Marilena Crescente

Shear-Activated Nanotherapeutics for Drug Targeting to Obstructed Blood Vessels

Bio-Inspired Drug Delivery Noting that platelets naturally migrate to narrowed blood vessels characterized by high fluid shear stress, Korin et al. (p. 738, published online 5 July; see the Perspective by Lavik and Ustin) developed a nanoparticle-based therapeutic that uses a similar targeting mechanism to deliver a drug to vessels obstructed by blood clots. Aggregates of nanoparticles coated with the clot-dissolving drug tPA (tissue plasminogen activator) were designed to fall apart and release the drug only when encountering high fluid shear stress. In preclinical models, the bio-inspired therapeutic dissolved clots and restored normal blood flow at lower doses than free tPA, suggesting that this localized delivery system may help reduce the risk of side effects such as excessive bleeding. Nanoparticles carrying a drug that dissolves blood clots disintegrate at sites of stenosis. Obstruction of critical blood vessels due to thrombosis or embolism is a leading cause of death worldwide. Here, we describe a biomimetic strategy that uses high shear stress caused by vascular narrowing as a targeting mechanism—in the same way platelets do—to deliver drugs to obstructed blood vessels. Microscale aggregates of nanoparticles were fabricated to break up into nanoscale components when exposed to abnormally high fluid shear stress. When coated with tissue plasminogen activator and administered intravenously in mice, these shear-activated nanotherapeutics induce rapid clot dissolution in a mesenteric injury model, restore normal flow dynamics, and increase survival in an otherwise fatal mouse pulmonary embolism model. This biophysical strategy for drug targeting, which lowers required doses and minimizes side effects while maximizing drug efficacy, offers a potential new approach for treatment of life-threatening diseases that result from acute vascular occlusion.

Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level. Functional and modelling studies.

While resveratrol and quercetin possess antiplatelet activity, little is known on the effect of gallic acid on platelets. We studied the interactions of these three different polyphenols among themselves and with aspirin, at the level of platelet cyclooxygenase-1 (COX-1). Both functional (in vitro and in vivo) and molecular modelling approaches were used. All three polyphenols showed comparable antioxidant activity (arachidonic acid [AA]-induced intraplatelet ROS production); however, resveratrol and quercetin, but not gallic acid, inhibited AA-induced platelet aggregation. Gallic acid, similarly to salicylic acid, the major aspirin metabolite, prevented inhibition of AA-induced platelet function by aspirin but, at variance with salicylic acid, also prevented inhibition by the other two polyphenols. Molecular modelling studies, performed by in silico docking the polyphenols into the crystal structure of COX-1, suggested that all compounds form stable complexes into the COX-1 channel, with slightly different but functionally relevant interaction geometries. Experiments in mice showed that gallic acid administered before aspirin, resveratrol or quercetin fully prevented their inhibitory effect on serum TxB(2). Finally, a mixture of resveratrol, quercetin and gallic acid, at relative concentrations similar to those contained in most red wines, did not inhibit platelet aggregation, but potentiated sub-inhibitory concentrations of aspirin. Gallic acid interactions with other polyphenols or aspirin at the level of platelet COX-1 might partly explain the complex, and possibly contrasting, effects of wine and other components of the Mediterranean diet on platelets and on the pharmacologic effect of low-dose aspirin.

PFA-100 closure time to predict cardiovascular events in aspirin-treated cardiovascular patients: a meta-analysis of 19 studies comprising 3,003 patients.

PFA-100 closure time to predict cardiovascular events in aspirin-treated cardiovascular patients: A meta-analysis of 19 studies comprising 3,003 patients -

OFFgel-based multidimensional LC-MS/MS approach to the cataloguing of the human platelet proteome for an interactomic profile.

The proteome of quiescent human platelets was analyzed by a shotgun proteomics approach consisting of enzymatic digestion, peptide separation based on isoelectric point by the use of OFFgel fractionation and, finally, RP nanoscale chromatography coupled to MS/MS detection (nano‐LC‐MS/MS). OFFgel fractionation in the first dimension was effective in providing an additional dimension of separation, orthogonal to RP nano‐LC, thus generating an off‐line multidimensional separation platform that proved to be robust and easy to set up. The analysis identified 1373 proteins with high confidence (false discovery rate<0.25%). The core set of 1373 human platelet proteins was investigated by Ingenuity Pathway Analysis software from which ten canonical pathways and eight networks have been validated, to suggest that platelets behave either as inflammatory or immune cells, and plasma membrane and cytoskeleton proteins play a fundamental role in their function. Moreover, toxicity pathway in agreement with network analysis, supports the concept that platelet life span is governed by an apoptotic mechanism.

