Graphene Oxide Significantly Modifies Cardiac Parameters and Coronary Endothelial Reactivity in Healthy and Hypertensive Rat Hearts Ex Vivo.

Interactions of graphene oxide (GO) with an ex vivo rat heart and its coronary vessels have not been studied yet. Moreover, the conflicting data on the "structure-properties" relationships do not allow for biomedical applications of GO. Herein, we study the impact of GO on the ex vivo isolated rat heart, normotensive and hypertensive, under the working heart and the constant-pressure perfusion (Langendorff) regimes. Four structural GO variants of the following initial morphology were used: few-layer (below 10-layer) GO1, O < 49%; predominantly single-layer GO2, O = 41-50%; 15-20-layer GO3, O < 11%; and few-layer (below 10-layer) NH4 +-functionalized GO4, O < 44%, N = 3-6%. The aqueous GO dispersions, sonicated and stabilized with bovine serum albumin in Krebs-Henseleit-like solution-uniformized in terms of the particle size-were eventually size-monodisperse as revealed by dynamic light scattering. To study the cardiotoxicity mechanisms of GO, histopathology, Raman spectroscopy, analysis of cardiac parameters (coronary and aortic flows, heart rate, aortic pressure), and nitric oxide (NO-)-dependent coronary flow response to bradykinin (blood-vessel-vasodilator) were used. GO1 (10 mg/L) exerted no effects on cardiac function and preserved an increase in coronary flow in response to bradykinin. GO2 (10 mg/L) reduced coronary flow, aortic pressure in normotensive hearts, and coronary flow in hypertensive hearts, and intensified the response to bradykinin in normal hearts. GO3 (10 mg/L) reduced all parameters in hypertensive hearts and coronary response to bradykinin in normal hearts. At higher concentrations (normotensive hearts, 30 mg/L), the coronary response to bradykinin was blocked. GO4 (10 mg/L) reduced the coronary flow in normal hearts, while for hypertensive hearts, all parameters, except the coronary flow, were reduced and the coronary response to bradykinin was blocked. The results showed that a low number of GO layers and high O-content were safer for normal and hypertensive rat hearts. Hypertensive hearts deteriorated easier upon perfusion with low-O-content GOs. Our findings support the necessity of strict control over the GO structure during organ perfusion and indicate the urgent need for personalized medicine in biomedical applications of GO.

Stabilization of Graphene Oxide Dispersion in Plasma-like Isotonic Solution Containing Aggregating Concentrations of Bivalent Cations.

Graphene oxide's (GO) intravascular applications and biocompatibility are not fully explored yet, although it has been proposed as an anticancer drug transporter, antibacterial factor or component of wearable devices. Bivalent cations and the number of particles' atom layers, as well as their structural oxygen content and pH of the dispersion, all affect the GO size, shape, dispersibility and biological effects. Bovine serum albumin (BSA), an important blood plasma protein, is expected to improve GO dispersion stability in physiological concentrations of the precipitating calcium and magnesium cations to enable effective and safe tissue perfusion. METHODS: Four types of GO commercially available aqueous dispersions (with different particle structures) were diluted, sonicated and studied in the presence of BSA and physiological cation concentrations. Nanoparticle populations sizes, electrical conductivity, zeta potential (Zetasizer NanoZS), structure (TEM and CryoTEM), functional groups content (micro titration) and dispersion pH were analyzed in consecutive preparation stages. RESULTS: BSA effectively prevented the aggregation of GO in precipitating concentrations of physiological bivalent cations. The final polydispersity indexes were reduced from 0.66-0.91 to 0.36-0.43. The GO-containing isotonic dispersions were stable with the following Z-ave results: GO1 421.1 nm, GO2 382.6 nm, GO3 440.2 nm and GO4 490.1 nm. The GO behavior was structure-dependent. CONCLUSION: BSA effectively stabilized four types of GO dispersions in an isotonic dispersion containing aggregating bivalent physiological cations.

Prehospital Ultrasound: A Narrative Review.

