Clopidogrel use and smoking cessation result in lower coated-platelet levels after stroke.

Coated-platelets are a subset of highly procoagulant platelets elevated in patients with non-lacunar ischemic stroke and associated with stroke recurrence. Cross-sectional studies in controls have shown that smoking is associated with higher coated-platelet levels while chronic use of serotonin reuptake inhibitors (SSRIs), statins or aspirin is associated with lower coated-platelet levels. We now investigate if initiation of treatment with SSRIs, statins, clopidogrel, aspirin or oral anticoagulants and smoking cessation impacts coated-platelet levels at 90 days after ischemic stroke. Coated-platelet levels, reported as percent of cells converted to coated-platelets, were measured in 87 consecutive patients with stroke at baseline and repeated at 90 days. Repeated-measure ANOVA was used to determine if initiation of treatment with individual medications or smoking cessation impacted coated-platelet levels. Decreased coated-platelets levels at 90 days as compared to baseline were observed after initiation of treatment with clopidogrel (p = .0001, partial η2 = 0.17) and smoking cessation (p = .014, partial η2 = 0.10). Initiation of treatment with SSRIs, statins, aspirin or oral anticoagulants did not result in significant changes in coated-platelet potential. These novel longitudinal data suggest that clopidogrel therapy and smoking cessation attenuate coated-platelet potential at 90 days after ischemic stroke.

Higher Coated-Platelet Levels in Acute Stroke are Associated with Lower Cognitive Scores at Three Months Post Infarction.

BACKGROUND: Coated-platelets are a subset of highly procoagulant platelets observed after dual agonist stimulation with collagen and thrombin. Coated-platelet levels are increased in acute stroke compared to controls, and higher levels are associated with stroke recurrence. We examined whether coated-platelet levels measured at the time of the stroke correlate with cognitive scores at 3 months following the brain infarction. METHODS: Coated-platelets were assayed in consecutive patients with nonlacunar stroke. Cognitive screening was performed using the Mini-Mental State Examination (MMSE) at 3 months after discharge. Linear regression, with adjustment for individual covariates, was used to model the association between coated-platelet levels and MMSE scores. RESULTS: One hundred and twenty-eight patients with a mean MMSE score of 26 points (range 14-30, standard deviation [SD] 3.1) and mean coated-platelet levels of 40.9% (range 5.2-76.2, SD 13.3), completed cognitive screening. An inverse linear association was found between coated-platelet levels and MMSE score, with higher levels seen in patients with lower MMSE scores (r = -.34, R2 = .12, P < .0001). This association remained despite adjustment for potential confounding factors. In the final model, higher coated-platelet levels (coefficient -.078, 95% confidence interval [CI]: -.12 to -.041, P < .0001), presence of hypertension (coefficient -2.42, 95% CI: -3.90 to -.95, P = .0015), and anticoagulant use at discharge (coefficient -1.48, 95% CI: -2.56 to -.39, P = .0079) were predictive of lower MMSE. CONCLUSIONS: These findings support a link between increased platelet procoagulant potential at the time of the stroke and development of cognitive impairment following cerebral infarction.

Lower Coated-Platelet Levels Are Associated With Increased Mortality After Spontaneous Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Coated-platelets are highly procoagulant platelets observed on dual-agonist stimulation with collagen and thrombin. Coated-platelet levels are decreased in patients with spontaneous intracerebral hemorrhage when compared with controls and inversely correlated with bleed volume. We sought to investigate whether coated-platelets are associated with increased mortality at 30 days after spontaneous intracerebral hemorrhage. METHODS: Coated-platelet levels were assayed in 95 consecutive patients with spontaneous intracerebral hemorrhage. The main outcome was mortality at 30 days according to coated-platelet levels at enrollment. Subjects were grouped into tertiles of the observed coated-platelet level distribution. Groups defined by tertile of coated-platelet level were compared using either ANOVA or a Kruskal-Wallis test for small group size for continuous measures and an exact Cochrane-Armitage trend test for categorical measures. Logistic regression was used to estimate the adjusted odds of death within 30 days associated with coated-platelet levels. RESULTS: Cumulative mortality at 30 days was 23% (22 subjects). Mortality at 30 days differed among the coated-platelet tertiles: 44% for the first tertile (lowest coated-platelet levels), 19% for the second tertile, and 6% for the third tertile (trend test; P=0.0004). Logistic regression examining the association between mortality and coated-platelet levels showed that the odds of death at 30 days in those with levels <27% (n=47) were 6.83× the odds for patients with levels ≥27% (95% confidence interval, 2.10-22.23). CONCLUSIONS: These results support a link between impaired coated-platelet potential and outcome in intracerebral hemorrhage.

