Enrichment of plasma in platelets and extracellular vesicles by the counterflow to erythrocyte settling.

In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = -0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.[Figure: see text].

Cryopreserved buffy-coat-derived platelets reconstituted in platelet additive solution: A safe and available product with sufficient haemostatic effectiveness.

BACKGROUND: Platelets (PLTs) stored at 20-24 °C have a short shelf life of only 5 days, which can result in their restricted availability. PLT cryopreservation extends the shelf life to 2 years. METHODS: We implemented a method of PLT freezing at -80 °C in 5-6% dimethyl sulfoxide. Buffy-coat-derived leucodepleted fresh PLTs blood group O (FP) were used for cryopreservation. Cryopreserved pooled leucodepleted PLTs (CPP) were thawed at 37 °C, reconstituted in PLT additive solution SSP + and compared to FP regarding PLT content, PLT concentration, pH, volume, PLT loss, anti-A/B antibody titre, total protein, plasma content, and PLT swirling. Clot properties were evaluated via rotational thromboelastometry. PLT microparticle number and surface receptor phenotype were assessed via flow cytometry. RESULTS: CPP met the required quality parameters. The mean freeze-thaw PLT loss was 22.24 %. Anti-A/B antibody titre and plasma content were significantly lower in CPP. CPP were characterised by faster clot initiation and form stable PLT clots. The number of PLT microparticles increased 25 times in CPP and there were more particles positive for the activation marker CD62 P compared to FP. CONCLUSION: Thawing and reconstitution are easy and fast processes if platelet additive solution is used. Low anti-A/B antibody titre and plasma content make possible the use of CPP of blood group O reconstituted in SSP + as universal ABO products, including clinical situations where washed PLTs are required. Clot properties evaluated via rotational thromboelastometry demonstrated that CPP retain a significant part of their activity compare to FP and are haemostatically effective.

Platelet microparticles-derived miR-25-3p promotes the hepatocyte proliferation and cell autophagy via reducing B-cell translocation gene 2.

Platelets are critical regulators of liver regeneration, but the mechanisms are still not fully understood. Platelets have been shown to contain a wide variety of microRNAs (miRNAs) and play an important role in many diseases. However, the mechanism that how the platelet microparticles (PMPs)-derived miRNA regulate the hepatocyte proliferation is not very clear. In this study, we have successfully isolated and identified PMPs. We also found that PMPs, which could be well integrated into the HHL-5 cells, could upregulate the level of miR-25-3p in HHL-5 cells. Meanwhile, we found that PMPs-derived miR-25-3p promoted HHL-5 cells proliferation by accelerating cells into the S phase, and enhanced the autophagy by increasing the LC3II expression and reducing the P62 expression. Then, we proved that the miR-25-3p could target the B-cell translocation gene 2 (BTG2) and downregulate the expression levels of the BTG2 gene in HHL-5 cells. In addition, the overexpression of BTG2 significantly inhibited the proliferation and autophagy abilities of HHL-5 cells, while cotransfected miR-25-3p mimics or PMPs could partially rescue HHL-5 cells proliferation and autophagy. Furthermore, we proved that PMPs accelerated hepatocyte proliferation by regulating autophagy pathways. Therefore, PMPs-derived miR-25-3p promoted HHL-5 cell proliferation and autophagy by targeting BTG2, which may be a new therapeutic method for liver regeneration.

Rac1 regulates platelet microparticles formation and rheumatoid arthritis deterioration.

Platelets play important roles in blood clotting, hemostasis and wound repair, while more and more research show that platelets also have significant contributions in the process of inflammation. Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease. Platelet microparticles, which are membrane vesicles shed by activated platelets, are reported to amplify inflammation in Rheumatoid arthritis. Here we show that either platelet-specific deletion of Rac1 (Rac1-/-) or Rac1-specific inhibitor NSC23766 dramatically inhibit platelet-derived microparticles formation. As we all know, collagen-induced arthritis (CIA) mouse model is the most common autoimmune model of rheumatoid arthritis. Interestingly, NSC23766 alleviated the process of collagen-induced arthritis of DBA mice in vivo, including the reduced hind paw thickness and ankle stiffness, the reduction of arthritic scores and incidence of arthritis. Our work also found that NSC23766-treated CIA mouse spleen is less swollen and contains less enlarged white pulp than PBS control. The histological analysis shows that NSC23766-treated but not solvent control improve the cartilage erosion symptom in the joint of CIA mouse. Interestingly, platelet microparticles in the peripheral blood of NSC23766-treated CIA mice were decreased significantly compared with PBS-treated CIA mice. In conclusion, our work demonstrated that Rac1 inhibition alleviates collagen-induced arthritis through the decrease of platelet microparticles' release. In short, Rac1 aggravate the rheumatoid arthritis deterioration through the regulation of platelet microparticles formation.

