Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

Do immature platelet levels in chest pain patients presenting to the emergency department aid in the diagnosis of acute coronary syndrome?

INTRODUCTION: Early and accurate identification of acute coronary syndrome (ACS) vs. noncardiac chest pain in patients presenting to the emergency department (ED) is problematic and new diagnostic markers are needed. Previous studies reported that elevated mean platelet volume (MPV) is associated with ACS and predictive of cardiovascular risk. MPV is closely related to the immature platelet fraction (IPF), and recent studies have suggested that IPF may be a more sensitive marker of ACS than MPV. The objective of the present study was to determine whether the measurement of IPF assists in the diagnosis of ACS in patients presenting to the ED with chest pain. METHODS: In this single-center, prospective, cross-sectional study, adult patients presenting to the ED with chest pain and/or suspected ACS were considered for enrollment. Blood samples from 236 ACS-negative and 44 ACS-positive patients were analyzed in a Sysmex XE-2100 for platelet count, MPV, IPF, and the absolute count of immature platelets (IPC). RESULTS: Total platelet counts, MPV, IPF, and IPC were not statistically different between ACS-negative and ACS-positive patients. The IPF was 4.6 ± 2.7% and 5.0 ± 2.8% (mean ± SD, P = 0.24), and the IPC was 10.0 ± 4.6 and 11.5 ± 7.5 × 10(3) /µL (P = 0.27) for ACS-negative and ACS-positive patients, respectively. CONCLUSION: In 280 patients presenting to the ED with chest pain and/or suspected ACS, no differences in IPF, IPC or MPV were observed in ACS-negative vs. ACS-positive patients, suggesting that these parameters do not assist in the diagnosis of ACS.

A low-dimensional deformation model for cancer cells in flow.

A low-dimensional parametric deformation model of a cancer cell under shear flow is developed. The model is built around an experiment in which MDA-MB-231 adherent cells are subjected to flow with increasing shear. The cell surface deformation is imaged using differential interference contrast microscopy imaging techniques until the cell releases into the flow. We post-process the time sequence of images using an active shape model from which we obtain the principal components of deformation. These principal components are then used to obtain the parameters in an empirical constitutive equation determining the cell deformations as a function of the fluid normal and shear forces imparted. The cell surface is modeled as a 2D Gaussian interface which can be deformed with three active parameters: H (height), σ(x) (x-width), and σ(y) (y-width). Fluid forces are calculated on the cell surface by discretizing the surface with regularized Stokeslets, and the flow is driven by a stochastically fluctuating pressure gradient. The Stokeslet strengths are obtained so that viscous boundary conditions are enforced on the surface of the cell and the surrounding plate. We show that the low-dimensional model is able to capture the principal deformations of the cell reasonably well and argue that active shape models can be exploited further as a useful tool to bridge the gap between experiments, models, and numerical simulations in this biological setting.

Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells.

The routine observation of tumor emboli in the peripheral blood of patients with carcinomas raises questions about the clinical relevance of these circulating tumor cells. Thrombosis is a common clinical manifestation of cancer, and circulating tumor cells may play a pathogenetic role in this process. The presence of coagulation-associated molecules on cancer cells has been described, but the mechanisms by which circulating tumor cells augment or alter coagulation remains unclear. In this study we utilized suspensions of a metastatic adenocarcinoma cell line, MDA-MB-231, and a non-metastatic breast epithelial cell line, MCF-10A, as models of circulating tumor cells to determine the thrombogenic activity of these blood-foreign cells. In human plasma, both metastatic MDA-MB-231 cells and non-metastatic MCF-10A cells significantly enhanced clotting kinetics. The effect of MDA-MB-231 and MCF-10A cells on clotting times was cell number-dependent and inhibited by a neutralizing antibody to tissue factor (TF) as well as inhibitors of activated factor X and thrombin. Using fluorescence microscopy, we found that both MDA-MB-231 and MCF-10A cells supported the binding of fluorescently labeled thrombin. Furthermore, in a model of thrombus formation under pressure-driven flow, MDA-MB-231 and MCF-10A cells significantly decreased the time to occlusion. Our findings indicate that the presence of breast epithelial cells in blood can stimulate coagulation in a TF-dependent manner, suggesting that tumor cells that enter the circulation may promote the formation of occlusive thrombi under shear flow conditions.