Bacillus anthracis peptidoglycan activates human platelets through FcγRII and complement.

Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbß3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa-blocking antibody IV.3, suggesting they are mediated by PGN-anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections.

Coated-platelets improve prediction of stroke and transient ischemic attack in asymptomatic internal carotid artery stenosis.

BACKGROUND AND PURPOSE: Coated-platelets, a subset of procoagulant platelets observed on dual agonist stimulation with collagen and thrombin, support a robust prothrombinase activity and provide a unique measure of platelet thrombotic potential. Coated-platelet levels are increased in large artery stroke, and higher levels are associated with early stroke recurrence, suggesting a potential role for risk stratification in asymptomatic patients with carotid artery stenosis. METHODS: Three-hundred twenty-nine consecutive patients with technically adequate carotid Doppler evaluation without stroke or transient ischemic attack (TIA) in the previous 6 months were enrolled as part of a prospective cohort study conducted during a 40-month period. The main outcome was occurrence of stroke or TIA according to coated-platelet levels and internal carotid stenosis severity at enrollment. The optimal cutoff value of coated-platelet levels was determined by recursive partitioning analysis. Event-free survival was estimated using Kaplan-Meier and Cox proportional hazards regression analyses. RESULTS: A cutoff of ≥45% for coated-platelet levels in combination with stenosis≥50% yielded a sensitivity of 0.78 (95% confidence interval, 0.51-1.0), specificity of 0.92 (0.89-0.95), positive predictive value of 0.21 (0.07-0.34), and a negative predictive value of 0.99 (0.98-1.0) for ipsilateral stroke or TIA. The incidence rate of ipsilateral stroke or TIA for patients with ≥50% stenosis and ≥45% coated-platelets was 21.5 per 100 person-years versus 1.27 per 100 person-years for patients with ≥50% stenosis and <45% coated-platelets (P<0.0001). CONCLUSIONS: Coated-platelet levels identify asymptomatic carotid stenosis patients at high risk for stroke or TIA, which suggests a role for coated-platelets in risk stratification before revascularization.

Elevated coated-platelets in symptomatic large-artery stenosis patients are associated with early stroke recurrence.

Coated-platelets are a subset of platelets with increased procoagulant potential observed upon dual agonist stimulation with collagen and thrombin. These prothrombotic platelets are elevated in patients with non-lacunar ischemic stroke and decreased in patients with spontaneous intracerebral hemorrhage compared to controls. We now investigated coated-platelet synthesis in patients with symptomatic large-artery stenosis and explored the association between coated-platelet levels and stroke recurrence at 3 months in this population. Coated-platelet levels were determined in 60 patients with either acute stroke or transient ischemic attack due to large-artery stenosis and 60 controls. Recurrent stroke incidence at 3 months was stratified by tertiles of coated-platelet levels and compared among groups using a log-rank test. Large-artery stenosis patients had significantly higher coated-platelet levels than controls (mean ± SD, 42.0 ± 15.5% vs. 29.4 ± 13.5%, p < 0.0001). The 3-month cumulative incidence of recurrent stroke was 41% for the highest, 6% for the middle, and 5% for the lowest tertile of coated-platelet levels (p = 0.0045). These results show that elevated coated-platelet levels in patients with symptomatic large-artery stenosis are associated with early stroke recurrence.

Coated-platelet levels are persistently elevated in patients with mild traumatic brain injury.

OBJECTIVE: Coated-platelets are procoagulant platelets that are elevated in patients with large-vessel ischemic stroke and are associated with stroke recurrence. Because of recent reports showing an increased risk for stroke following traumatic brain injury (TBI), we undertook a pilot study to investigate coated-platelet synthesis in veterans with TBI. DESIGN: Cross-sectional study. PARTICIPANTS: Forty patients with a diagnosis of mild TBI (mTBI) and 40 controls without a history of TBI and matched for age, gender, and ethnicity/race were enrolled in the study. MAIN MEASURE: Coated-platelet levels were determined in patients with mTBI and controls. The time period since most recent injury ranged from 6 months to 9 years. RESULTS: Coated-platelet levels were significantly higher for mTBI patients than for controls (mean ± SD = 52.0% ± 14.0% vs 35.4% ± 13.0%; P < .0001). No relationship between these levels and the length of time since the last injury was found (P = .5). CONCLUSIONS: Coated-platelet levels are markedly and persistently elevated in individuals with mTBI. These data suggest a link to previous findings of increased stroke risk and chronic inflammation among individuals who sustained a TBI.

Cerebral microbleeds in nonlacunar brain infarction are associated with lower coated-platelet levels.

