Platelet function following induced hypoglycaemia in type 2 diabetes.

AIM: Strict glycaemic control has been associated with an increased mortality rate in subjects with type 2 diabetes (T2DM). Here we examined platelet function immediately and 24hours following induced hypoglycaemia in people with type 2 diabetes compared to healthy age-matched controls. METHODS: Hyperinsulinaemic clamps reduced blood glucose to 2.8mmol/L (50mg/dl) for 1hour. Sampling at baseline; euglycaemia 5mmol/L (90mg/dl); hypoglycaemia; and at 24 post clamp were undertaken. Platelet function was measured by whole blood flow cytometry. RESULTS: 10 subjects with T2DM and 8 controls were recruited. Platelets from people with T2DM showed reduced sensitivity to prostacyclin (PGI2, 1nM) following hypoglycaemia. The ability of PGI2 to inhibit platelet activation was significantly impaired at 24hours compared to baseline in the T2DM group. Here, inhibition of fibrinogen binding was 29.5% (10.3-43.8) compared to 50.8% (36.8-61.1), (P<0.05), while inhibition of P-selectin expression was 32% (16.1-47.6) vs. 54.4% (42.5-67.5) (P<0.05). No significant changes in platelet function were noted in controls. CONCLUSION: Induced hypoglycaemia in T2DM enhances platelet hyperactivity through impaired sensitivity to prostacyclin at 24hours.

Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets.

BACKGROUND: Dissecting the signaling events that contribute to platelet activation will increase our understanding of platelet function and aid in the development of new antiplatelet agents. However, high-throughput methodology for the quantitative analysis of platelet signaling events is still lacking. OBJECTIVE: To develop a high-throughput assay for the analysis of platelet signaling events in whole blood. METHODS AND RESULTS: We developed a fluorescent barcoding protocol to facilitate multiplexing and enable large-scale signaling profiling in platelets in whole blood. The methodology allowed simultaneous staining and acquisition of 24-96 samples in a single analysis tube with a standard flow cytometer. This approach significantly reduced experimental numbers, data acquisition time, and antibody consumption, while providing automated statistically rich quantitative data on signaling events. Using vasodilator-stimulated phosphoprotein (VASP), an established marker of platelet inhibition and antiplatelet drug therapy, we demonstrated that the assay could detect subtle changes in phosphoVASP-Ser157/239 in response to cAMP-elevating agents of varying potency and known modulators of the cAMP signaling cascade. The assay could be used with washed platelets or whole blood, analyzed immediately or frozen, without any significant change in assay performance. To demonstrate the usefulness of the assay as a drug discovery platform, we examined a prostaglandin screening library. Our screen of 70 prostaglandin derivatives revealed three previously uncharacterized lipids that stimulated phosphorylation of VASP-Ser157. Follow-up analyses demonstrated that these agents elevated intraplatelet cAMP and inhibited collagen-induced platelet aggregation. CONCLUSIONS: This novel method enables rapid, large-scale quantitative signaling profiling and compound screening in human platelets present in whole blood.

Acute hypertriglyceridemia induces platelet hyperactivity that is not attenuated by insulin in polycystic ovary syndrome.

BACKGROUND: Atherothrombosis is associated with platelet hyperactivity. Hypertriglyceridemia and insulin resistance (IR) are features of polycystic ovary syndrome (PCOS). The effect of induced hypertriglyceridemia on IR and platelet function was examined in young women with PCOS. METHODS AND RESULTS: Following overnight fasting, 13 PCOS and 12 healthy women were infused with saline or 20% intralipid for 5 hours on separate days. Insulin sensitivity was measured using a hyperinsulinemic euglycaemic clamp in the final 2 hours of each infusion. Platelet responses to adenosine diphosphate (ADP) and prostacyclin (PGI2) were measured by flow cytometric analysis of platelet fibrinogen binding and P-selectin expression using whole blood taken during each infusion (at 2 hours) and at the end of each clamp. Lipid infusion increased triglycerides and reduced insulin sensitivity in both controls (median, interquartile range ) (5.25 [3.3, 6.48] versus 2.60 [0.88, 3.88] mg kg(-1) min(-1), P<0.001) and PCOS (3.15 [2.94, 3.85] versus 1.06 [0.72, 1.43] mg kg(-1) min(-1), P<0.001). Platelet activation by ADP was enhanced and ability to suppress platelet activation by PGI2 diminished during lipid infusion in both groups when compared to saline. Importantly, insulin infusion decreased lipid-induced platelet hyperactivity by decreasing their response to 1 µmol/L ADP (78.7% [67.9, 82.3] versus 62.8% [51.8, 73.3], P=0.02) and increasing sensitivity to 0.01 µmol/L PGI2 (67.6% [39.5, 83.8] versus 40.9% [23.8, 60.9], P=0.01) in controls, but not in PCOS. CONCLUSION: Acute hypertriglyceridemia induced IR, and increased platelet activation in both groups that was not reversed by insulin in PCOS subjects compared to controls. This suggests that platelet hyperactivity induced by acute hypertriglyceridemia and IR could contribute athero-thrombotic risk. CLINICAL TRIAL REGISTRATION URL: www.isrctn.org. Unique Identifier: ISRCTN42448814.

Midline spikes.

Midline spikes are characterized by spike foci recorded at Cz, Fz, or Pz with amplitude ranging from 20 to 350 microvolts. Out of 7,929 EEGs performed at the Neurodiagnostics Laboratory, Kaiser Permanente Medical Center, Anaheim, California, between 1996 and 2006, 17 EEGs (0.21%) were identified as having interictal midline spikes with or without other epileptiform discharges. Eight EEGs showed midline spikes at Cz, 2 at Fz, 2 at Cz and Fz, 1 at Cz and C3, 1 at Cz, C3, and P3, 1 at Cz and F8, 1 at Cz and T4, and 1 at Cz with 2 Hz generalized spike and slow wave complex. Midline spikes were recorded in 10 males and 7 females. The age ranged from 4 days to 38-years-old with a mean age of 10.8 years. Twelve patients (70.6%) were children. Twelve patients (70.6%) had generalized tonic-clonic seizures and 5 had partial motor seizures. Of the 17 patients, 14 had no known causes, 1 had an agenesis of corpus callosum, 1 had a left frontal arteriovenous malformation, and 1 had a left frontal area stroke. We postulate that the mechanism for the genesis of midline spikes may be heterogeneous. Midline spikes may be triggered by thalamocortical network in a generalized tonic-clonic seizure, or may originate in the parasagittal cortex in a partial motor seizure.