Background: Point-of-care ultrasound is rapidly becoming more prevalent in the prehospital environment. Though considered a relatively new intervention in this setting, there is growing literature that aims to explore the use of prehospital ultrasound by EMS personnel.Methods: To better understand and report the state of the science on prehospital ultrasound, we conducted a narrative review of the literature.Results: Following a keyword search of MEDLINE in Ovid from inception to August 2, 2022, 2,564 records were identified and screened. Based on review of abstracts and full texts, with addition of seven articles via bibliography review, 193 records were included. Many included studies detail usage in air medical and other critical care transport environments. Clinicians performing prehospital ultrasound are often physicians or other advanced practice personnel who have previous ultrasound experience, which facilitates implementation in the prehospital setting. Emerging literature details training programs for prehospital personnel who are novices to ultrasound, and implementation for some study types appears feasible without prior experience. Unique use scenarios that show promise include during critical care transport, for triage in austere settings, and for thoracic evaluation of patients at risk of life-threatening pathology.Conclusion: There is a growing mostly observational body of literature describing the use of ultrasound by prehospital personnel. Prehospital ultrasound has demonstrated feasibility for specific conditions, yet interventional studies evaluating benefit to patient outcomes are absent.

The Pharmacological Effects of Silver Nanoparticles Functionalized with Eptifibatide on Platelets and Endothelial Cells.

Purpose: In the search for new drug delivery platforms for cardiovascular diseases and coating of medical devices, we synthesized eptifibatide-functionalized silver nanoparticles (AgNPs-EPI) and examined the pharmacological activity of AgNPs-EPI on platelets and endothelial cells in vitro and ex vivo. Methods: Spherical AgNPs linked to eptifibatide were synthesized and characterized. Cytotoxicity was measured in microvascular endothelial cells (HMEC-1), platelets and red blood cells. Platelet mitochondrial respiration was measured using the Oxygraph-2k, a high-resolution modular respirometry system. The effect of AgNPs-EPI on the aggregation of washed platelets was measured by light aggregometry and the ex vivo occlusion time was determined using a reference laboratory method. The surface amount of platelet receptors such as P-selectin and GPIIb/IIIa was measured. The influence of AgNPS-EPI on blood coagulation science was assessed. Finally, the effect of AgNPs-EPI on endothelial cells was measured by the levels of 6-keto-PGF1alpha, tPa, cGMP and vWF. Results: We describe the synthesis of AgNPs using eptifibatide as the stabilizing ligand. The molecules of this drug are directly bonded to the surface of the nanoparticles. The synthesized AgNPs-EPI did not affect the viability of platelets, endothelial cells and erythrocytes. Preincubation of platelets with AgNPs-EPI protected by mitochondrial oxidative phosphorylation capacity. AgNPs-EPI inhibited aggregation-induced P-selectin expression and GPIIb/IIIa conformational changes in platelets. AgNPs-EPI caused prolongation of the occlusion time in the presence of collagen/ADP and collagen/adrenaline. AgNPs-EPI regulated levels of 6-keto-PGF1alpha, tPa, vWf and cGMP produced in thrombin stimulated HMEC-1 cells. Conclusion: AgNPs-EPI show anti-aggregatory activity at concentrations lower than those required by the free drug acting via regulation of platelet aggregation, blood coagulation, and endothelial cell activity. Our results provide proof-of-principle evidence that AgNPs may be used as an effective delivery platform for antiplatelet drugs.

In vitro effects of cobalt and chromium nanoparticles on human platelet function.