BACKGROUND: Coated-platelets are a subset of platelets with high procoagulant potential observed on dual-agonist stimulation with collagen and thrombin. Coated-platelet levels are elevated in patients with nonlacunar ischemic stroke compared with controls, although the presence of early hemorrhagic transformation is associated with lower coated-platelet levels. In contrast to infarction, patients with spontaneous intracerebral hemorrhage have lower coated-platelet levels, and these levels inversely correlate with bleed size. Cerebral microbleeds (CMBs) represent previous small hemorrhagic occurrences. We undertook a pilot study to investigate coated-platelet production and the presence of CMBs in patients with nonlacunar ischemic stroke. METHODS: Coated-platelet levels were determined in 110 consecutive patients with a diagnosis of nonlacunar stroke. Microbleeds were identified using the published criteria by an experienced stroke neurologist. Coated-platelet levels were compared statistically between patients with and without CMBs using the nonparametric Wilcoxon rank sum test. RESULTS: Coated-platelet levels (median [interquartile range]) for all patients were 44.1% [34%-51.2%]. CMBs were detected in 22 patients (20%); these patients had significantly lower coated-platelet levels compared with those without CMBs (35.6% [22.6%-47.2%] versus 45.1% [36.1%-51.5%]; P = .025), whereas other demographic and clinical factors did not differ significantly. CONCLUSIONS: The presence of CMBs in patients with nonlacunar ischemic stroke is associated with lower levels of coated-platelets. Larger prospective studies are needed to better establish the potential connection between altered coated-platelet synthesis, microbleeds, cerebral infarction, and possible hemorrhage-prone vascular changes.

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4.

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.

Higher coated-platelet levels are associated with chronic hypertension in patients with transient ischemic attack.

Coated-platelets are procoagulant platelets observed upon dual stimulation with collagen and thrombin. We previously reported that coated-platelet levels are elevated in patients with transient ischemic attack (TIA) compared to controls and that these levels correlate with ABCD2 scores, a validated tool for identifying the short-term risk for stroke occurrence in TIA patients. We now investigate the effect of individual elements of the ABCD2 score on coated-platelet levels in TIA. Coated-platelet levels were measured in 124 TIA patients. A nine-way ANOVA evaluated the impact of components of the ABCD2 score (age, blood pressure (BP), clinical features, symptom duration, and diabetes), smoking, pertinent medications, race, and gender on coated-platelet levels. In the initial model, the only significant main effect was for BP; patients with BP ≥ 140/90 had higher coated-platelet levels than those without (mean ± SEM; 44.0 ± 2.1% vs. 35.4 ± 2.3%, p = 0.0007). Because the diagnosis of hypertension (HTN) requires multiple readings of elevated BP, we re-analyzed the data by replacing BP with HTN. In the second model, there were two significant main effects: HTN - with higher coated-platelet levels in patients with vs. those without HTN (46.3 ± 2.1% vs. 33.6 ± 2.1%, p < 0.0001), and symptom duration - with higher coated-platelet levels in patients with duration ≥60 minutes vs. those with duration <60 minutes (42.5 ± 2.0% vs. 37.4 ± 2.1%, p = 0.031). These data suggest a link between chronic HTN and platelet thrombotic potential.

AMPKalpha1 deletion shortens erythrocyte life span in mice: role of oxidative stress.

AMP-activated protein kinase (AMPK) is an energy sensor essential for maintaining cellular energy homeostasis. Here, we report that AMPKalpha1 is the predominant isoform of AMPK in murine erythrocytes and mice globally deficient in AMPKalpha1 (AMPKalpha1(-/-)), but not in those lacking AMPKalpha2, and the mice had markedly enlarged spleens with dramatically increased proportions of Ter119-positive erythroid cells. Blood tests revealed significantly decreased erythrocyte and hemoglobin levels with increased reticulocyte counts and elevated plasma erythropoietin concentrations in AMPKalpha1(-/-) mice. The life span of erythrocytes from AMPKalpha1(-/-) mice was less than that in wild-type littermates, and the levels of reactive oxygen species and oxidized proteins were significantly increased in AMPKalpha1(-/-) erythrocytes. In keeping with the elevated oxidative stress, treatment of AMPKalpha1(-/-) mice with the antioxidant, tempol, resulted in decreased reticulocyte counts and improved erythrocyte survival. Furthermore, the expression of Foxo3 and reactive oxygen species scavenging enzymes was significantly decreased in erythroblasts from AMPKalpha1(-/-) mice. Collectively, these results establish an essential role for AMPKalpha1 in regulating oxidative stress and life span in erythrocytes.

