Self-Reporting Theranostic: Nano Tool for Arterial Thrombosis.

Arterial thrombosis (AT) originates through platelet-mediated thrombus formation in the blood vessel and can lead to heart attack, stroke, and peripheral vascular diseases. Restricting the thrombus growth and its simultaneous monitoring by visualisation is an unmet clinical need for a better AT prognosis. As a proof-of-concept, we have engineered a nanoparticle-based theranostic (combined therapy and monitoring) platform that has the potential to monitor and restrain the growth of a thrombus concurrently. The theranostic nanotool is fabricated using biocompatible super-paramagnetic iron oxide nanoparticles (SPIONs) as a core module tethered with the anti-platelet agent Abciximab (ReoPro) on its surface. Our in vitro feasibility results indicate that ReoPro-conjugated SPIONS (Tx@ReoPro) can effectively prevent thrombus growth by inhibiting fibrinogen receptors (GPIIbIIIa) on the platelet surface, and simultaneously, it can also be visible through non-invasive magnetic resonance imaging (MRI) for potential reporting of the real-time thrombus status.

Innate Immune Invisible Ultrasmall Gold Nanoparticles-Framework for Synthesis and Evaluation.

Nanomedicine is seen as a potential central player in the delivery of personalized medicine. Biocompatibility issues of nanoparticles have largely been resolved over the past decade. Despite their tremendous progress, less than 1% of applied nanosystems can hit their intended target location, such as a solid tumor, and this remains an obstacle to their full ability and potential with a high translational value. Therefore, achieving immune-tolerable, blood-compatible, and biofriendly nanoparticles remains an unmet need. The translational success of nanoformulations from bench to bedside involves a thorough assessment of their design, compatibility beyond cytotoxicity such as immune toxicity, blood compatibility, and immune-mediated destruction/rejection/clearance profile. Here, we report a one-pot process-engineered synthesis of ultrasmall gold nanoparticles (uGNPs) suitable for better body and renal clearance delivery of their payloads. We have obtained uGNP sizes of as low as 3 nm and have engineered the synthesis to allow them to be accurately sized (almost nanometer by nanometer). The synthesized uGNPs are biocompatible and can easily be functionalized to carry drugs, peptides, antibodies, and other therapeutic molecules. We have performed in vitro cell viability assays, immunotoxicity assays, inflammatory cytokine analysis, a complement activation study, and blood coagulation studies with the uGNPs to confirm their safety. These can help to set up a long-term safety-benefit framework of experimentation to reveal whether any designed nanoparticles are immune-tolerable and can be used as payload carriers for next-generation vaccines, chemotherapeutic drugs, and theranostic agents with better body clearance ability and deep tissue penetration.

A Pilot Study on Probing of Imatinib Induced Platelet Dysfunction in Patients with Chronic Myeloid Leukemia-Chronic Phase and Absence of Associated Bleeding Manifestation: Trying to Solve an Enigma.

Imatinib, the first Tyrosine Kinase Inhibitor (TKI) used for the treatment of chronic myeloid leukaemia (CML) has revolutionized the management by inhibiting BCR-ABL tyrosine kinase. According to earlier reports there are concerns regarding the adverse effect of imatinib on haemostasis by causing platelet dysfunction. Here we studied platelet function using platelet aggregometry, in 19 CML chronic phase (CML-CP) patients on imatinib therapy, in complete haematologic response (CHR). The median duration of imatinib therapy before performing the test was 154 days. This study reveals that there are large inter-individual variations in platelet functions among imatinib treated patients and different levels of variability have been seen for different agonists. Most common aggregation abnormality (< 50% aggregation) was seen with low dose collagen (1 µg/ml) in 31.57% patients. Despite in-vitro platelet aggregation defects, none of the patients showed any bleeding symptoms. This enigma can possibly be explained by the fact that platelet specific agonists, epinephrine and collagen act in synergy for platelet aggregation compared against individual low dose agonists, supported by ex-vivo experiments in normal healthy control group (n = 5) (p value < 0.0004 for epinephrine, p value < 0.0001 for collagen). This experiment was also confirmed in a CML-CP patient. In future, more studies are needed to find out the exact mechanism of this inhibition.

Varying effects of tyrosine kinase inhibitors on platelet function-A need for individualized CML treatment to minimize the risk for hemostatic and thrombotic complications?

