISSUE DAMAGEIn the present experiments attempts have been made to prevent in vivothrombus formation or tissue damage induced by the blood cell aggregation

processes. Microthrombi in lungs and respiratory death were produced in therat according to the technique of Nordoy (1964). Adenosine diphosphate (ADP)was injected at a dose of t7 mg/kg into the femoral vein of 24 animals, twelveof which were pretreated with 2-4 BMK. The mortality rate of the ADP controlanimals was 91t 2, that of the drug pretreated group was 3/12. Histological sectionsof the lungs showed platelets and red cell aggregates plugging the small vesselsin almost all animals of the first group, and only occasionally in rats pretreatedWith 2-4 BMK.Sludge-induced vein thrombosis was provoked in cats using the method ofBorgstrom [202]. This technique is a combination of ligation of both femoral


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A Concept of Anti-Thrombotic Therapy 539veins (to produce venous stasis) with trauma-induced intravascular red cellaggregation. Venous thrombi developed in the ligated veins after 24 hours. Theexperiments were performed on three groups of ten animals each. In the firstgroup vein ligatiou only was performed, and two thrombi were found. In thesecond group vein ligation was combined with trauma, and t8 thrombi werefound. The animals of the last group were given 2-4 BMK before and t2 hoursafter being exposed to the two previous procedures: only nine thrombi werefound: Observations of the peripheral circulation confirmed the presence of intra-vascular red cell aggregation in animals of the second group only.

The induction of intravascular red cell aggregation causes anoxic myocardialdamage in the atherosclerotic rabbit Et 57] demonstrable in the ECG and histo-logical sections. Pretreatment of the atherosclerotic rabbits with 2-4 BMK beforethe sludge induction prevented this effect.

A new ultramicro-oxygen electrode (2 ~ tip) has been developed. The externalsurface of a glass micropipette is coated with a thin layer of platinum that actsas an oxygen cathode when covered, but for the tip, by a second layer of anoxygen impervious material Et 541. A reference Ag-AgC1 electrode can be placedeither externally in the animal tissue or in the KC1 filled glass micropipette.Using this electrode, it can be shown that the compensatory response of thecirculation to brain cortex anoxia has two phases, a quick phase that returnscortex PO~ to pre-anoxic levels (reoxygenation time), and a second slow over-shoot phase wherein the PO~ exceeds the pre-anoxic base line. Induction ofintravascular red cell aggregation by the intravenous administration of highmolecular weight dextran dampens the second phase of the response, holding itunder the baseline PO~ levels, and considerably prolonging the reoxygenationtime . The intravenous injection of 2-4 BMK, 200 mg/kg, counteracts the effectsof intravascular red cell aggregation and restores the reoxygenation time tonormal values.

THROMBOTIC PROCESSThe classic concept of thrombosis--the intravascular formation of fibrin,

leading to blood cell entrapment, vessel occlusion and tissue death should beinterpreted in view of the increased amount of evidence describing the behaviorof the blood cells flowing in the small peripheral vessels. These vessels are ulti-mately responsible for the delivery of amounts of oxygen adequate to keep thetissue alive. A likely sequence of events for the thrombotic process based onthe current literature and the preceding facts follows (see Fig. 1).

Phase a: Intravascular Red Cell Aggregation (Combined With Atherosclerosisor Transient Hypoxia). The absence of intravascular red cell aggregation duringhealth has been confirmed in man E813]. Based on microscopic observations ofmicro~hrombotic occlusions in capillary vessels showing marked sludging of bloodcells, a relation between sludge and thrombosis has been suggested. Experimentalstudies ~202] and clinical observations ~19t, 606] have confirmed this relationship.Since sludge is present in thrombotic disease, the obvious question is: Is ita consequence or the beginning of the thrombotic process ?Gelin [521] summarized the possible pathogenetic mechanisms by whichsludge could induce tissue injury. Our present experiments show that when sludge


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is combined with atherosclerosis or a period of transient hypoxia, anoxic tissuedamage is the consequence, both in brain and heart.Phase b: Anoxic Damage to Vascular Endothelium In the postcapillary venules

and the venous limb of the capillary the delivery of oxygen is normally veryprecarious. It is in this region that hypoxic damage to vessel walls by the re-stricted blood flow of vasoconstriction or intravascular agglutination, or by both ofthese plus atherosclerosis is more easily evidenced. Knisely E7t2~ has describedthis damage that leads to an endothelial lesion and protein leak. Recently, ReneauEt 2241 developed a mathematical model in which the factors influencing oxygendiffusion to tissue were analyzed and predicted the formation of minute hypoxicareas at the venous ends of brain capillaries under pathological conditions.