Quantitation of coated platelet potential during collection, storage, and transfusion of apheresis platelets.

BACKGROUND: Coated platelets (PLTs), a subpopulation of PLTs observed upon dual agonist stimulation with collagen and thrombin, are known to retain several procoagulant α-granule proteins on their surface. By formation of a highly active membrane-bound prothrombinase complex, these PLTs represent an important step in the coagulation cascade as a consequence of their ability to generate thrombin at the site of vascular injury. Various clinical observations suggest that higher levels of coated PLTs are associated with thrombosis while a deficiency of coated PLTs results in a bleeding diathesis. Current quality control guidelines for in vitro PLT storage measure PLT viability but no routine evaluation of the hemostatic function of stored PLTs and particularly no estimation of coated PLT potential is performed. Our primary objective was to evaluate if the process of apheresis and storage of PLT units alters the levels of coated PLTs. In addition, we sought to determine how transfusion of stored PLTs into patients with thrombocytopenia affects the patient's coated PLT levels. STUDY DESIGN AND METHODS: Coated PLT levels were analyzed in 13 voluntary PLT donors before donation, in the fresh apheresis product (Trima, CaridianBCT) and in the stored apheresis product just before transfusion. In addition, 10 patients with thrombocytopenia were analyzed for coated PLTs before and after transfusion of a stored PLT product. RESULTS: Coated PLT levels were significantly decreased after the process of apheresis (17% relative decline; p < 0.01) and with prolonged storage (1 to 5 days; 53% relative decline; p < 0.001). Transfusion of stored PLT units did not result in significant increment of coated PLT levels in patients with thrombocytopenia as expected considering the low level of coated PLTs in stored PLT units. Furthermore, there was no suggestion of regeneration of coated PLT potential upon reinfusion. CONCLUSIONS: Isolation and storage of apheresis PLTs by standard blood bank procedures results in a significant decline in coated PLT potential. Reinfusion of stored apheresis PLTs into patients with thrombocytopenia resulted in a predictable change in coated PLT potential with no suggestion of regeneration of lost coated PLT potential.

Increased procoagulant platelet levels are predictive of death in COVID-19.

Prior research has identified abnormal platelet procoagulant responses in COVID-19. Coated-platelets, a form of procoagulant platelets, support thrombin formation and are elevated in ischemic stroke patients with increased risk for recurrent infarction. Our goal was to examine changes in coated-platelet levels over the course of COVID-19 infection and determine their association with disease severity, thrombosis, and death. Coated-platelet levels were assayed after admission and repeated weekly in COVID-19 patients, and in COVID-19 negative controls. Receiver operator characteristic (ROC) analysis was used to calculate area under the curve (AUC) values for a model including baseline coated-platelets to predict death. Kaplan-Meier and Cox proportional hazards analysis was used to predict risk for death at 90 days. We enrolled 33 patients (22 with moderate and 11 with severe infection) and 20 controls. Baseline coated-platelet levels were lower among moderate (mean ± SD; 21.3 ± 9.8%) and severe COVID-19 patients (28.5 ± 11.9%) compared to controls (38.1 ± 10.4%, p < 0.0001). Coated-platelet levels increased during follow-up in COVID-19 patients by 7% (relative) per day from symptom onset (95% CI 2-12%, p = 0.007). A cut-off of 33.9% for coated-platelet levels yielded 80% sensitivity and 96% specificity for death at 90 days, with resulting AUC of 0.880 (95% CI 0.680-1.0, p = 0.0002). The adjusted hazard ratio for death in patients with coated-platelet levels > 33.9% was 40.99 when compared to those with levels ≤ 33.9% (p < 0.0001). Platelet procoagulant potential is transiently decreased in most patients during COVID-19; however, increased baseline platelet procoagulant levels predict death. Defining the mechanisms involved and potential links with aging may yield novel treatment targets.

Coated platelet levels correlate with bleed volume in patients with spontaneous intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Coated platelets are produced on dual agonist stimulation with collagen and thrombin. These highly procoagulant platelets are critical to normal hemostasis, and an earlier study demonstrated decreased coated platelet production in patients with spontaneous intracerebral hemorrhage. We have expanded this observation to investigate if coated platelet levels correlate with bleed volume in spontaneous intracerebral hemorrhage. METHODS: Coated platelet levels and bleed volume were determined in 45 patients with a diagnosis of spontaneous intracerebral hemorrhage. RESULTS: There was an inverse relationship between coated platelet levels and bleed volume (r=-0.38, P=0.01). CONCLUSIONS: These data support a link between decreased coated platelet synthesis and the severity of spontaneous intracerebral hemorrhage.

