Mark I.M. Noble^*1

1. Department of Medicine and Therapeutics, Polwarth Building, Foresterhill, Aberdeen AB25 2ZH, University of Aberdeen, UK

^*Corresponding author:Mark I.M. Noble, Department of Medicine and Therapeutics, Polwarth Building, Foresterhill, Aberdeen AB25 2ZH, University of Aberdeen, UK, Tel: +44 1224 272000;

Abstract

Over many years, laboratory testing of platelet aggregability have been carried out inattempts to develop drugs that would prevent thrombosis in arteries. The problems encounteredincluded the question of methodology. Blood samples have to be anticoagulated in order to studythe platelets. Anti-coagulation with citrate and tests on derived platelet rich plasma did not correlateat all well with thrombus growth in the stenosed coronary arteries of experimental animals andcitrate removes the calcium ions which are vital for platelet function. Anticoagulation with heparinalso interfered with platelet function, so that now, hirudins are the preferred anticoagulant.

However it was observed that if, in stead of stimulating platelet aggregation with adrenaline or ADP,serotonin was applied to the the preparation, very little aggregation took place in spite of serotonin5HT2A antagonists being the most potent inhibitors of thrombus growrh in experimental animals.Another indicator that primary platelet agggregation is not a predictor of in vivo efficacy was thefinding that 5HT2A antagonism inhibited aggregate growth. In a stenosed artery the platelets areactivated by increased shear stress and blood turbulance with release of platelet serotonin causing positive feedback activation of more platelets. At present, there does not seem to be a bench in vitrotest that accuratly predicts in vivo efficacy in stenosed artery occlusive thrombosis.

Introduction

In the obituary of Gustav Born[1], one finds the following statement about his work onblood platelets, “When stimulated by blood vessel damage or blood clotting, these tiny cells“aggregate” together, plugging the damaged vessel and thereby preventing any further escape ofblood. To measure this quantitatively, Gus devised and developed a simple, but extremely effective,device (known as the Born aggregometer) and, in doing so, launched an entirely new sub-disciplineof haematology often referred to as “platelet aggregometry” (a term he hated). The aggregometer,used in laboratories and hospitals around the world, revolutionised the diagnosis of platelet-related diseases as well as providing a tool eminently suited to the study of the basic biology of these cells and their response to drugs”. Very true and Born deserves all the credit he achieved, but is the statement still true today?

The Limitations of Born Aggregometry

The method involved anticoagulating blood samples with citrate and using a centrifuge toseparate out a layer of platelet-rich plasma (PRP). When aggregating agents such as collagen areadded to PRP in a cuvette, the platelets aggregate and settle at the bottom of the cuvette causing anincrease in light transmission through the PRP which can be recorded on a chart or computer [2].

The first problem with this is that citrate anticoagulates the blood sample by removing the calciumions (Ca2+). However, (Ca2+) is essential for normal platelet function!An equally obvious problem is that such in vitro bench tests take place in very differentcircumstances than real life where one has whole blood flowing through vessels. John Folts had theidea that platelet aggregation would be best studied in partially obstructed arteries in ananaesthetised animal [3,4,5], and showed the various potentcies of platelet aggregation inhibitors[5].

How well does PRP Aggregometry Correlate with In Vivo Platelet Thrombosis?

Torret al. [2] compared the results of ex vivo classical aggregometry of PRP prepared fromexperimental animals with the number of flow reductions in a stenosed coronary artery ofanaesthetised dogs. Measurements were made before and after administraion of a putative plateletaggregation inhibitor ritanserin. They used collagen and ADP as aggregation stimulants, collagen at10μM and 50μM, also 5μM plus adrenaline, ADP at 2μg plus adrenaline, 4μg and 6μg. Theinhibition by ritanserin in number and slopes of the flow reductions in the animals was very much greater than the inhibition of ex vivo platelet aggregation.

Clearly the platelet aggregation results were a very poor way of assessing the inhibition ofthrombus growth in the artery by ritanserin. Why use ritanserin? This drug was an inhibitor of theplatelet serotonin 5HT2A receptor and was a way of testing the theory that platelet activation inarteries results from the haemodynamic consequences of partial obstruction of the artery.

Arterial Stenosis

At an arterial narrowing (stenosis),∂P = a1Q + a2Q2 where ∂P is the pressure drop across the stenosis, Q is the flow through the artery, a1 and a2 are proportionality parameters. This means that the relationship between the variables ∂P and Q is quadratic and stenotic resistance increases with flow a1Q expresses viscous resistance, while a2Q2 expresses turbulance. Another quantitative expression of a stenosis is the area ratio As/Ao, where Asis the cross-sectional area of the stenosis and Ao is the cross-sectional are of the open, normal artery;this can be expressed as a percentage as (1 – As/Ao)x100. As the same flow has to go through both the normal section, Ao and the much smaller As of the stenosis, the blood has to go faster, i.e.,velocity of blood flow increases; this is called convective acceleration. One can easily observe this phenomenon by watching a placid full river running into a gorge.Acceleration is determined by force according to Newton’s second law of motion,F = ma, where F is force, m is mass and a is acceleration. So we envisage a mass of fluid accelerating into an arrowing of the artery exerting greater force, and just as objects in a river gorge feel force, so do blood cells in a stenosis. This effect is called shear stress because it is equal to the force divided bythe area of the surface of the object.