Nanoparticles (NPs) are released from orthopedic and neurosurgical prostheses and can interact with a number of blood components once in the bloodstream. Potential toxic effects of Co and Cr NPs on blood platelets have not been thoroughly investigated. The aim of this study was to analyze the effect of Co and Cr NPs on platelet function in vitro. The ability of the tested NPs to induce platelet activation and aggregation was measured using light transmission aggregometry, flow cytometry, and quartz crystal balance with dissipation (QCM-D). This was confirmed by transmission electron microscopy (TEM), scanning electron microscopy, and optical and immunofluorescence microscopy. Perfusion of QCM-D sensor crystals with platelet-rich-plasma in the presence of Co 28 nm, CoO 50 nm, Co2O3 50 nm, Co3O4 30-50nm, Cr 35-45nm, Cr2O3 60 nm NPs (0.5-5.0 µg/mL) resulted in significant changes in frequency and dissipation, indicating that these NPs caused platelet microaggregation. Transmission electron microscopy also revealed that Cr NPs led to platelet swelling and lysis. Our study shows that both Co and Cr NPs affect platelet function in vitro with two distinct mechanisms. While Co NPs result in standard platelet aggregation, Cr NPs cause both platelet aggregation and decreased platelet membrane integrity and lysis. Based on these findings, monitoring serum NP levels and platelet-mediated hemostasis can be advised in patients with metal-on-metal Co-Cr prostheses.

Use of Ultrasound in Introducing Anatomical Pathology to Preclinical Medical Students, in Correlation with Physical Exam Curricula.

Introduction: Point-of-care ultrasound has become an important diagnostic tool in many clinical settings. Many medical schools have responded by incorporating instruction on ultrasound into the curriculum for medical students in their clinical years. The curriculum presented here will assist preclinical medical students in distinguishing between normal and pathologic sonographic anatomical findings. Methods: The course consisted of four, approximately 30-minute case-based PowerPoint slide shows introducing pathologic anatomical findings on ultrasound through clinical case-based scenarios. Twelve preclinical (first- and second-year) medical students attended each weekly session. An emergency medicine resident created and presented the course content as an adjunct to an established course instructing students on how ultrasound correlates to the normal physical exam. Upon completion of the course, the instructors emailed the students an online, seven-question survey. Results: Survey results showed positive feedback, with 71% of respondents answering strongly agree to the survey question that addressed the primary educational objective, which was to enable students to distinguish between normal and pathologic anatomical findings on ultrasound. Open response feedback highlighted that the course complemented the existing course well and suggested that the course be continued. Discussion: A curriculum which presents pathologic anatomical findings on ultrasound can be a useful tool in the education of preclinical medical students. Such a course enabled learners to more easily distinguish between normal and pathologic exam findings and introduced them to the many clinical uses of ultrasound at an earlier stage in their training, allowing them to develop this important skillset.

Novel Barbiturate-Nitrate Compounds Inhibit the Upregulation of Matrix Metalloproteinase-9 Gene Expression in Intestinal Inflammation through a cGMP-Mediated Pathway.

Matrix metalloproteinase-9 is upregulated in inflammatory bowel disease. Barbiturate nitrate hybrid compounds have been designed to inhibit MMP secretion and enzyme activity. In this study, we investigated the mechanism of action of barbiturate-nitrate hybrid compounds and their component parts using models of intestinal inflammation in vitro. Cytokine-stimulated Caco-2 cells were used in all in vitro experiments. The NO donors SNAP and DETA-NONOate were used to study the effect of NO on MMP-9 mRNA. Mechanistic elucidation was carried out using the soluble guanylate cyclase (sGC) inhibitor, ODQ, and the cGMP analogue, 8-Bromo-cGMP. Further experiments were carried out to elucidate the role of NF-κB. NO donors exerted an inhibitory effect on MMP-9 mRNA in cytokine-stimulated cells. While the non-nitrate barbiturates had a limited effect on MMP-9 expression, the hybrid compounds inhibited MMP-9 expression through its NO-mimetic properties. No effect could be observed on mRNA for MMP-1 or MMP-2. The sGC inhibitior, ODQ, abolished the nitrate-barbiturate inhibition of MMP-9 gene expression, an effect which was reversed by 8-Br-cGMP. This study shows that the barbiturate scaffold is suitable for hybrid design as an MMP-9 inhibitor in cytokine-stimulated Caco-2 cells. The inhibition of MMP-9 levels was largely mediated through a reduction in its mRNA by a sGC/cGMP pathway mediated mechanism.

Effects of functionalized silver nanoparticles on aggregation of human blood platelets.

