Quantification of platelet-surface interactions in real-time using intracellular calcium signaling.

Platelets get easily activated when in contact with a surface. Therefore in the design of microfluidic blood analysis devices surface activation effects have to be taken into account. So far, platelet-surface interactions have been quantified by morphology changes, membrane marker expression or secretion marker release. In this paper we present a simple and effective method that allows quantification of platelet-surface interactions in real-time. A calcium indicator was used to visualize intracellular calcium variations during platelet adhesion. We designated cells that showed a significant increase in cytosolic calcium as responding cells. The fraction of responding cells upon binding was analyzed for different types of surfaces. Thereafter, the immobilized platelets were chemically stimulated and the fraction of responding cells was analyzed. Furthermore, the time between the binding or chemical stimulation and the increased cytosolic calcium level (i.e. the response delay time) was measured. We used surface coatings relevant for platelet-function testing including Poly-L-lysine (PLL), anti-GPIb and collagen as well as control coatings such as Bovine Serum Albumin (BSA) and mouse immunoglobulin (IgG). We found that a lower percentage of responding cells upon binding, results in a higher percentage of responding cells upon chemical stimulation after binding. The measured delay time between platelet binding under sedimentation and calcium response was the lowest on a PLL-coated surface, followed by an anti-GPIb and collagen-coated surface and IgG-coated surface. The presented method provides real-time information of platelet-surface interactions on a single cell as well as on a cell ensemble level. For future in-vitro diagnostic tests, this real-time single-cell function analysis can reveal heterogeneities in the biological processes of a cell population.

Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets.

Essentials Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF). Salivary EVs expose CD24, a ligand of P-selectin. CD24 and coagulant TF co-localize on salivary EVs. TF+ /CD24+ salivary EVs bind to activated platelets and trigger coagulation. SUMMARY: Background Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF-exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell-derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin. Objectives We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P-selectin. Methods We investigated the presence of two ligands of P-selectin on salivary EVs, PSGL-1 and CD24. Results Salivary EVs expose CD24 but PSGL-1 was not detected. Immune depletion of CD24-exposing EVs completely abolished the TF-dependent coagulant activity of cell-free saliva, showing that coagulant TF and CD24 co-localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24. Conclusions A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P-selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action.

High platelet reactivity is associated with myocardial infarction in premenopausal women: a population-based case-control study.

SUMMARY BACKGROUND: Platelets are involved in the occlusion of coronary arteries after rupture of an atherosclerotic plaque. Furthermore, activated platelets release large quantities of growth factors, chemokines and interleukins that regulate inflammatory reactions. Therefore, we hypothesized that high basal platelet reactivity may contribute to an increased risk of myocardial infarction (MI) in premenopausal women. METHODS: We assessed the relation between high platelet reactivity and MI in a population-based case-control study among premenopausal women (aged < 50 years). We used multivariable logistic regression to quantify the effect of high platelet reactivity, adjusted for potential confounders. Platelet reactivity was estimated by plasma levels of neutrophil activating peptide 2 (NAP-2), CXC chemokine ligand (CXCL)4, soluble glycoprotein 1b (sGPIb) and soluble P-selectin. RESULTS: High platelet reactivity (i.e. levels >or= 90th percentile control subjects) was associated with a 2- to 3-fold increased incidence of MI: the adjusted odds ratios (ORs) were 3.0 [95% confidence interval (CI) 1.4-6.4] for NAP-2, 2.2 (0.9-5.1) for CXCL4, 1.9 (0.7-4.6) for sP-selectin and 2.5 (1.1-5.7) for sGPIb. The incidence of MI dose-dependently increased when more markers were elevated. High platelet reactivity according to both NAP-2 and sGPIb was associated with an up to tenfold increased incidence (9.9, 95% confidence interval 2.0-48.3). CONCLUSIONS: High basal platelet reactivity was associated with a 2- to 3-fold higher incidence of MI compared with normal platelet reactivity in premenopausal women. Our results suggest that high basal platelet reactivity may contribute to a higher risk of MI.