The development of new drugs is often limited or even halted by their side effects on platelet function. In drug discovery, identifying compounds that might have secondary pharmacological effects on platelet function can contribute to de-risking and help with the progression of potential new drugs.
Half-maximal inhibitory concentration (IC50 or pIC50 (-log10IC50)), the concentration of an antagonist (or inhibitor) that blocks a given predefined stimulus by 50 percent, is commonly used to measure antagonist potency. This type of analysis has the advantage of allowing a large number of compounds to be assessed. However, due to the nature of platelets and their inherent variability, generating accurate and reliable pIC50 values in platelet functional assays can be challenging.
In this blog, we highlight the use of a Schild type analysis, as a more robust method for assessing secondary pharmacology effects of compounds on platelet function.
Although pIC50 type analyses are widely used in drug discovery programs, the resultant pIC50 estimations are critically dependent on the agonist concentration used. Typically, when doing a pIC50 type analysis, an approximately 80% maximal agonist response (EC80) is used to investigate the effects of increasing antagonist concentrations. However, as shown in Figure 1, agonist responses in platelets can be quite variable making pIC50 type analyses difficult to perform in platelet functional studies. This can lead to failed experiments and/or lead to under or overestimation of antagonistic effects and inaccurate ranking of antagonist potencies.
Figure 1: Variability of platelet aggregation in platelet rich plasma induced by (A) collagen related peptide (CRP) EC80 (1 µg/mL) and (B) thrombin receptor activating peptide (TRAP) EC80 (16 µM) across a 96-well plate within one volunteer.
Schild analysis, first described by Schild in 1949, allows estimation of the affinity (potency) of an antagonist for a particular receptor. At Platelet Services, we undertake a Schild type analysis by generating a concentration-response curve to a known platelet agonist in the absence of the antagonist. Further sets of agonist concentration-response curves are obtained in the presence of a series of increasing antagonist concentrations, as illustrated in Figure 2A. Antagonist potency is estimated as a pA2 value (the negative logarithm of the molar concentration of the antagonist, which causes a two-fold shift to the right of the control agonist concentration-response), using the equation shown in Figure 2B.
Figure 2: (A) Antagonism of PAR-1-AP induced platelet aggregation in platelet rich plasma by SCH79797. (B) pA2 equation.
In this instance, the pA2 of 6.1 (0.79 µM) was estimated using the dose ratio measured with 2 µM of SCH79797. Because the Schild-type analysis assesses the antagonist’s potential to induce a right-shift of the agonist concentration curve, it is not affected by the variability that can be observed in the EC80 of an agonist.
Schild type analysis also allows pIC50 values to be calculated from the agonist concentration-response curve data. This is illustrated in Figure 3 with ibrutinib, an FDA-approved tyrosine kinase inhibitor (TKI) for the treatment of mantle cell lymphoma, but which has known bleeding side effects. Using a CRP concentration of 0.1 µg/ml the effects of the various concentrations of ibrutinib on the response to CRP can be measured (see red box in Figure 3A) and plotted to calculate a pIC50 (Figure 3B).
Figure 3: (A) Antagonism of CRP-induced platelet aggregation in platelet rich plasma by ibrutinib. (B) % inhibition of platelet aggregation in platelet rich plasma induced by ibrutinib.
Schild type analysis has the potential added benefit over pIC50 type analysis of providing information on compounds’ mechanism of action. Figure 2 illustrates that SCH79797 produces parallel concentration-dependent rightward shifts of the agonist responses. Such behaviour is consistent with simple competitive behaviour, that is, SCH79797 is a PAR-1 competitive antagonist. In contrast, Figure 3 indicates that ibrutinib is a non-competitive inhibitor of platelet aggregation stimulated by CRP. In this case, ibrutinib both depresses the maximum agonist response and causes ‘bunching’ of the agonist concentration-effect curves at concentrations of 3 µM and above. By revealing such behaviour, Schild type analysis can potentially suggest further experiments to explore the compound’s mechanism of action on platelet function.
Although there are many advantages to doing a Schild type analysis when investigating secondary pharmacology effects of antagonists on platelet function, this type of analysis is lower throughput compared to pIC50 analysis and therefore fewer compounds can be assessed per experiment. A balance between assay throughput and robustness of potency estimates will be client-led, but we highly recommend performing Schild type analysis when assessing lead compounds or ranking compound potencies on platelet function.
Here at Platelet Services, our team can advise you on every aspect of platelet function and help you determine if your compounds in development have an effect on platelets. Find out more about our services here.
