Activation of villous trophoblastic p38 and ERK1/2 signaling pathways in preterm preeclampsia and HELLP syndrome.

Preterm preeclampsia is associated with the failure of trophoblast invasion, placental hypoxic/ischemic injury and the release of toxic substances, which promote the terminal pathway of preeclampsia. In term preeclampsia, factors yet unknown trigger the placenta to induce the terminal pathway. The contribution of the villous trophoblast to these pathologic events has not been fully elucidated. Here we aimed to study how stress and signaling pathways influence trophoblastic functions in various subforms of preeclampsia. Tissue microarrays (TMAs) were constructed from placentas obtained from pregnant women in the following groups: 1-2) preterm preeclampsia with (n = 8) or without (n = 7) HELLP syndrome; 3) late-onset preeclampsia (n = 8); 4-5) preterm (n = 5) and term (n = 9) controls. TMA slides were stained for phosphorylated Akt-1, ERK1/2, JNK, and p38 kinases, and trophoblastic immunostainings were semi-quantitatively evaluated. BeWo cells were kept in various stress conditions, and the expression of FLT1, GCM1, LEP, and PGF was profiled by qRT-PCR, while Akt-1, ERK1/2, JNK, and p38 kinase activities were measured with phospho-kinase immunoassays. We found that: 1) Placental LEP and FLT1 expression was up-regulated in preterm preeclampsia with or without HELLP syndrome compared to controls; 2) Mean pp38 immunoscore was higher in preterm preeclampsia, especially in cases with HELLP syndrome, than in controls. 3) Mean pERK1/2 immunoscore was higher in preterm preeclampsia with HELLP syndrome than in controls. 4) In BeWo cells, ischemia up-regulated LEP expression, and it increased JNK and decreased ERK1/2 activity. 5) Hypoxia up-regulated FLT1 and down-regulated PGF expression, and it increased ERK1/2, JNK and p38 activity. 6) IL-1ß treatment down-regulated PGF expression, and it increased JNK and p38 activity. 7) The p38 signaling pathway had the most impact on LEP, FLT1 and PGF expression. In conclusion, hypoxic and ischemic stress, along with unknown factors, activates trophoblastic p38 signaling, which has a key role in villous trophoblastic functional changes in preterm preeclampsia. The activation of ERK1/2 signaling may induce additional trophoblastic functional changes in HELLP syndrome, while distinct mechanisms may promote late-onset preeclampsia.

Sepsis- and endotoxemia-generated cytokines do not trigger activation of human platelets.

OBJECTIVE: To analyze the effect of cytokines generated in sepsis and endotoxemia (tumor necrosis factor [TNF]-alpha and interleukins [IL]-1beta, -6, and -8) on activation of human platelets and to study the effect of cytokines on platelet activation in the presence of alpha-thrombin, a potent inducer of coagulation and platelet activation generated in sepsis and endotoxemia. DESIGN: flow cytometric study of platelet activation induced by cytokines and/or thrombin in the whole blood and platelet-rich plasma (PRP) of healthy volunteers. SETTING: Research laboratory in a Canadian hospital. SUBJECTS: Nine healthy volunteers recruited from laboratory staff. MEASUREMENTS AND MAIN RESULTS: Venous blood samples were obtained into acid-citrate-dextrose anticoagulant. Whole blood and PRP were diluted with appropriate buffer optimized for analyzing platelet activation by flow cytometry. TNF-alpha, IL-1beta, IL-6, and IL-8 were added to blood or PRP in concentrations ranging from 1 to 100 ng/mL and incubated for 15 mins at 37 degrees C in the presence or absence of a submaximal concentration of human alpha-thrombin (0.025 units/mL). Samples were stained with fluorescent antibodies against markers of platelet activation (P-selectin [CD62], lysosomal protein [CD63], and fibrinogen and von Willebrand factor receptors [CD41 and CD42b, respectively]) and analyzed by flow cytometry. The data obtained show that none of these cytokines trigger activation of resting platelets in whole blood or PRP and do not modulate the effect of thrombin on platelet activation as measured by quantitation of CD62, CD63, and CD42b markers on the platelet surface. CONCLUSIONS: Cytokines TNF-alpha, IL-1beta, IL-6, and IL-8, which are extensively produced in sepsis and endotoxemia, do not trigger activation of resting human platelets directly or indirectly by mediating processes in white or red blood cells. The cytokines did not affect thrombin-induced platelet activation.

