Increased levels of circulating platelet-derived microparticles in psoriasis:Possible implications for the associated cardiovascular risk

AIM To evaluate platelet activation markers in psoriasis patients, compared to controls, and investigate their association with the inflammatory burden of psoriasis.METHODS Forty psoriatic patients without cardiovascular disease,and 12 healthy controls were subjected to measurement of baseline platelet CD62 P, CD63 and CD42 b expression, platelet-leukocyte complexes, i.e., platelet-monocyte complexes(PMC), platelet-neutrophil complexes(PNC) and platelet-lymphocyte complexes, and concentrations of platelet-derived microparticles(PMPs) using flow cytometry. Both larger-size(0.5-0.9 μm) and smallersize( 0.5 μm) PMPs were determined. Serum interleukin(IL)-12 and IL-17 levels were also measured by enzyme-linked immunosorbent assay. The severity of psoriasis was evaluated by the Psoriasis Area Severity Index(PASI).RESULTS PMP concentrations were significantly higher in psoriasis patients than controls [mean±standard error of mean(SEM): 22±5/μL vs 11±6/μL; P=0.018), for both smaller-size(10±2/μL vs 4±2/μL; P=0.033) and larger-size(12±3/μL vs 6±4/μL; P=0.014) PMPs. Platelet CD62 P, CD63 and CD42 b expression and circulating PMC and PNC were similar between the two groups. Lower circulating PLC were observed in psoriasis patients compared to controls(mean±SEM: 16%±3% vs 23%±6%; P=0.047). Larger-size PMPs were related with IL-12 levels(P0.001) and smaller-size PMPs with both IL-12 and IL-17 levels(P0.001). Total PMPs also correlated with IL-12(P0.001). CD63 expression was positively correlated with both IL-12 and IL-17(P0.05). Increased PASI score was associated with increased levels of larger-size PMPs(r=0.45; P=0.011) and increased CD63 expression(r=0.47; P0.01).CONCLUSION PMPs, known to be predictive of cardiovascular outcomes, are increased in psoriasis patients, and associated with high inflammatory disease burden. Enhanced platelet activation may be the missing link leading to cardiovascular events in psoriatic patients.     

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow

Summary. Background: Microparticles (MPs) released by activated or apoptotic cells increase in number in the blood of subjects with vascular or metabolic diseases and may contribute to thrombotic complications. Objectives: In this study, we investigated whether MPs promoted platelet recruitment to endothelial cells in flow conditions, and by which mechanism. Methods: Human umbilical vein endothelial cells (HUVECs) grown in microslide perfusion chambers were exposed to MPs prepared in vitro from HUVECs, monocytes or platelets. Results: Videomicroscopy of DIOC‐labelled blood perfused at arterial rate on human umbilical vein ECs demonstrated that, irrespective of their cell origin, MPs promoted the formation of platelet strings at the surface of HUVECs. This platelet/endothelial cell interaction was dependent on von Willebrand factor (VWF) expression at the HUVEC surface and involved Glycoprotein Ib and P‐selectin. Interestingly, HUVECs internalized MPs within a few hours through a process involving anionic phospholipids, lactadherin and αvβ3 integrin. This uptake generated the production of reactive oxygen species via the xanthine/xanthine oxidase system (inhibited by allopurinol and the ROCK inhibitor Y‐27632) and the NADPH oxidase (inhibited by SOD). Reactive oxygen species appeared essential for VWF expression at the endothelial cell surface and subsequent platelet/endothelial cell interaction under flow. The pathophysiological relevance of this process is underlined by the fact that circulating MPs from Type I diabetic patients induced platelet/endothelial cell interaction under flow, with an intensity correlated with the severity of the vasculopathy.     

Improving Protein Delivery from Microparticles Using Blends of Poly(DL lactide co-glycolide) and Poly(ethylene oxide)-poly(propylene oxide) Copolymers

Purpose. Microparticles containing ovalbumin as a model for protein drugs were formulated from blends of poly(DL lactide-co-glycolide) and poly(ethylene oxide)-poly(propylene oxide) copolymers (Pluronic). The objectives were to achieve uniform release characteristics and improved protein delivery capacity. Methods. The water- in oil -in oil emulsion/solvent extraction technique was used for microparticle production. Results. A protein loading level of over 40% (w/w) was attained in microparticles having a mean diameter of approximately 5 µm. Linear protein release profiles over 25 days in vitro were exhibited by certain blend formulations incorporating hydrophilic Pluronic F127. The release profile tended to plateau after 10 days when the more hydrophobic Pluronic L121 copolymer was used to prepare microparticles. A delivery capacity of 3 µg OVA/mg particles/ day was achieved by formulation of microparticles using a 1:2 blend of PLG:Pluronic F127. Conclusions. The w/o/o formulation approach in combination with PLG:Pluronic blends shows potential for improving the delivery of therapeutic proteins and peptides from microparticulate systems. Novel vaccine formulations are also feasible by incorporation of Pluronic L121 in the microparticles as a co-adjuvant.     

