Epi-fluorescent video microscopy was used to evaluate the effect of ethanol and two nipecotoylpiperazine based antiplatelet agents (BPAT-117 and BPAT-143) on platelet mural thrombus formation. In addition, a series of novel radio frequency glow discharge (RFGD) modified surfaces with varying nitrogen content were evaluated for blood biocompatibility.
Whole blood, treated with the indicated agent, was perfused over collagen coated or RFGD modified glass coverslips in a parallel-plate flow chamber at a shear rate of 1000/s. Digital image processing and photodiode measurements were used to analyze the dynamics of thrombus growth on these surfaces. RFGD modified samples were also evaluated with ESCA and SEM for chemical composition and cell morphology, respectively.
Ethanol concentrations as low as 0.02% v/v were found to inhibit 45 $\pm$ 23% ($\pm$ S.D.) of normal platelet accumulation on the slide while 0.2% v/v ethanol effected an 82 $\pm$ 15% inhibition of mural thrombus formation. While platelet adhesion to the collagen surface appeared unaffected by ethanol concentrations up to 0.1% v/v, 0.2% v/v ethanol had an effect on adhesion as well as aggregation. These results imply that low ethanol concentrations inhibit the formation of mural thrombi in a model of a damaged blood vessel at physiological shear rates. This inhibition would not be detected in systems which measure bulk aggregation, e.g. in aggregometric determinations.
At a concentration of 50 $\mu$M, BPAT-117 (the considerably more hydrophobic molecule) inhibited platelet accumulation by a striking 90 $\pm$ 2% ($\pm$ S.E.), while it took 2- to 4-fold higher concentrations of BPAT-143 to register meaningful to comparable effects (52 $\pm$ 6% and 80 $\pm$ 4%, respectively). These results corroborate the substantial impact of hydrophobic features, within the matrix of appropriately structured molecules, on their ability to alter platelet function.
The levels of adhesion observed on RFGD modified slides were highest (22.2 platelets/1000 $\mu$m$\sp2$) for the surfaces with an intermediate nitrogen content of 10.8%. Adhesion on all of the RFGD modified surfaces was significantly lower than that on collagen coated slides. SEM images show activated, spread platelets on 0% and 10.8% nitrogen surfaces, while 14.6% nitrogen surfaces support very little adhesion and activation. Flow cytometric analysis of effluent blood shows that the RFGD modified surfaces do not induce any significant bulk aggregation.