PURPOSE: We studied the effects of silver nanoparticles (AgNPs) on human blood platelet function. We hypothesized that AgNPs, a known antimicrobial agent, can be used as blood-compatible, "ideal material'' in medical devices or as a drug delivery system. Therefore, the aim of the current study was to investigate if functionalized AgNPs affect platelet function and platelets as well as endothelial cell viability in vitro. METHODS: AgNPs, functionalized with reduced glutathione (GSH), polyethylene glycol (PEG) and lipoic acid (LA) were synthesized. Quartz crystal microbalance with dissipation was used to measure the effect of AgNPs on platelet aggregation. Platelet aggregation was measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast microscopy. Flow cytometry was used to detect surface abundance of platelet receptors. Lactate dehydrogenase test was used to assess the potential cytotoxicity of AgNPs on human blood platelets, endothelial cells, and fibroblasts. Commercially available ELISA tests were used to measure the levels of thromboxane B2 and metalloproteinases (MMP-1, MMP-2) released by platelets as markers of platelet activation. RESULTS: 2 nm AgNPs-GSH, 3.7 nm AgNPs-PEG both at 50 and 100 µg/mL, and 2.5 nm AgNPs-LA at 100 µg/mL reduced platelet aggregation, inhibited collagen-mediated increase in total P-selectin and GPIIb/IIIa, TXB2 formation, MMP-1, and MMP-2 release. The tested AgNPs concentrations were not cytotoxic as they did not affect, platelet, endothelial cell, or fibroblast viability. CONCLUSION: All tested functionalized AgNPs inhibited platelet aggregation at nontoxic concentrations. Therefore, functionalized AgNPs can be used as an antiplatelet agent or in design and manufacturing of blood-facing medical devices, such as vascular grafts, stents, heart valves, and catheters.

Cardioprotective effect of MMP-2-inhibitor-NO-donor hybrid against ischaemia/reperfusion injury.

Hypoxic injury of cardiovascular system is one of the most frequent complications following ischaemia. Heart injury arises from increased degradation of contractile proteins, such as myosin light chains (MLCs) and troponin I by matrix metalloproteinase 2 (MMP-2). The aim of the current research was to study the effects of 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate (MMP-2-inhibitor-NO-donor hybrid) on hearts subjected to ischaemia/reperfusion (I/R) injury. Primary human cardiac myocytes and Wistar rat hearts perfused using Langendorff method have been used. Human cardiomyocytes or rat hearts were subjected to I/R in the presence or absence of tested hybrid. Haemodynamic parameters of heart function, markers of I/R injury, gene and protein expression of MMP-2, MMP-9, inducible form of NOS (iNOS), asymmetric dimethylarginine (ADMA), as well as MMP-2 activity were measured. Mechanical heart function, coronary flow (CF) and heart rate (HR) were decreased in hearts subjected to I/R Treatment of hearts with the hybrid (1-10 µmol/L) resulted in a concentration-dependent recovery of mechanical function, improved CF and HR. This improvement was associated with decreased tissue injury and reduction of synthesis and activity of MMP-2. Decreased activity of intracellular MMP-2 led to reduced degradation of MLC and improved myocyte contractility in a concentration-dependent manner. An infusion of a MMP-2-inhibitor-NO-donor hybrid into I/R hearts decreased the expression of iNOS and reduced the levels of ADMA. Thus, 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate protects heart from I/R injury.

