Fine and Ultrafine Particles Differentially Affect the PFA-100-Times and Raise Proinflammatory Cytokines in Human Blood.

INTRODUCTION: Peaks of ambient air pollution with increased concentrations of particulate matter (i.e. PM_2.5: Fine particles of < 2.5 μm diameter and UFP: Ultrafine particles of <0.1 μm diameter) easily reach levels of 100’000 particles/cm³ and are associated with increased cardiovascular morbidity and mortality. Especially the UFP-fraction rapidly crosses cellular membranes by non-phagocytotic mechanisms and is translocated into the circulation. The absorbed microparticles may affect the coagulation directly or indirectly via monocyte/macrophage activation. The data on prothrombotic and inflammatory effects are controversial, depending on the type, size and charge of particles and the experimental models chosen.

HYPOTHESIS: We therefore hypothesized that typically sized particles differentially affect human platelets and coagulation either directly or indirectly by the proinflammatory response.

METHODS AND RESULTS: Three microparticle sources were mixed with citrated human blood at concentrations of 5 and 50 μg/ml for one hour:

a) PM_2.5 polystyrene beads (PS) of 0.66 μm diameter. b) UFP ultrafine carbon black (ufCB, 14 nm, Printex), and c) residual oil fly ash (ROFA, with contents of Al, Zu, Cu-ions, and residual polyaromatic hydrocarbons).

We calculated that >800 μg of microparticles may be absorbed by the human lung daily and thus a dose of appr. 5 μg/ml blood could be achieved easily.

Platelet aggregation with ADP, collagen, arachidonate and ristocetin at different concentrations was not affected by any of the three types of microparticles and neither was PT, aPTT, fibrinogen, DDimer or soluble P-selectin. Direct microscopical observation did not show platelet aggregates, nor rosetting of platelets with leucocytes, nor mixed platelet aggregates with microparticles. PFA-100 CADP, however, was prolonged in the presence of PS beads from 85 ± 3 sec to 101 ± 6 (n = 3, p < 0.04); ROFA, on the other hand, shortened it by more than 20 sec to 78 ± 2 vs 110 ± 6 sec (n = 3, p < 0.05) and ufCB/UFP did not affect it. The standardized endogenous thrombin potential as measured by the thrombinoscope did not exhibit significant changes under the conditions chosen.

Preliminary data of a highly standardized assay of cytokine release after 24 hours of stimulation with low dose LPS (1μg/ml blood) indicates a 2 - 3 fold increase of interferon, interleukine-1 and TNFa, but not of interleukine-8.

CONCLUSION: Our data demonstrate a direct shortening effect on shear induced platelet aggregation (PFA-100) by ROFA and a prolongation by positively charged PS beads. None of the three particle types showed a direct effect in any of the other coagulation assays evaluated. The strong proinflammatory cytokine response to all three microparticle preparations is likely responsible for the procoagulant / prothrombotic effects which have been observed in experimental mouse and hamster models.