Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.

Pentobarbital Anesthesia Suppresses the Glucose Response to Acute Intermittent Hypoxia in Rat.

A key feature of sleep disordered breathing syndromes, such as obstructive sleep apnea is intermittent hypoxia. Intermittent hypoxia is well accepted to drive the sympathoexcitation that is frequently associated with hypertension and diabetes, with measurable effects after just 1 h. The aim of this study was to directly measure the glucose response to 1 h of acute intermittent hypoxia in pentobarbital anesthetized rats, compared to conscious rats. However, we found that while a glucose response is measurable in conscious rats exposed to intermittent hypoxia, it is suppressed in anesthetized rats. Intermittent hypoxia for 1, 2, or 8 h increased blood glucose by 0.7 ± 0.1 mmol/L in conscious rats but had no effect in anesthetized rats (-0.1 ± 0.2 mmol/L). These results were independent of the frequency of the hypoxia challenges, fasting state, vagotomy, or paralytic agents. A supraphysiological challenge of 3 min of hypoxia was able to induce a glycemic response indicating that the reflex response is not abolished under pentobarbital anesthesia. We conclude that pentobarbital anesthesia is unsuitable for investigations into glycemic response pathways in response to intermittent hypoxia in rats.

Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium.

Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils' importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.