New Oxidovanadium(IV) Coordination Complex Containing 2-Methylnitrilotriacetate Ligands Induces Cell Cycle Arrest and Autophagy in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Pancreatic cancer is characterized by one of the lowest five-year survival rates. In search for new treatments, some studies explored several metal complexes as potential anticancer drugs. Therefore, we investigated three newly synthesized oxidovanadium(IV) complexes with 2-methylnitrilotriacetate (bcma3-), N-(2-carbamoylethyl)iminodiacetate (ceida3-) and N-(phosphonomethyl)-iminodiacetate (pmida4-) ligands as potential anticancer compounds using pancreatic cancer cell lines. We measured: Cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and lactate dehydrogenase (LDH) assay; antiproliferative activity by bromodeoxyuridine BrdU assay; reactive oxygen species (ROS) generation and cell cycle analysis by flow cytometry; protein level by Western blot and cellular morphology by confocal laser scanning microscopy. The results showed that these oxidovanadium(IV) complexes were cytotoxic on pancreatic cancer cells (PANC-1 and MIA PaCa2), but not on non-tumor human immortalized pancreas duct epithelial cells (hTERT-HPNE) over the concentration range of 10⁻25 µM, following 48 h incubation. Furthermore, molecular mechanisms of cytotoxicity of [4-NH2-2-Me(Q)H][VO(bcma)(H2O)]2H2O (T1) were dependent on antiproliterative activity, increased ROS generation, cell cycle arrest in G2/M phase with simultaneous triggering of the p53/p21 pathway, binucleation, and induction of autophagy. Our study indicates that oxidovanadium(IV) coordination complexes containing 2-methylnitrilotriacetate ligand are good candidates for preclinical development of novel anticancer drugs targeting pancreatic cancer.

Feasibility of Out-of-Hospital Cardiac Arrest Ultrasound by EMS Physicians.

INTRODUCTION: Point-of-care ultrasound (POCUS) has been suggested as a useful tool to predict survival and guide interventions in out-of-hospital cardiac arrest (OHCA). While POCUS has been deployed in prehospital settings, a minimal amount of data exists on prehospital use, particularly by personnel with limited ultrasound experience. We aimed to characterize the feasibility and barriers to prehospital POCUS during OHCA by emergency medicine services (EMS) physicians in training. METHODS: We deployed the SonoSite iViz portable ultrasound device for use by EMS physicians for OHCA in an urban EMS system. All physicians received POCUS education as part of their graduate medical training and were provided an instructional video on use of the SonoSite iViz device. POCUS use was limited to identifying cardiac motion during pulse checks, without interrupting resuscitation, and the results could be used to supplement management at the physicians' discretion. Data were recorded prospectively by saving images on the device and through a custom electronic form within the patient care report. The primary measure was the frequency of use of POCUS during OHCA. Secondarily, we characterized agreement by expert (ultrasound fellowship trained) faculty (using a kappa statistic) and identified reported barriers to the use of prehospital POCUS. RESULTS: From November 2016 to March 2017, 348 physician field responses were reviewed, including 127 cases of OHCA. There were 106 patients remaining in arrest on physician arrival, with 56 (52.8%) cases of POCUS use. Still or video images were recorded in 48 cases; video in 34 cases. From video images, agreement in identifying cardiac motion between the EMS physician and expert reviewer occurred in 91% of cases (K = 0.82). Reasons cited for not using POCUS included return of circulation soon before or after arrival, prioritizing clinical interventions, not having the ultrasound device, mechanical failure, and cessation of resuscitation per advanced directives. CONCLUSION: Use of POCUS by EMS physicians to detect cardiac activity in OHCA is feasible and correlates with expert interpretation. Several avoidable barriers were identified and should be considered in the future implementation of prehospital POCUS. Larger studies are needed to determine what role POCUS may play in prehospital cardiac arrest management.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BACKGROUND: The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia. OBJECTIVES: To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock. METHODS: This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane). RESULTS: There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques. CONCLUSIONS: Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.

The Role of Mucin in the Toxicological Impact of Polystyrene Nanoparticles.

The development of novel oral drug delivery systems is an expanding area of research and both new approaches for improving their efficacy and the investigation of their potential toxicological effect are crucial and should be performed in parallel. Polystyrene nanoparticles (NPs) have been used for the production of diagnostic and therapeutic nanosystems, are widely used in food packaging, and have also served as models for investigating NPs interactions with biological systems. The mucous gel layer that covers the epithelium of the gastrointestinal system is a complex barrier-exchange system that it is mainly constituted by mucin and it constitutes the first physical barrier encountered after ingestion. In this study, we aimed to investigate the effect of polystyrene NPs on mucin and its potential role during NP⁻cell interactions. For this purpose, we evaluated the interaction of polystyrene NPs with mucin in dispersion by dynamic light scattering and with a deposited layer of mucin using a quartz crystal microbalance with dissipation technology. Next, we measured cell viability and the apoptotic state of three enterocyte-like cell lines that differ in their ability to produce mucin, after their exposure to the NPs. Positive charged NPs showed the ability to strongly interact and aggregate mucin in our model. Positive NPs affected cell viability and induced apoptosis in all cell lines independently of their ability of produce mucin.

