Satellite Epidemic of Covid-19 Associated Mucormycosis in India: A Multi-Site Observational Study.

BACKGROUND: Sudden upsurge in cases of COVID-19 Associated Mucormycosis (CAM) following the second wave of the COVID-19 pandemic was recorded in India. This study describes the clinical characteristics, management and outcomes of CAM cases, and factors associated with mortality. METHODS: Microbiologically confirmed CAM cases were enrolled from April 2021 to September 2021 from ten diverse geographical locations in India. Data were collected using a structured questionnaire and entered into a web portal designed specifically for this investigation. Bivariate analyses and logistic regression were conducted using R version 4.0.2. RESULTS: A total of 336 CAM patients were enrolled; the majority were male (n = 232, 69.1%), literate (n = 261, 77.7%), and employed (n = 224, 66.7%). The commonest presenting symptoms in our cohort of patients were oro-facial and ophthalmological in nature. The median (Interquartile Range; IQR) interval between COVID diagnosis and admission due to mucormycosis was 31 (18, 47) days, whereas the median duration of symptoms of CAM before hospitalization was 10 (5, 20) days. All CAM cases received antifungal treatment, and debridement (either surgical or endoscopic or both) was carried out in the majority of them (326, 97.02%). Twenty-three (6.9%) of the enrolled CAM cases expired. The odds of death in CAM patients increased with an increase in HbA1c level (aOR: 1.34, 95%CI: 1.05, 1.72) following adjustment for age, gender, education and employment status. CONCLUSION: A longer vigil of around 4-6 weeks post-COVID-19 diagnosis is suggested for earlier diagnosis of CAM. Better glycemic control may avert mortality in admitted CAM cases.

A prospective observational multicentric clinical trial to evaluate microscopic examination of acid-fast bacilli in sputum by artificial intelligence-based microscopy system.

Microscopy-based tuberculosis (TB) diagnosis i.e., Ziehl-Neelsen (ZN) stained smear screening still remains the primary diagnostic method in resource poor and high TB burden countries, however itrequires considerable experience and is bound to human errors. In remote areas, wherever expert microscopist is not available, timely diagnosis at initial level is not possible. Artificial intelligence (AI)-based microscopy may be a solution to this problem. A prospective observational multi-centric clinical trial to evaluate microscopic examination of acid-fast bacilli (AFB) in sputum by the AI based system was done in three hospitals in Northern India. Sputum samples from 400 clinically suspected cases of pulmonary tuberculosis were collected from three centres. Ziehl-Neelsen staining of smears was done. All the smears were observed by 3 microscopist and the AI based microscopy system. AI based microscopy was found to have a sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of 89.25%, 92.15%, 75.45%, 96.94%, 91.53% respectively. AI based sputum microscopy has an acceptable degree of accuracy, PPV, NPV, specificity and sensitivity and thus may be used as a screening tool for the diagnosis of pulmonary tuberculosis.

Endogenous SOD2 (Superoxide Dismutase) Regulates Platelet-Dependent Thrombin Generation and Thrombosis During Aging.