Since their introduction, tyrosine kinase inhibitors (TKIs, eg, imatinib, nilotinib, dasatinib, bosutinib, ponatinib) have revolutionized the treatment of chronic myeloid leukemia (CML). However, long-term treatment with TKIs is associated with serious adverse events including both bleeding and thromboembolism. Experimental studies have shown that TKIs can cause platelet dysfunction. Herein, we present the first side-by-side investigation comparing the effects of currently used TKIs on platelet function and thrombin generation when used in clinically relevant concentrations. A flow cytometry multiparameter protocol was used to study a range of significant platelet activation events (fibrinogen receptor activation, alpha granule, and lysosomal exocytosis, procoagulant membrane exposure, and mitochondrial permeability changes). In addition, thrombin generation was measured in the presence of TKIs to assess the effects on global hemostasis. Results show that dasatinib generally inhibited platelet function, while bosutinib, nilotinib, and ponatinib showed less consistent effects. In addition to these general trends for each TKI, we observed a large degree of interindividual variability in the effects of the different TKIs. Interindividual variation was also observed when blood from CML patients was studied ex vivo with whole blood platelet aggregometry, free oscillation rheometry (FOR), and flow cytometry. Based on the donor responses in the side-by-side TKI study, a TKI sensitivity map was developed. We propose that such a sensitivity map could potentially become a valuable tool to help in decision-making regarding the choice of suitable TKIs for a CML patient with a history of bleeding or atherothrombotic disease.

Probing ADP Induced Aggregation Kinetics During Platelet-Nanoparticle Interactions: Functional Dynamics Analysis to Rationalize Safety and Benefits.

Platelets, one of the most sensitive blood cells, can be activated by a range of external and internal stimuli including physical, chemical, physiological, and/or non-physiological agents. Platelets need to respond promptly during injury to maintain blood hemostasis. The time profile of platelet aggregation is very complex, especially in the presence of the agonist adenosine 5'-diphosphate (ADP), and it is difficult to probe such complexity using traditional linear dose response models. In the present study, we explored functional analysis techniques to characterize the pattern of platelet aggregation over time in response to nanoparticle induced perturbations. This has obviated the need to represent the pattern of aggregation by a single summary measure and allowed us to treat the entire aggregation profile over time, as the response. The modeling was performed in a flexible manner, without any imposition of shape restrictions on the curve, allowing smooth platelet aggregation over time. The use of a probabilistic framework not only allowed statistical prediction and inference of the aggregation signatures, but also provided a novel method for the estimation of higher order derivatives of the curve, thereby allowing plausible estimation of the extent and rate of platelet aggregation kinetics over time. In the present study, we focused on the estimated first derivative of the curve, obtained from the platelet optical aggregometric profile over time and used it to discern the underlying kinetics as well as to study the effects of ADP dosage and perturbation with gold nanoparticles. In addition, our method allowed the quantification of the extent of inter-individual signature variations. Our findings indicated several hidden features and showed a mixture of zero and first order kinetics interrupted by a metastable zero order ADP dose dependent process. In addition, we showed that the two first order kinetic constants were ADP dependent. However, we were able to perturb the overall kinetic pattern using gold nanoparticles, which resulted in autocatalytic aggregation with a higher aggregate mass and which facilitated the aggregation rate.

Nanoimaging in cardiovascular diseases: Current state of the art.

Nanotechnology has been integrated into healthcare system in terms of diagnosis as well as therapy. The massive impact of imaging nanotechnology has a deeper intervention in cardiology i.e. as contrast agents , to target vulnerable plaques with site specificity and in a theranostic approach to treat these plaques, stem cell delivery in necrotic myocardium, etc. Thus cardiovascular nanoimaging is not limited to simple diagnosis but also can help real time tracking during therapy as well as surgery. The present review provides a comprehensive description of the molecular imaging techniques for cardiovascular diseases with the help of nanotechnology and the potential clinical implications of nanotechnology for future applications.

Surface tunability of nanoparticles in modulating platelet functions.

Metallic nanoparticles are attractive candidates as MRI contrast agents and mediators for drug delivery, diagnostics, and therapy. Direct contact and exposure to blood circulation is common in many such applications. The consequent thrombotic response may therefore be important to study. The main objective of the present work was to study how platelet functions were changed in the presence of different nano-surface or surface capping, which may provide a measure for the safety of a nanoparticle, and also assess the use of such nanoparticles in platelet modulation. Aggregometry, ATP release reaction, flow cytometry and immune-blotting studies were performed to study platelet response to different nano-particles (iron oxide, gold and silver). For each nanoparticle surface conjugation (capping) was varied. It was found that citric acid functionalized iron oxide nanoparticles have anti-platelet activity, with a decrease in aggregation, tyrosine phosphorylation level, and granule release. On the other hand in other cases (e.g. gold nanoparticles) pro-aggregatory response was observed in the presence of nanoparticles and, in some cases, the nanoparticles behaved neutrally (e.g. for starch-coated iron oxide nanoparticles). Therefore, nanoparticles can induce antiplatelet or a pro-aggregatory response, or remain neutral depending on surface capping. A related observation is that antiplatelet drugs can be made more potent by nanoparticle conjugation.