Platelet microaggregation in sepsis examined by quartz crystal microbalance with dissipation technology.

Antiplatelet therapies remain an area of potential interest for the treatment of sepsis; however, studies of platelet aggregation in sepsis have yielded conflicting results. We examined platelet aggregation patterns in patients with septic shock using quartz crystal microbalance with dissipation technology, a microfluidic device capable of measuring platelet microaggregate formation under flow conditions. Platelet aggregation was increased in the washed platelet samples of septic patients. Conversely, these same platelets aggregated less than healthy controls when examined in their plasma.

Metal nanoparticles in dermatology and cosmetology: Interactions with human skin cells.

Nanotechnology is a rapidly developing branch of science, which studies control of phenomena and materials sized below 100 nm. Nanotechnology is applicable in many areas of life and medicine including skin care and personal hygiene. The nanoparticles (NPs) of metals and metal oxides are increasingly used in dermatology and cosmetology, especially in prevention and treatment of bacterial and fungal infections, in protection against the harmful effects of the sun and in preparations reducing the visibility of scars by accelerating the repair processes of skin cells. NPs may also be used for skin care and dermatological treatments to improve the quality of life of patients. Nanodermatology and nanocosmetology offer effective, safe, fast-acting product formulations, thus minimizing the side effects of the products used so far. The unique properties of NPs: high surface area relative to the size as well as the ability to penetrate biological membranes and barriers greatly reduces systemic dose thus potential side effects and toxicity. Recent studies show very promising clinical potential of NPs to serve as controlled release and delivery systems for drugs/active substances. In addition, NPs can be used in diagnostic imaging of skin diseases. However, NPs may also carry a risk of cytotoxicity and side effects. The present review focuses on the use of metal and metal oxide NPs in dermatology and cosmetology and their interactions with skin cells.

Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation.

AIMS: In addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor. METHODS AND RESULTS: Utilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbß3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation. CONCLUSION: These findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.

MMP-2, MMP-9, and TIMP-4 and Response to Aspirin in Diabetic and Nondiabetic Patients with Stable Coronary Artery Disease: A Pilot Study.

BACKGROUND: High on-aspirin treatment platelets reactivity (HPR) is a significant problem in long-term secondary prevention of cardiovascular events. We hypothesize that imbalance between platelets MMPs/TIMPs results in cardiovascular disorders. We also explored whether chronically elevated blood glucose affects MMP-2/TIMP-4 release from platelets. MATERIALS AND METHODS: Seventy patients with stable coronary artery disease, supplemented with aspirin, participated in this pilot study. The presence of HPR and/or diabetes mellitus was considered as the differentiating factor. Light aggregometry, impedance aggregometry, and ELISA tests for TXB2, MMP-2, MMP-9, and TIMP-4 were performed in serum, plasma, platelet-rich plasma, and platelets-poor plasma, as appropriate. RESULTS: Aspirin-HPR did not affect plasma MMP-2, MMP-9, and TIMP-4. Arachidonic acid-induced aggregation of platelets from aspirin-HPR patients did not lead to increased release of MMP-2, MMP-9, and TIMP-4. Studying patients at the lowest TXB2 serum concentration quartile revealed that high concentration of plasma TIMP-4 and TIMP-4 negatively correlated with TXB2 and platelet aggregation. Diabetics showed an increased plasma MMP-2 as well as an increased MMP-2 in supernatants after platelet aggregation. However, diabetes mellitus did not affect MMP-9 and TIMP-4. CONCLUSION: Aspirin-HPR did not affect the translocation and release of MMPs and TIMP-4 from platelets. TIMP-4 may serve as a marker of TXA2-mediated platelet aggregation. Chronically elevated plasma glucose increases plasma MMP-2, and HPR potentiates this phenomenon.