Activation of platelets by exposure to increased shear stress and turbulance causes release ofserotonin which is present in high quantities in the dense granules [6]. The serotonin is released upon activation and activates more platelets via the 5HT2A receptors, thus maintaining thrombus growth. Thrombus growth is completely abolished by 5HT2A platelet receptor antagonism [7]. The drug used by McAuliffe et al [7] was trialed in stable coronary disease patients and found to cause no change in bleeding time and to have no cerebral or other significant side effects [8].

The Effect of Serotonin on Platelet Aggregation

This subject has been neglected because serotonin is said to be weak agonist for plateletaggregation [9]. At least these authors [9] did not use PRP, but whole blood using flow cytometry,impedance aggregometry and filtragometry. They concluded that their data suggest that 5-HT per sedoes not activate platelets, but dose-dependently enhances platelet activation induced by ADP and,in particular, thrombin in whole blood. This effect is mediated by 5-HT2A receptors. While it is truethat the coronary thrombosis stimulated by adrenaline is also abolished by 5HT2A antagonism [7],there is a very great difference between the high potency of inhibition of thrombus growth not sostimulated, with the weakness of serotonin in in vitro testing [9].

Another problem with whole blood aggregometry is the activation of platelets by heparin[10] making heparins unsuitable for in vitro testing. The mst suitable anticoagulants for in vitrowork are the hirudins, because the effects of hirudins on platelet function are different from those ofunfractionated heparin. Spontaneous aggregation was rarely observed in hirudinized PRP comparedto citrate-PRP. In comparison with citrate-PRP, two hirudins reduced the maximal response toepinephrine, but had no influence on the maximal response to ADP. Spontaneous aggregation israrely observed in hirudinized PRP compared to citrate-PRP. In comparison with citrate-PRP, bothhirudins reduced the maximal response to adrenaline, but had no influence on the maximal responseto ADP [11]. Other authors agree that of the anticoagulants investigated, the selective thrombininhibitor hirudin is the most suitable anticoagulant for studies of platelet aggregation in vitro inwhole blood, e.g., [12].Menys [13], studying platelet aggregation to collagen, used hirudin to maintain normocalcaemia, thereby avoiding erroneous findings often obtained with citrated blood. Plateletaggregation was quantified by optical aggregometry, and aggregate growth by single platelet counting, i.e., as the aggregates grew, fewer single platlets remained. He concluded that 5-HTcontributes to collagen induced aggregate growth and 5-HT2 receptor antagonism with ICI 170809retards the rate of growth. This could explain the efficacy of 5-HT2 antagonists in limiting coronarythrombosis despite the limited role of 5-HT in primary aggregation. ICI 170809 is the same drug asthat used by McAuliffe [7] in the Folts model and by Noble et al in patients [8]. So the classicalplatelet aggregation tests measure the wrong thing. It is the aggrgation of platelets that leads tothrombus growth, not the primary aggregation.

It is the reliance on platelet aggregation tests that has led to the development of the presentdual anti-platelet therapy of aspirin (antagonising thromboxane) and clopidogrel (antagonisingADP), both of which are ubiquitously present in tissues and antagonism of them inevitably leads toescessive bleeding. Serotonin, on the other hand is only present to any extent in brain and plateletsand not in tissue. Excess bleeding is therefore absent [8].

What Happens at the Vessel Wall?

The endothelial layer is covered by a layer of gel called the glycocalyx, dysfunction of which has been postulated to be the first step in the development of atheromatous lesions [14,15].Such dysfunction occurs by oxidised LDL[16] and with hyperglycaemia [17] leading to celladhesion to the endothlium [18] and thrombosis [17]. However, in arteries with normal luminal cross sectional area, this does not result in thrombotic occlusion. That only occurs in stenosis where very high concentration of serotoin occur in a confined space y the mechanism described above. A literature search for a bench model of hirudinised whole blood being forced through astenosis failed to find any such attempt at a more realistic test for predicting clinical efficacy.

What Test Must One Use?

Unfortunately, none of the laboratory tests on platelets realistically simulates the in vivo situation of obstructive arterial thrombosis. The only way at present to test potential treatments is by using the Folts model, i.e., animal experimentation. This has been a reliable predictor of clinical efficacy, and has brought to light the importance of serotonin antagonism in spite of the fact that seerotonin is a weak agonist in bench tests [19 -30].

References

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Received: June 11, 2018.
Accepted: June 28, 2018.
Published: July 03, 2018.

To cite this article : Mark Noble IM. Are platelet aggregation tests the best way to assess new drugs for arterial disease?.British Heart Diseases. 2018: 1:1.

© Mark Noble IM. 2018.