BACKGROUND: Reactive oxygen species (ROS) contribute to platelet hyperactivation during aging. Several oxidative pathways and antioxidant enzymes have been implicated; however, their mechanistic contributions during aging remain elusive. We hypothesized that mitochondria are an important source of platelet ROS and that mitochondrial SOD2 (superoxide dismutase) protects against mitochondrial ROS-driven platelet activation and thrombosis during aging. METHODS: We studied littermates of platelet-specific SOD2-knockout (SOD2fl/flPf4Cre, pSOD2-KO) and control (SOD2fl/fl) mice at young (4-5 months) or old (18-20 months) ages. We examined agonist-induced platelet activation, platelet-dependent thrombin generation potential, and susceptibility to in vivo thrombosis. RESULTS: Platelet αIIbß3 activation, aggregation, and adhesion were increased to similar extents in aged mice of both genotypes compared with young mice. In contrast, the age-dependent increases in mitochondrial and total cellular ROS, calcium elevation, and phosphatidylserine exposure were augmented in platelets from pSOD2-KO mice compared with control mice. Aged pSOD2-KO mice showed increased platelet-dependent thrombin generation compared with aged control mice. In vivo, aged pSOD2-KO mice exhibited enhanced susceptibility to carotid artery and pulmonary thrombosis compared to aged control mice. Adoptive transfer of platelets from aged pSOD2-KO but not aged control mice increased thrombotic susceptibility in aged host mice, suggesting a prothrombotic effect of platelet pSOD2 deficiency. Treatment with avasopasem manganese (GC4419), a SOD mimetic, decreased platelet mitochondrial pro-oxidants, cellular ROS levels, and inhibited procoagulant platelet formation and arterial thrombosis in aged mice. CONCLUSIONS: Platelet mitochondrial ROS contributes to age-related thrombosis and endogenous SOD2 protects from platelet-dependent thrombin generation and thrombosis during aging.

Noncanonical Sonic Hedgehog signaling amplifies platelet reactivity and thrombogenicity.

Sonic Hedgehog (Shh) is a morphogen in vertebrate embryos that is also associated with organ homeostasis in adults. We report here that human platelets, though enucleate, synthesize Shh from preexisting mRNAs upon agonist stimulation, and mobilize it for surface expression and release on extracellular vesicles, thus alluding to its putative role in platelet activation. Shh, in turn, induced a wave of noncanonical signaling in platelets leading to activation of small GTPase Ras homolog family member A and phosphorylation of myosin light chain in activated protein kinase-dependent manner. Remarkably, agonist-induced thrombogenic responses in platelets, which include platelet aggregation, granule secretion, and spreading on immobilized fibrinogen, were significantly attenuated by inhibition of Hedgehog signaling, thus, implicating inputs from Shh in potentiation of agonist-mediated platelet activation. In consistence, inhibition of the Shh pathway significantly impaired arterial thrombosis in mice. Taken together, the above observations strongly support a feed-forward loop of platelet stimulation triggered locally by Shh, similar to ADP and thromboxane A2, that contributes significantly to the stability of occlusive arterial thrombus and that can be investigated as a potential therapeutic target in thrombotic disorders.

DNase 1 Protects From Increased Thrombin Generation and Venous Thrombosis During Aging: Cross-Sectional Study in Mice and Humans.

Background Human aging is associated with increased risk of thrombosis, but the mechanisms are poorly defined. We hypothesized that aging induces peroxide-dependent release of neutrophil extracellular traps that contribute to thrombin generation and thrombosis. Methods and Results We studied C57BL6J mice and littermates of glutathione peroxidase-1 transgenic and wild-type mice at young (4 month) and old (20 month) ages and a healthy cohort of young (18-39 years) or middle-aged/older (50-72 years) humans. In plasma, we measured thrombin generation potential and components of neutrophil extracellular traps (cell-free DNA and citrullinated histone). Aged wild-type mice displayed a significant increase in thrombin generation that was decreased in aged glutathione peroxidase-1 transgenic mice. Both aged wild-type and aged glutathione peroxidase-1 transgenic mice demonstrated similar elevation of plasma cell-free DNA compared with young mice. In contrast, plasma levels of citrullinated histone were not altered with age or genotype. Release of neutrophil extracellular traps from neutrophils in vitro was also similar between young and aged wild-type or glutathione peroxidase-1 transgenic mice. Treatment of plasma or mice with DNase 1 decreased age-associated increases in thrombin generation, and DNase 1 treatment blocked the development of experimental venous thrombi in aged C57BL6J mice. Similarly, thrombin generation potential and plasma cell-free DNA, but not citrullinated histone, were higher in middle-aged/older humans, and treatment of plasma with DNase 1 reversed the increase in thrombin generation. Conclusions We conclude that DNase 1 limits thrombin generation and protects from venous thrombosis during aging, likely by hydrolyzing cell-free DNA.