Multistability in platelets and their response to gold nanoparticles.

The nanoparticle (NP) response of platelets is shown to be critically dependent on extent of preactivation of platelets by an agonist like ADP. A transition from de-aggregatory to aggregatory state is triggered in the presence of gold NPs (AuNP) only in such critical conditions. Adhered and suspended platelets respond differentially to NPs. Preactivation in the adhered state induced by shear force explains such observation. The NP effect is associated with enhanced release reaction, tyrosine phosphorylation and CD62P expression level. Unlike cancer cells, whose response is maximal when NP size is optimal (within the range 50 - 70 nm), the platelet response monotonically increases with reduction of the AuNP size. The uptake study, using quenching of quinacrine hydrochloride fluorescence by AuNP, indicates that accumulation 18 nm AuNP is several-fold higher than the 68 nm AuNP. It is further shown that AuNP response can provide a simple measure for thrombotic risk associated with nano-drugs. FROM THE CLINICAL EDITOR: Platelet aggregation can be triggered in the presence of gold nanoparticles (AuNP). Platelet response monotonically increases with reduction of the AuNP size. AuNP response can provide a simple measure for thrombotic risk associated with nano-drugs.

Vitamin C prevents cigarette smoke induced atherosclerosis in guinea pig model.

AIM: Cigarette smoking is a major risk for developing atherosclerosis; however, the underlying mechanism is poorly understood. This paucity of knowledge is largely attributed to the lack of an animal model; therefore, our efforts were targeted towards establishing cigarette smoke (CS)-induced atherosclerosis in guinea pig. To understand the mechanism, we investigated apoptosis, an event implicated in atherosclerosis, in the aorta of CS-exposed animals. Since a major deleterious effect of CS is oxidative stress, we also examined the effect of vitamin C, an antioxidant, on CS- induced atherosclerosis. METHODS AND RESULTS: Guinea pigs on a diet with or without vitamin C supplement were exposed to CS for different time periods. Aortal sections from these animals were examined for atherosclerotic changes by staining with H&E and Oil red O. Atherogenic changes were observed in sections obtained from CS-exposed guinea pigs only. TUNEL assay showed the occurrence of apoptosis in CS-exposed guinea pig aorta. Our results revealed that CS-induced apoptosis could contribute to the progression but not to the initiation of the disease. Immunohistochemical analysis documents that CS-induced apoptosis in aortal sections is mediated at least in part by an increased Bax/Bcl2 ratio. In contrast, CS-exposed guinea pigs fed with vitamin C-supplemented diet exhibit little or no atherogenic changes. This anti-atherosclerotic activity of vitamin C can be attributed partly to its ability to inhibit CS-induced apoptosis and platelet activation. CONCLUSION: Exposure of guinea pigs to cigarette smoke causes the development of atherosclerosis, which can be prevented by vitamin C supplement.

Dual antiplatelet therapy in ACS: time-dependent variability in platelet aggregation during the first week.

AIMS AND OBJECTIVES: Platelets play an important role in the pathogenesis of Acute Coronary Syndrome (ACS). Most of the complications of ACS occur during the initial hours of presentation. We tried to gain an insight into the platelet function during the initial phase of ACS in patients on dual antiplatelet therapy. MATERIALS AND METHODS: Platelet aggregation study was performed by light transmittance aggregometry in 64 ACS patients 48 hour and 7 days after initiation of dual antiplatelet therapy with aspirin and clopidogrel. RESULTS: Epinephrine, ADP and collagen induced platelet aggregation was significantly higher at 48 hours, following initiation of dual antiplatelet therapy, in comparison to the profile observed on the 7th day. Diabetics demonstrated a significantly higher aggregation at both the time points and aggregation was also somewhat higher in smokers though it did not reach statistical significance. CONCLUSION: This study conceptualizes the hypothetical role of alpha-2 adrenoreceptor blockers during the early hours following ACS and also warrants further investigations exploring the optimum loading dose of antiplatelet agents, especially clopidogrel in patients with ACS.

Dual antiplatelet drug resistance in patients with acute coronary syndrome.