Increased platelet procoagulant potential predicts recurrent stroke and TIA after lacunar infarction.

BACKGROUND: Mean levels of coated-platelets, a subset of highly procoagulant platelets, are decreased in patients with lacunar as compared to those with non-lacunar stroke. Elevated coated-platelets are associated with increased risk for recurrent infarction in non-lacunar stroke and predict incident stroke after transient ischemic attack (TIA). OBJECTIVE: We investigated if coated-platelet levels are predictive of recurrent cerebral ischemia following lacunar stroke. METHODS: Coated-platelet levels were assayed in consecutive patients with acute lacunar stroke, who were followed for up to 12 months. Cox proportional hazards regression analysis was used to estimate the combined risk of stroke and TIA at 12 months according to initial coated-platelet levels. RESULTS: We enrolled a total of 109 lacunar stroke patients. Eight events were recorded over a mean follow-up period of 10.8 months. A cut-off of 42.6% for coated-platelet levels yielded a sensitivity of 0.75 (0.35-0.97; 95% confidence interval [CI]), specificity of 0.92 (0.85-0.97), positive predictive value of 0.43 (0.26-0.62), and a negative predictive value of 0.98 (0.93-0.99) for recurrent stroke/TIA. The adjusted hazard ratio for recurrent stroke/TIA in patients with coated-platelet levels ≥ 42.6% was 23.9 (95% CI: 4.26-134.4) when compared to those with levels < 42.6%. CONCLUSIONS: Identification of increased platelet procoagulant potential may improve our ability to identify patients at higher risk of recurrent stroke/TIA following a lacunar stroke. Further study of mechanisms involved is warranted and may yield novel targets for prevention and treatment.

Xylosyltransferase II is a significant contributor of circulating xylosyltransferase levels and platelets constitute an important source of xylosyltransferase in serum.

Circulating glycosyltransferases including xylosyltransferases I (XylT1) and II (XylT2) are potential serum biomarkers for various diseases. Understanding what influences the serum activity of these enzymes as well as the sources of these enzymes is important to interpreting the significance of alterations in enzyme activity during disease. This article demonstrates that in the mouse and human the predominant XylT in serum is XylT2. Furthermore, that total XylT levels in human serum are approximately 200% higher than those in plasma due in part to XylT released by platelets during blood clotting in vitro. In addition, the data from Xylt2 knock-out mice and mice with liver neoplasia show that liver is a significant source of serum XylT2 activity. The data presented suggest that serum XylT levels may be an informative biomarker in patients who suffer from diseases affecting platelet and/or liver homeostasis.

Coated-platelet levels are low in patients with spontaneous intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Coated-platelets are a subset of platelets with high procoagulant potential observed on dual agonist stimulation with collagen and thrombin. Failure to produce coated-platelets in animals results in a bleeding diathesis. With this background, we undertook a pilot study to investigate coated-platelet production in patients with spontaneous intracerebral hemorrhage (SICH). METHODS: Coated-platelet levels were determined in 26 patients with a diagnosis of SICH and 52 controls. RESULTS: The patient population had significantly lower coated-platelet levels than the controls (mean+/-SD, 24.8+/-9.7% versus 32.9+/-12.6%, P=0.0035). CONCLUSIONS: Decreased coated-platelet synthesis may be linked to the mechanisms involved in the events leading to SICH.

Differences in coated-platelet production between frontotemporal dementia and Alzheimer disease.

OBJECTIVE: Coated-platelets are a subset of platelets produced by dual-agonist activation with collagen and thrombin. These platelets retain full-length amyloid precursor protein on their surface and correlate inversely with disease severity in Alzheimer disease (AD). We have now investigated coated-platelet production and its relationship with disease severity in frontotemporal dementia (FTD) patients to determine whether our earlier observations were unique to AD. METHODS: Coated-platelet levels were assayed in 40 FTD, 40 AD patients, and 40 controls. Both patient groups were equally divided between mild-stage (Clinical Dementia Rating < or =1) and advanced stage dementia (Clinical Dementia Rating >1). RESULTS: Coated-platelet levels were not significantly different between patients with early-stage and advanced stage FTD (P=0.9), whereas early-stage AD patients had significantly higher levels than advanced stage AD (P<0.001). In addition, coated-platelet production was significantly elevated in early-stage AD versus early-stage FTD patients (P=0.01). CONCLUSIONS: In contrast to AD, there is no significant relationship between disease severity and coated-platelet levels in FTD. Differences in coated-platelet levels between early-stage AD and early-stage FTD patients warrant further investigation for potential clinical applications in helping to differentiate between these 2 disorders.