Phase c: Platetet to Vessel Wall Reaction (Ptatetet Adhesiveness Phase). Contactis established through a leaky endothelium between platelet and tissue factors(collagen, etc.) that enhance platelet adhesiveness, thus initiating a more acti~zechain of events in the thrombotic process. Platelet adhesiveness has been cor-related with collagen-induced platelet aggregation. Native collagen fibrils aggregateplatelets in citrated plasma. The fibrils release considerable amounts of ADPfrom the platelets, and the aggregation is inhibited by AMP [712]. viden ehas also been obtained that collagen releases serotonin from platelets and rendersplatelet factor 3 available [13891. The electron microscopic studies of Hovig E712]indicated that the platelet membrane remains intact during interaction withcollagen, while the intracellular granules disappear. The release is thereforeprobably due either to an increased membrane permeability to ADP and sero-tonin or to an extrusion of platelet granular material without membrane rupture.Recent work by Johnson ~75t] has provided further details of this complicatedprocess. The release of these factors leads into the next steps of thrombus for-mation.

Phase d: Platdet Aggregation. The importance of platelet aggregation forhemostasis and for the pathogenesis of thrombotic processes is well established.It is associated with release of more endogenous platelet aggregation factors(EPAF), platelet factor III, minor red cell trapping, and enhanced local anoxia.Changes in platelet reactivity to ADP, the chemical agent most frequently con-sidered to be the natural platelet aggregation-inducer, has been determined ina number of hemorrhagic and thrombotic conditions E1070~. with the aid ofmethods that lend themselves to a quantitative determination of platelet function~Platelet clump embolism is also considered to be part of the mechanism leadingto atherosclerosis, according to Duguids theory [41 t 1.

Once the process is initiated, more endogenous ADP and other aggregationfactors are liberated, and the process tends to perpetuate itself. Our experimentsdemonstrated that red cells are trapped in the platelet aggregates, becominga major factor to increase the mass of the microemboli and probably the localhypoxia.

At this point, coagulation factors liberated from tissue (thromboplastin),platelets (factors III, V) and red cells probably initiate changes in plasma proteinswhich lead to:

Phase e: Cascade or Thrombin Activation Process o[ Fibrin Formation.These well known theories [898, t335~, not discussed herein, demonstrate the


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A Concept of Anti-Thrombotic Therapymanner in which the needed coagulation factors are made available by the hypoxiaaggregation process, which can, being reversible in nature, regulate the amountsand availability of these factors.

Phase [: Formation o] Red Thrombus . French E496] has described how theactual length of a thrombus is determined by the mass of red cells trapped in it.This is similar to the trapping of phase c, but the cells are incorporated in amore major way into the fibrin mesh.

SITE OF ACTIVITY OF ANTI-ADHESIVE DR UGSAccording to this scheme of thrombus formation, we propose that the anti-

adhesive drugs exert their anti-thrombotic action on phases a, b, c, d and fof the thrombotic sequence (marked with X in Fig. 1).X Phase a

X PhaseX PhaseX Phase

PhaseX Phase

Intravascular red cell aggregation combined with therosclerosis ndlortransient hypoxiaAnoxie damage to vascular endotheliuml~latelet to vessel mall reaction-/idDP and serotonin release (epaf)

Platelet aggregation further epaf, platelet factor III liberation, minoriVibri~ formationMay or blood cell trapping

X-Phases prevented by the action of antiadhesive drugsFig. 1. The thrombotic process


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Universit~tsdruckerei H. Stfirtz AG. Wfirzburg

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0050_A CONCEPT OF ANTI-THROMBOTIC THERAPY BASED ON THE PHARMACOLOGICAL EFFECTS - 02.pdf