AIMS AND OBJECTIVES: Antiplatelet therapy is a cornerstone in the management of the atherosclerotic vascular disease. Aspirin and clopidogrel are the two most commonly used antiplatelet drugs in its management. Recently, there has been a concern about the development of resistance to one or both antiplatelet agents with potentially devastating consequences. In this study we tried to assess the in vitro resistance to antiplatelet agents in patients presenting with acute coronary syndrome (ACS). MATERIALS AND METHODS: 144 patients presenting with ACS, who were not on any antiplatelet therapy prior to hospital admission were evaluated in this study. Baseline clinical data was obtained before giving the oral loading dose of aspirin and clopidogrel. Patients received a loading dose of 325 mg of aspirin and 300 mg of clopidogrel followed by a daily dose of 150 mg. of aspirin and 75 mg.of clopidogrel. After 7 days of dual antiplatelet therapy, platelet aggregation pattern was analyzed using optical aggregometer (chrono-log). Response to aspirin and clopidogrel was assessed by interaction with collagen (2microg/ml) and Adenosine diphosphate (ADP) (10micro/ml) respectively. The results were analyzed. Response to doubling the dose of antiplatelet agents was also observed in 6 aspirin resistant patients, 12 clopidogrel resistant patients and in 6 patients resistant to the effect of dual antiplatelet agents. RESULTS: There were 22 patients (15.27%) who showed poor response to aspirin, 28 patients (19.44%) to clopidogrel (primary non-responder) and 18 patients (12.5%) showed a primary non-responsiveness to both the antiplatelet agents in the usual doses. After dose doubling, all 6 aspirin resistant patients showed adequate response but 4 out of 12 clopidogrel resistant patients showed inadequate response. CONCLUSIONS: This pilot study brings out a disquieting picture of 12.5% patients suffering from ACS showing resistance to the antiplatelet effects of both aspirin and clopidogrel in the conventional dose. A long-term prospective randomized controlled trial is required to give an insight into this problem and its clinical consequences.

Platelet responsiveness to yohimbine hydrochloride and MRS2179 in the context of the interaction between collagen and epinephrine in acute coronary syndrome.

Acute coronary syndrome (ACS) covers a spectrum of clinical conditions ranging from unstable angina, Non-ST segment elevation myocardial infarction (NSTEMI), or ST segment elevation myocardial infarction (STEMI). This study encompasses patients with acute coronary syndrome, who were receiving the dual antiplatelet therapy of aspirin and clopidogrel. The focus of the study was to gain insight into the role of selective P2Y1 antagonism using MRS2179 in such cases as well as its effects, if any, on collagen-epinephrine interaction. All the cases showed greater potency of inhibition of the interaction when yohimbine hydrochloride (YH), a blocker of alpha2A-adrenoreceptor, was used compared to MRS2179, a P2Y1 antagonist, although there was variability in responsiveness to the antiplatelet drugs. These findings indicate that alpha2A-adrenoreceptors of platelets in this group play a major role in precipitating the interactive effect of collagen and epinephrine. The dose-response effect as studied by platelet aggregometry showed that the required molar concentration to block the interactive effect in the case of YH was less than that of MRS2179. Hence, it is postulated that although there may be an impairment of collagen-induced aggregation by MRS2179, the interactive effect of collagen-epinephrine may not be impaired by MRS2179 as efficaciously as YH.

Antiplatelet drug resistance in patients with recurrent acute coronary syndrome undergoing conservative management.

AIMS AND OBJECTIVES: Recurrent ischemic events continue to occur despite combination anti-platelet therapy. Currently aspirin, clopidogrel and dual resistance are increasingly recognized entities. The relationship of such resistance to recurrent ischemic events is largely unknown. In this study, we tried to gain an insight into the role of antiplatelet drug resistance with recurrent Acute Coronary Syndrome (ACS). MATERIALS AND METHODS: The antiplatelet effect of aspirin and clopidogrel was studied in 40 recurrent ACS patients and 170 patients with first episode of ACS, after > or = 7 days of dual antiplatelet therapy. Platelet aggregation study was done with optical aggregometer. Resistance to aspirin and clopidogrel was defined as > or = 50% aggregation with collagen and ADP respectively. RESULTS: Aspirin, clopidogrel and dual drug resistance were encountered respectively in 35%, 72.5% and 32.5% patients with recurrent ACS. The corresponding figures for the patients with first episode of ACS were 25.3%, 42.3% and 18.8% respectively. P values for the comparisons were 0.237 for aspirin, 0.0007 for clopidogrel and 0.084 for dual drugs. Patients with recurrent ACS were relatively younger and had a higher prevalence of conventional risk factors like hypertension, diabetes and elevated LDL. CONCLUSION: Antiplatelet drug resistance is likely to play an important role in recurrent ACS alongside other conventional risk factors. Further research is required in this field to have a definitive conclusion.