Inhibiting platelet-stimulated blood coagulation by inhibition of mitochondrial respiration

Abstract

Platelets are important mediators of blood coagulation that lack nuclei, but contain mitochondria. Although the presence of mitochondria in platelets has long been recognized, platelet mitochondrial function remains largely unaddressed. On the basis of a small amount of literature that suggests platelet mitochondria are functional, we hypothesized that the inhibition of platelet mitochondria disrupts platelet function and platelet-activated blood coagulation. To test this hypothesis, members of the tetrazole, thiazole, and 1,2,3-triazole families of small molecule heterocycles were screened for the ability to inhibit isolated mitochondrial respiration and coagulation of whole blood. The families of heterocycles screened were chosen on the basis of the ability of the heterocycle family to inhibit a biomimetic model of cytochrome c oxidase (CcO). The strength of mitochondrial inhibition correlates with each compound's ability to deter platelet stimulation and platelet-activated blood clotting. These results suggest that for this class of molecules, inhibition of blood coagulation may be occurring through a mechanism involving mitochondrial inhibition.

Conflict of interest statement

Conflict of interest statement: A patent application has been filed by the authors on the compounds described in this paper.

Figures

Fig. 1.

(A) Schematic of a biomimetic model of CcO tethered to a gold electrode via a self-assembled monolayer (SAM). (B) Linear sweep voltammograms showing the model's electrocatalytic O2 reduction (solid black line), its inhibition by a 1-mM solution of tetrazole (red line), and the recovery of its catalysis after removing the solution of tetrazole (dotted black line). (C) Percent inhibition of peak catalytic current by 1-mM solutions of representative compounds from different families of heterocycles.

Fig. 2.

Heterocycles screened for inhibition of mitochondrial function and blood coagulation.

Fig. 3.

Percent inhibition of mitochondrial respiration versus concentration of tetrazole (black circles). Separating tetrazole from the mitochondria by centrifugation after incubating the mitochondria in a 450-mM solution restores respiration to very near its original value (89 ± 7% of initial value, denoted by red triangle).

Fig. 4.

Correlation between strength of mitochondrial inhibition and decrease in platelet clumping activity. IC50 values for mitochondrial respiration were determined using a curve fit to a plot of percent mitochondrial inhibition versus heterocycle concentration as shown in Fig. 3 (remainder of data shown in Table S1). The concentrations required to increase clumping time by 50% were determined using a curve fit to plots of time required for platelet clumping versus heterocycle concentration (data shown in Table S2). Although consistent with the trend, 3a is not shown on the plot due to the difficulty of encompassing all of the data on the same graphical scale.