Platelet proteome in healthy volunteers who smoke

Smoking accelerates atherosclerosis and is a well-known risk factor for acute cardiovascular complications; however, the mechanisms of these effects have not been completely clarified. Recently developed proteomic approaches may offer new clues when combined with well-established functional tests. Platelet proteome of healthy smokers and non-smokers was resolved by two-dimensional difference gel electrophoresis, compared by Decyder software and identified by mass spectrometry analysis (nano-LC-MS/MS). In smokers, three proteins (Factor XIII-A subunit, platelet glycoprotein IIb and beta-actin) were significantly up-regulated, whereas WDR1 protein and chaperonine HSP60 were down-regulated. Furthermore, the highest scored network derived by Ingenuity Pathway Analysis using the modulated proteins as input showed the involvement of several proteins to be related to inflammation and apoptosis. Platelet function tests and the levels of markers of platelet and leukocyte activation were not different in smokers vs. non-smoker subjects. The platelet proteomic approach confirms that cigarette smoking triggers several inflammatory reactions and may help clarify some of the molecular mechanisms of smoke effect on cellular systems relevant for vascular integrity and human health.

ADAMTS13 exerts a thrombolytic effect in microcirculation

Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl(3)) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop on-going thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl(3) injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.

Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases

Objective— Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. Approach and Results— Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). Conclusions— Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide–sensitive fusion protein attachment receptor/Munc13-4–dependent vesicular–plasma membrane fusion.

Epoprostenol inhibits human platelet-leukocyte mixed conjugate and platelet microparticle formation in whole blood

Circulating platelet-leukocyte mixed conjugates and platelet microparticles are potential markers of inflammation in the atherothrombotic disease. Epoprostenol is a synthetic salt of PGI2 (prostacyclin) clinically used in pulmonary hypertension and transplantation as a potent inhibitor of platelet aggregation. In this study the in vitro effect of this drug was investigated on the interaction of platelets with leukocytes and on markers of leukocyte and platelet activation, including platelet microparticle formation. The analyses were performed by flow cytometry on citrated whole blood collected from healthy subjects and challenged by a mixture of collagen-ADP. Preliminarily, the epoprostenol antiplatelet effect was confirmed by both aggregometry and PFA-100 and by evaluation of intraplatelet VASP phosphorylation. Epoprostenol, at nanomolar concentrations, prevented the formation of platelet mixed conjugates with PMN or monocytes, platelet PAC-1 and P-selectin expression and platelet microparticle generation. The reference drugs PGE1, aspirin and the novel ADP-receptor antagonist, cangrelor, were only effective at micromolar concentrations. No effect of epoprostenol was detected on leukocyte activation markers. Our data suggest a possible additional mechanism of action of epoprostenol in reducing the inflammatory cell contribution to pulmonary hypertension and thrombosis.

Incomplete inhibition of platelet function as assessed by the platelet function analyzer (PFA-100) identifies a subset of cardiovascular patients with high residual platelet response while on aspirin

Sixty-six patients with a history of ischemic events (myocardial infarction, unstable angina, or stroke) on chronic aspirin therapy were studied by different platelet function tests: 37 patients had suffered a recurrent event while on aspirin and 29 were without recurrences. Based on results from light transmission aggregometry (LTA) induced by arachidonic acid (AA) and serum TxB2 both COX-1-dependent methods, only one patient could be identified as aspirin “resistant”. However, when methods only partially-dependent on platelet COX-1 activity were considered, the prevalence of aspirin non-responders ranged, according to the different tests, from 0 to 52%. No difference was observed between patients with recurrences and those without. Among patients with recurrent events, those with an incomplete inhibition of platelet function, as assessed by the PFA-100, had significantly higher residual serum TxB2 (2.4 ± 2.4 ng/mL vs 0.4 ± 0.1 ng/mL, p = 0.03), residual LTA-AA (9.2 ± 10.6% vs 2.0 ± 1.6%, p = 0.008), LTA-Coll (49.3 ± 14.6% vs 10.2 ± 8.3%, p = 0.007) and LTA-ADP (50.9 ± 16.2% vs 34.3 ± 11.0%, p = 0.04). In conclusion, laboratory tests solely exploring the AA-mediated pathway of platelet function, while being the most appropriate to detect the effect of aspirin on its pharmacologic target (platelet COX-1), may fail to reveal the functional interactions between minimal residual TxA2 and additional stimuli or primers potentially leading to aspirin-insensitive platelet aggregation. High residual platelet response in platelet function tests only partially dependent on COX-1 may reveal a condition of persistent platelet reactivity in a subset of aspirin-treated patients characterizing them as a subgroup at higher vascular risk.

Pharmacological actions of nobiletin in the modulation of platelet function

The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin, in the modulation of platelet function.