Prolonged circulation of activated platelets following plasmapheresis

The extent and duration of in vivo platelet activation were determined in 12 volunteer donors undergoing automated plasmapheresis. Expression of P-selectin, activated GpIIb/IIIa, and platelet microparticle formation were measured by flow cytometry on peripheral blood samples obtained immediately before and after plasmapheresis and at 24 hour intervals thereafter for up to 3 days. Although no adverse effects were noted in any donor, immediately after apheresis 3 87% of circulating platelets expressed P-selectin: by 48 hours. 0.5 50% expressed P-selectin; and by 72 hours, all donors studied had fewer than 5% P-selectin expression on circulating platelets. Results were similar for the expression of the activated conformation of GpIIb/IIIa. There was a positive correlation with in vitro P-selectin expression in response to ADP in the pre-apheresis sample and the number of platelet microparticles detected in the donor following plasmapheresis. In addition, the percent expression of P-selectin and activated GpIIb/IIIa in response to ADP was reproducible in each individual studied on five separate occasions (CV ≤ 8%). Platelets activated during plasmapheresis using an automated device may circulate for at least 48 hours, and pre-plasmapheresis response of platelets to the agonist ADP correlated with platelet microparticle formation post-plasmapheresis. © 1994 Wiley-Liss, Inc.     

Augmentation of ultrasound-induced clot disruption by nongas-filled microparticles

The augmentation of ultrasound-induced clot disruption by echocardiographic contrast microbubbles may be a direct mechanical erosive effect of the clot by the microparticles. To further assess this hypothesis, we evaluated the rate and extent of clot disruption by using external ultrasound and nongas-filled microparticles (HAEMACCEL and HAES). Human blood clots were used in this in vitro study. The percent clot reduction using the combination of ultrasound and microparticles was dose dependent and significantly higher than that with microparticles alone.     

Supercritical fluids crystallization of budesonide and flunisolide

Purpose: Budesonide and flunisolide anhydrate were crystallized using the solution enhanced dispersion by supercritical fluids (SEDS) technique. The aim was to investigate the possibility of preparing different pure polymorphs. Methods: 0.25% w/v solutions of each drug were prepared from acetone and methanol. Operating conditions were 40-80°C and 80-200 bars. The flow rate of drug solution was 0.3 mL/min and that of CO₂ was 9-25 mL/min. Sample characterizations included differential scanning calorimetry, X-ray powder diffraction, variable temperature X-ray diffraction, scanning electron microscopy, and solubility studies. Results: The particle morphology of budesonide was dependent on the nature of the solvent. SEDS processing of flunisolide with acetone at 100 bars resulted in the formation of polymorphic mixtures at 80°C and a new polymorph III at 60 C and 40°C. With methanol at 100 bars another new polymorph IV was formed with different particle morphology at 80°C and a polymorphic mixture at 60°C. Conclusion: Using the SEDS, microparticles of crystalline budesonide were prepared and new polymorphs of flunisolide were produced. Particle characteristics were controlled by the temperature, pressure and relative flow rates of drug solution and CO₂.     

Formation and Isolation of Spherical Fine Protein Microparticles Through Lyophilization of Protein-Poly(ethylene Glycol) Aqueous Mixture

Purpose. Preparation of spherical fine protein microparticles by the lyophilization of a protein-poly(ethylene glycol) (PEG) aqueous mixture was investigated. The main objective was to establish a method for preparing protein microparticles suitable for pharmaceutical production. Methods. Aqueous solutions containing bovine serum albumin (BSA) and PEG at various mixing ratios were freeze-dried. The lyophilizates were dispersed in methylene chloride and subjected to particle size analysis. Analogous studies were performed using several model proteins. A phase diagram of the PEG-BSA aqueous system was obtained by the titration method. Results. The particle size of BSA decreased as the PEG-BSA ratio increased. A bending point was observed in this relationship, at which the PEG-BSA ratio coincided with that of the critical point on the phase diagram of the PEG-BSA system. These results were explained by the freezing-induced condensation, followed by phase separation in the PEG-BSA system. Conclusions. Spherical fine protein microparticles were successfully obtained at high yield and without any activity loss under optimum conditions. This new technology could be applicable to proteins with a wide range of molecular weights, and is expected to be developed for dry powder inhalations or long-term sustained release microsphere formulations.     

Survival and stability of bifidobacteria loaded in alginate poly-l-lysine microparticles

Bifidobacteria-loaded alginate microparticles were prepared by spraying a mixture of alginate and bifidobacteria culture using an air atomization method. Survival and stability of bifidobacteria loaded in microparticles were then evaluated. Survival of bifidobacteria from alginate poly-l-lysine microparticles was significantly increased when MRS broth or yeast extract was added in simulated intestinal fluid (pH 6.8). The number of bifidobacteria gradually increased for 8 h (10(8) cfu/g) and then reached about 10(9)-10(10) cfu/g when incubated over 12 h in intestinal fluid containing 0.5% yeast extract and 0.05% L-cysteine. The survival of bifidobacteria was highly dependent on the pH of the exposing media. When the bifidobacteria was immobilized with alginate or even poly-l-lysine treatment, the survival of bifidobacteria was highly enhanced in the low pH conditions (ca. > 10(8) vs. < 10(3) cfu/g). The stability of free flowing bifidobacteria-loaded alginate poly-l-lysine microparticles was significantly improved during storage at 4 degrees C in a refrigerator when compared to bifidobacteria cultures. The bifidobacteria-loaded alginate poly-l-lysine microparticles could be applied to various dairy products.