The Role of Oxidative and Nitrosative Stress of Silver Nanoparticles in Human Parasitic Helminth Brugia malayi: A Mechanistic Insight.

PURPOSE: Silver nanoparticles (AgNPs) mediated apoptosis is well-known but its rationale is yet to be elucidated. This study explored the mechanistic underpinning of the apoptosis in the Brugia malayi parasitic model. METHOD: Silver nanoparticles were synthesized and tested against B. malayi microfilariae (Mf) to explore the role of oxidative and nitrosative stress in its apoptotic effect. RESULTS: AgNPs caused significant decrease in reduced glutathione (GSH) level and increase in both protein carbonylation and nitric oxide (NO) level indicating oxidative as well as nitrosative stress. Both GSH and nitric oxide synthase (NOS) inhibitors exhibited marked reversal. Nanoparticles and NO-donor in combination but not the NO-donor alone showed significant antiparasitic effect implying the requisite of combined oxidative and nitrosative stress to induce apoptosis. Synthetically prepared peroxynitrite from NaNO2 to H2O2 showed marked antiparasitic effect in very low dose which could be achieved neither by NaNO2 or H2O2 alone. GSH reversed the effect of peroxynitrite similar to its specific inhibitor, acetaminophen. GSH also reversed the plummet in mitochondrial oxygen consumption by AgNPs. CONCLUSION: We conclude that apoptosis by AgNPs is possibly mediated through peroxynitrite dependent depletion of GSH; this provides a significant insight into the pharmacological as well as toxicological impact of AgNPs.

AMPK inhibition protects against arterial thrombosis while sparing hemostasis through differential modulation of platelet responses.

AMP-activated protein kinase (AMPK) is a metabolic master switch that has critical role in wide range of pathologies including cardiovascular disorders. As AMPK-α2 knockout mice exhibit impaired thrombus stability, we asked whether pharmacological inhibition of AMPK with a specific small-molecule inhibitor, compound C, could protect against arterial thrombosis without affecting hemostasis. Mice pre-administered with compound C exhibited decreased mesenteric arteriolar thrombosis but normal tail bleeding time compared to vehicle-treated animals. Compound C potently restricted platelet aggregation, clot retraction and integrin activation induced by thrombin and collagen. It impaired platelet spreading on both immobilized fibrinogen and collagen matrices; it, however, had no significant effect on thrombin-induced phosphatidylserine exposure that is characteristic of procoagulant platelets. In parallel, compound C brought about significant drop in thrombin-induced phosphorylation of myosin light chain (MLC) and MLC phosphatase (MYPT1) as well as abrogated rise in level of RhoA-GTP in thrombin-stimulated platelets. Thus, effects of compound C on agonist-induced platelet responses could be at least in part attributed to modulation of cytoskeletal changes mediated by RhoA-MYPT1-MLC signaling. An ideal antithrombotic drug would spare hemostatic responses that maintain vascular integrity while preferentially protecting against thrombosis. The present study suggests that AMPK could be one such potential therapeutic target.

Memantine Protects From Exacerbation of Ischemic Stroke and Blood Brain Barrier Disruption in Mild But Not Severe Hyperhomocysteinemia.