Hemolink, an o-raffinose cross-linked haemoglobin-based oxygen carrier, does not affect activation and function of human platelets in whole blood in vitro.

Haemoglobin-based oxygen carriers (HBOCs) are anticipated to be safe and efficient alternatives to RBC transfusions. Haemoglobin (Hb) raffimer (Hemolink; Hemosol, Toronto, ON, Canada) is polymerized human Hb, cross-linked with o-raffinose. As administration of cell-free Hb may affect blood cells and tissues, this study was focused on evaluating effects of Hb raffimer on human platelets in whole blood in vitro. Citrated blood from healthy donors was incubated with Hb raffimer to achieve raffimer concentrations of 2-50 vol percentage (2-50 g/l). Platelet activation, phosphatidylserine exposure and microparticle generation were measured by flow cytometry. Aperture closure time on collagen/ADP- and collagen/epinephrine-coated membranes was determined by a platelet function analyser (PFA-100). We found that addition of Hb raffimer to blood samples up to 50 vol % did not affect human platelets as measured by various markers of platelet activation (CD42b, CD41, PAC-1, CD62, CD63), procoagulant activity (annexin V) and microparticle formation; differences between Hb raffimer- and lactated Ringer's-diluted blood were not significant. Similarly, no adverse effect of Hb raffimer on closure time was observed at concentrations up to 50 vol %, in comparison with Ringer's solution. These data indicate that exposure of human blood to high concentrations of Hb raffimer in vitro did not cause platelet activation nor affect platelet function.

A rabbit model for monitoring in vivo viability of human platelet concentrates using flow cytometry.

BACKGROUND: Viability in vivo of novel platelet components cannot be readily determined in human transfusions. Elaboration of valid animal models may be useful for this purpose. STUDY DESIGN AND METHODS: Viability of platelet concentrates (PCs) WBC reduced before storage was determined by flow cytometry in rabbits whose reticuloendothelial system was inhibited by ethyl palmitate administration. PCs stored at 22 degrees C for 2 and 5 days (D2- and D5-PCs) or refrigerated PCs (3-6 days at 22 degrees C plus 1-4 days at 4 degrees C, RF-PCs) were transfused into rabbits. Five parameters of PC viability in vivo were calculated from human platelet survival curves: survival time, recovery 0.5 and 24 hours after transfusion (R0.5, R24), maximal recovery (Rmax), and total recovery for 0 to 24 hours (RSigma). RESULTS: No differences in viability of D2- and D5-PCs were found. In contrast, viability of RF-PCs was significantly lower than that of D2-PCs, as was revealed with diverse sensitivity by four parameters: RSigma > R24 > R0.5=survival time (p < 0.001, p < 0.01, and p < 0.05, respectively). CONCLUSION: The rabbit model elaborated is sufficiently sensitive to reveal differences in human platelet viability in vivo between conventional and cold-damaged PCs. It may be useful for comparing viability of different platelet components that cannot be readily tested in human transfusions.

The relationship of von Willebrand factor binding to activated platelets from healthy neonates and adults.

von Willebrand Factor (VWF) is important in platelet adhesion and shear-dependent platelet activation. We performed flow cytometric analyses of VWF binding to and activation of platelets from healthy neonates, children, and adults. Platelets from cord blood (n = 38; gestational age: 36-42 wk; birth weight: 2.4-5.1 kg), neonatal venous blood (n = 19; d 2-3 of life), children (n = 15; age: 1.5-16.3 y), and adults (n = 22; age: 18-55 y) were studied. Binding of VWF was assessed using an antihuman VWF polyclonal antibody and a FITC-conjugated secondary antibody. Platelet activation was determined by the expression of CD62P, CD63, CD41, CD42b, activated GPIIb/IIIa (PAC-1), procoagulant surface (as reflected by annexin V binding), and microparticle formation. Although the mean percentage of VWF-positive platelets was not significantly higher in unstimulated platelets from 2- to 3-d-old neonates, their platelets were more activated than those from adults, and there was a positive correlation of VWF binding with platelet activation (CD62P: r = 0.74, p < 0.001; annexin V: r = 0.46, p < 0.05). In adults, after in vitro activation of platelets with thrombin and ADP, VWF binding to platelets increased and correlated significantly with CD62P expression (r = 0.71, p < 0.001). VWF binding to unstimulated neonatal platelets was, however, higher than that to in vitro-stimulated platelets from adults at the same level of expression of platelet activation markers. Further studies are required to assess the mechanism and significance of VWF binding to activated platelets in the neonatal period.