Morphological Differences between GPIb Antibody-Induced and Shear Stress-Induced Platelet Aggregates

Abstract

We determined the morphological differences between NNKY5-5-induced and shear stress-induced platelet aggregates using confocal laser scanning microscopy, and investigated the effects of cytokines on these aggregates. As shear stress was increased from low to high, many aggregates were generated. Some platelets and aggregates already exhibited PAC-1 binding at low shear stress. And finally, aggregates were clearly stained by PAC-1 at high shear stress. Low shear stress-induced aggregates included fibrinogen, but not von Willebrand factor (vWF). In contrast, high shear stress-induced aggregates included both fibrinogen and vWF. NNKY5-5-induced platelet aggregates exhibited both PAC-1 binding and fibrinogen, but vWF was not found in them. The aggregate formation in NNKY5-5-treated platelet-rich plasma (PRP) at low shear stress was clearly more pronounced than that in untreated PRP. In addition, aggregates in NNKY5-5-treated PRP formed after 6 min under low shear stress were clearly stained by PAC-1 and included fibrinogen, but vWF was not found in them. In order to investigate the effects of cytokines on platelet activation under NNKY5-5 stimulation, changes in light transmission and light scatter were assessed with an AG-10. G-CSF, IL-6 and thrombopoietin (TPO) each enhanced light scatter compared to control PRP, although IL-3, GM-CSF, erythropoietin and stem cell factor did not. In particular, TPO significantly enhanced the ‘%-T’ and ‘S-Max’ of NNKY5-5-treated PRP. TPO clearly enhanced the aggregate formation with high shear stress or NNKY5-5 stimulation. These results suggest that the glycoprotein Ib (GPIb)-unmediated aggregates can be formed with only fibrinogen, but the GPIb-mediated aggregates by vWF and fibrinogen synergistically. In particular, the latter is formed more firmly, and both aggregates are enhanced by TPO.

Microparticle formation after exposure of blood to activated endothelium under flow

“…Studies have shown that high sheer stress alone can induce platelet activation and platelet MP formation (32–34), and we have previously shown that the in vitro model used in the current study does cause some activation of monocytes and neutrophils in diluted whole blood but that specific interaction with TNF‐activated EC preconditioned to laminar flow‐induced expression of TF and PAR‐2 on monocytes (11). Here, we have extended these studies and show that interactions with TNF‐activated EC under flow conditions can lead to changes in the proportion of circulating MP defined as <1.0 μm diameter particles within whole blood (25).…”

Aggregation Mechanism of Blood Platelets Studied by the Time-Resolved Light Scattering Method

“…Recently, a laser light-scattering aggregometer (Kowa PA-100) was developed with the intention of diagnostic uses, ,− where particular attention was paid to the sensitivity for detecting a very low concentration of AG S at the initial stage of the platelet aggregation process. The number concentration of aggregated particles was given solely as counts of scattering signals/time, while an attempt was made to estimate the particle size from scattering intensity on the basis of Mie's theory .…”

“…The number concentration of aggregated particles was given solely as counts of scattering signals/time, while an attempt was made to estimate the particle size from scattering intensity on the basis of Mie's theory . In these studies, − thus, a quantitative description for time-dependent platelet aggregation with respect to the size and the number concentration of aggregates had not yet been successful. As a result, the previous laser light-scattering aggregometer is excellent in diagnostic and biomedical uses, but for use in a quantitative study of the platelet aggregation there is still great room for improvement in the way of analyzing scattering data.…”