Background Hyperhomocysteinemia is a risk factor for ischemic stroke; however, a targeted treatment strategy is lacking partly because of limited understanding of the causal role of homocysteine in cerebrovascular pathogenesis. Methods and Results In a genetic model of cystathionine beta synthase (CBS) deficiency, we tested the hypothesis that elevation in plasma total homocysteine exacerbates cerebrovascular injury and that memantine, a N-methyl-D-aspartate receptor antagonist, is protective. Mild or severe elevation in plasma total homocysteine was observed in Cbs+/- (6.1±0.3 µmol/L) or Cbs-/- (309±18 µmol/L) mice versus Cbs+/+ (3.1±0.6 µmol/L) mice. Surprisingly, Cbs-/- and Cbs+/- mice exhibited similar increases in cerebral infarct size following middle cerebral artery ischemia/reperfusion injury, despite the much higher total homocysteine levels in Cbs-/- mice. Likewise, disruption of the blood brain barrier was observed in both Cbs+/- and Cbs-/- mice. Administration of the N-methyl-D-aspartate receptor antagonist memantine protected Cbs+/- but not Cbs-/- mice from cerebral infarction and blood brain barrier disruption. Our data suggest that the differential effect of memantine in Cbs+/- versus Cbs-/- mice may be related to changes in expression of N-methyl-D-aspartate receptor subunits. Cbs-/-, but not Cbs+/- mice had increased expression of NR2B subunit, which is known to be relatively insensitive to homocysteine. Conclusions These data provide experimental evidence that even a mild increase in plasma total homocysteine can exacerbate cerebrovascular injury and suggest that N-methyl-D-aspartate receptor antagonism may represent a strategy to prevent reperfusion injury after acute ischemic stroke in patients with mild hyperhomocysteinemia.

Correction to: Plasma Fibrinogen Is a Natural Deterrent to Amyloid ß-Induced Platelet Activation and Neuronal Toxicity.

Following publication of the original article [1], the author reported an error in Figure 1. The correct version of Figure 1 is as follows.

Nox2 NADPH oxidase is dispensable for platelet activation or arterial thrombosis in mice.

Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome.

The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.

Aerobic glycolysis fuels platelet activation: small-molecule modulators of platelet metabolism as anti-thrombotic agents.

Platelets are critical to arterial thrombosis, which underlies myocardial infarction and stroke. Activated platelets, regardless of the nature of their stimulus, initiate energy-intensive processes that sustain thrombus, while adapting to potential adversities of hypoxia and nutrient deprivation within the densely packed thrombotic milieu. We report here that stimulated platelets switch their energy metabolism to aerobic glycolysis by modulating enzymes at key checkpoints in glucose metabolism. We found that aerobic glycolysis, in turn, accelerates flux through the pentose phosphate pathway and supports platelet activation. Hence, reversing metabolic adaptations of platelets could be an effective alternative to conventional anti-platelet approaches, which are crippled by remarkable redundancy in platelet agonists and ensuing signaling pathways. In support of this hypothesis, small-molecule modulators of pyruvate dehydrogenase, pyruvate kinase M2 and glucose-6-phosphate dehydrogenase, all of which impede aerobic glycolysis and/or the pentose phosphate pathway, restrained the agonist-induced platelet responses ex vivo These drugs, which include the anti-neoplastic candidate, dichloroacetate, and the Food and Drug Administration-approved dehydroepiandrosterone, profoundly impaired thrombosis in mice, thereby exhibiting potential as anti-thrombotic agents.

Impact on Quality of Life in Vitiligo Patients Treated with Narrowband Ultraviolet B Phototherapy.

CONTEXT: Vitiligo is a psychosocial problem which significantly affects quality of life in Indian scenario. AIMS: The purpose of this study was to compare the changes in quality of life in patients of vitiligo before and after treatment with narrowband ultraviolet B (NBUVB) phototherapy. SUBJECTS AND METHODS: A total of 54 patients had completed the study. The age ranged between 16 and 70 years with a mean age of 26.77±14.2 years. The initial dose of NBUVB was 300 mJ/cm2 in adults and 150 mJ/cm2 in children twice weekly with 20% dose increment on subsequent visits. It was given for a maximum period of 6 months and was followed up for another 6 months to determine stability of repigmentation. RESULTS: The average number of exposure given to the patients was 45.63±12.74 while the mean irradiation cumulative dose was 39.8 J/cm2. Mean Dermatology Life Quality Index (DLQI) of the vitiligo patients was 8.64±4.32 while those patients with acrofacial vitiligo had a mean DLQI of 11.78±5.61. After treatment with NBUVB, mean DLQI of all vitiligo patients was significantly reduced to 5.86±2.15 (P<0.01). CONCLUSIONS: This study showed that phototherapy had a positive therapeutic outcome in vitiligo, especially in younger patients. Even a small, depigmented lesion in a child could be psychosocially devastating.

Dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases, inhibits platelet aggregation and arterial thrombosis.

Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) to generate adenosine triphosphate, whereas activated platelets exhibit a high level of aerobic glycolysis, suggesting the existence of metabolic flexibility in platelets. Mitochondrial pyruvate dehydrogenase kinases (PDK 1-4) play a pivotal role in metabolic flexibility by inhibiting pyruvate dehydrogenase complex. We determined whether metabolic reprogramming, diverting metabolism from aerobic glycolysis back to OXPHOS, would inhibit platelet function. PDKs activity in human and mouse platelets was inhibited with dichloroacetic acid (DCA), a potent inhibitor of all 4 forms of PDK. Human and mouse platelets pretreated with DCA exhibited decreased platelet aggregation to suboptimal doses of collagen, convulxin, thrombin, and adenosine diphosphate concomitant with decreased glucose uptake. Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Furthermore, DCA inhibited ATP secretion, thromboxane A2 generation, and tyrosine phosphorylation of Syk and PLCγ2 in response to collagen or convulxin in human and mouse platelets (P < .05 vs vehicle treated). In the flow chamber assay, human and mouse blood pretreated with DCA formed smaller thrombi when perfused over collagen for 10 minutes at an arterial shear rate of 1500 s-1 (P < .05 vs control). Wild-type mice pretreated with DCA were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models (P < .05 vs vehicle control), without altering hemostasis. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function.

Staphylococcal ß-Toxin Modulates Human Aortic Endothelial Cell and Platelet Function through Sphingomyelinase and Biofilm Ligase Activities.

Staphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE). IE is an endovascular infection of native and prosthetic valves and the lining of the heart; it is characterized by the formation of cauliflower-like "vegetations" composed of fibrin, platelets, other host factors, bacteria, and bacterial products. ß-Toxin is an S. aureus virulence factor that contributes to the microorganism's ability to cause IE. This cytolysin has two enzymatic activities: sphingomyelinase (SMase) and biofilm ligase. Although both activities have functions in a rabbit model of IE, the mechanism(s) by which ß-toxin directly affects human cells and is involved in the infectious process has not been elucidated. Here, we compared the in vitro effects of purified recombinant wild-type ß-toxin, SMase-deficient ß-toxin (H289N), and biofilm ligase-deficient ß-toxin (H162A and/or D163A) on human aortic endothelial cells (HAECs) and platelets. ß-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8) from these cells by both SMase and biofilm ligase activities. ß-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1). HAECs treated with ß-toxin displayed granular membrane morphology not seen in treatment with the SMase-deficient mutant. The altered morphology resulted in two possibly separable activities, cell rounding and redistribution of cell membranes into granules, which were not the result of endosome production from the Golgi apparatus or lysosomes. ß-Toxin directly aggregated rabbit platelets via SMase activity.IMPORTANCE Each year there are up to 100,000 cases of infective endocarditis (IE) in the United States. S. aureus is the most common pathogen in patients with health care-associated IE and the leading cause of community-associated IE in the developed world. Multiple clonal group strains as defined by the Centers for Disease Control and Prevention, particularly USA200 and other clones encoding ß-toxin, are highly associated with IE. Considering the strong association and established contribution of ß-toxin in animal models of IE, determining how ß-toxin directly affects human cell types, including endothelial cells and platelets, is important. In this study, we demonstrate that ß-toxin functions to modulate endothelial cells and platelets by both toxin sphingomyelinase and biofilm ligase activities. Our data suggest that these activities modulate inflammation and increase infection severity.

Plasma Fibrinogen Is a Natural Deterrent to Amyloid Beta-Induced Platelet Activation.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, characterized by extensive loss of neurons, and deposition of amyloid beta (Aß) in the form of extracellular plaques. Aß is considered to have critical role in synaptic loss and neuronal death underlying cognitive decline. Platelets contribute to 95% of circulating amyloid-precursor protein that releases Aß into circulation. We have recently demonstrated that, Aß active fragment containing amino acid sequence 25-35 (Aß25-35) is highly thrombogenic in nature, and elicits strong aggregation of washed human platelets in RhoA-dependent manner. In the present study we evaluated the influence of fibrinogen on Aß-induced platelet activation. Intriguingly, Aß failed to induce aggregation of platelets suspended in plasma but not in buffer. Fibrinogen brought about dose-dependent decline in aggregatory response of washed human platelets elicited by Aß25-35, which could be reversed by increasing doses of Aß. Fibrinogen also attenuated Aß-induced platelet responses like secretion, clot retraction, rise in cytosolic Ca+2 and reactive oxygen species (ROS). Fibrinogen prevented intracellular accumulation of full length amyloid beta peptide (Aß42) in platelets as well as neuronal cells. We conclude that fibrinogen serves as a physiological check against the adverse effects of Aß by preventing its interaction with cells.

Prion protein fragment (106-126) induces prothrombotic state by raising platelet intracellular calcium and microparticle release.

Prion diseases are neurodegenerative disorders where infectious prion proteins (PrP) accumulate in brain leading to aggregation of amyloid fibrils and neuronal cell death. The amino acid sequence 106-126 from prion proteins, PrP(106-126), is highly amyloidogenic and implicated in prion-induced pathologies. As PrP is known to be expressed in blood following leakage from brain tissue, we sought to investigate its biological effects on human platelets, which have been widely employed as 'peripheral' model for neurons. Our findings suggested that, PrP(106-126) (20µM) induced dramatic 30-fold rise in intracellular calcium (from 105±30 to 3425±525nM) in platelets, which was attributable to influx from extracellular fluid with comparatively less contribution from intracellular stores. Calcium mobilization was associated with 8-10-fold stimulation in the activity of thiol protease calpain that led to partial cleavage of cytoskeleton-associated protein talin and extensive shedding of microparticles from platelets, thus transforming platelets to 'activated' phenotype. Both proteolysis of talin and microparticle release were precluded by calpeptin, a specific inhibitor of calpain. As microparticles are endowed with phosphatidylserine-enriched surface and hence are pro-coagulant in nature, exposure to prion favored a thrombogenic state in the organism.

Amyloid ß peptide stimulates platelet activation through RhoA-dependent modulation of actomyosin organization.

Platelets contribute to 95% of circulating amyloid precursor protein in the body and have widely been employed as a "peripheral" model of neurons in Alzheimer's disease. We sought to analyze the effects of amyloid ß (Aß) on platelets and to understand the underlying molecular mechanism. The Aß active fragment containing amino acid sequence 25-35 (Aß(25-35); 10-20 µM) was found to induce strong aggregation of human platelets, granule release, and integrin activation, similar to that elicited by physiological agonists. Platelets exposed to Aß(25-35) retracted fibrin clot and displayed augmented adhesion to collagen under arterial shear, reflective of a switch to prothrombotic phenotype. Exposure of platelets to Aß peptide (20 µM) resulted in a 4.2- and 2.3-fold increase in phosphorylation of myosin light chain (MLC) and MLC phosphatase, respectively, which was reversed by Y27632, an inhibitor of Rho-associated coiled-coil protein kinase (ROCK). Aß(25-35)-induced platelet aggregation and clot retraction were also significantly attenuated by Y27632. Consistent with these findings, Aß(25-35) elicited a significant rise in the level of RhoA-GTP in platelets. Platelets pretreated with reverse-sequenced Aß fragment (Aß(35-25)) and untreated resting platelets served as controls. We conclude that Aß induces cellular activation through RhoA-dependent modulation of actomyosin, and hence, RhoA could be a potential therapeutic target in Alzheimer's disease and cerebral amyloid angiopathy.