Circulating microparticles as biomarkers of stroke: A focus on the value of endothelial- and platelet-derived microparticles.

Stroke is one of the leading causes of mortality and disability worldwide. Numerous pathophysiological mechanisms involving blood vessels, coagulation and inflammation contribute to the vascular occlusion. Perturbations in these pathways can be detected by numerous methods including changes in endoplasmic membrane remodeling and rearrangement leading to the shedding of microparticles (MPs) from various cellular origins in the blood. MPs are small membrane-derived vesicles that are shed from nearly all cells in the body in resting state or upon stimulation. MPs act as biological messengers to transfer information to adjacent and distant cells thus regulating various biological processes. MPs may be important biomarkers and tools for the identification of the risk and diagnosis of cerebrovascular diseases. Endothelial activation and dysfunction and altered thrombotic responses are two of the main features predisposing to stroke. Endothelial MPs (EMPs) have been recognized as both biomarkers and effectors of endothelial cell activation and injury while platelet-derived MPs (PMPs) carry a strong procoagulant potential and are activated in thrombotic states. Therefore, we reviewed here the role of EMPs and PMPs as biomarkers of stroke. Most studies reported high circulating levels of EMPs and PMPs in addition to other cell origins in stroke patients and have been linked to stroke severity, the size of infarction, and prognosis. The identification and quantification of EMPs and PMPs may thus be useful for the diagnosis and management of stroke.

Caspase 3/ROCK1 pathway mediates high glucose-induced platelet microparticles shedding.

BACKGROUND: Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. This study aimed to explore the underlying mechanisms of high glucose-induced PMPs generation. METHODS: Washed platelets, obtained from the plasma of healthy male Sprague-Dawley rats, were incubated with high glucose. PMPs were isolated using gradient centrifugation and counted by flow cytometry. Expression and activity of ROCK1 and caspase3 were evaluated by real-time PCR, Western blotting, and activity assay kit. RESULTS: High glucose enhanced PMPs shedding in the presence of collagen. The mRNA and protein levels of ROCK1, but not ROCK2, were increased in platelets incubated with high glucose. Y-27632, an inhibitor of ROCK, blocked the increased PMPs shedding induced by high glucose. Expression and activity of caspase3 were elevated in platelets under the high glucose conditions. Z-DVED-FMK, a caspase3 inhibitor, inhibited ROCK1 activity and decreased the PMPs generation under high glucose. CONCLUSION: High glucose increased PMPs shedding via caspase3-ROCK1 signal pathway.

Platelet microparticles contribute to aortic vascular endothelial injury in diabetes via the mTORC1 pathway.

Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. The present study aimed to investigate the effects of PMPs in diabetes on aortic vascular endothelial injury and to explore the underlying mechanisms. Peritoneal injection of streptozotocin was used to generate a diabetic rat model in vivo, and human umbilical vein endothelial cells (HUVECs) treated with PMPs were used in vitro. PMP levels in the circulation and aorta tissues were time-dependently increased in streptozotocin-induced diabetic rats at weeks 4, 8, and 12 (P < 0.05). Aspirin significantly inhibited the PMP levels at each time point (P < 0.05). In diabetic rats, the endothelial nitric oxide levels were decreased significantly combined with increased endothelial permeability. PMPs were internalized by HUVECs and primarily accumulated around the nuclei. PMPs inhibited endothelial nitric oxide levels to about 50% and caused approximately twofold increase in reactive oxygen species production. Furthermore, PMPs significantly decreased the endothelial glycocalyx area and expression levels of glypican-1 and occludin (P < 0.05). Interestingly, the PMP-induced endothelial injuries were prevented by raptor siRNA and rapamycin. In conclusion, increased PMPs levels contribute to aortic vascular endothelial injuries in diabetes through activating the mTORC1 pathway.

Platelet Microparticles Mediate Glomerular Endothelial Injury in Early Diabetic Nephropathy.

BACKGROUND: Glomerular endothelium dysfunction, which plays a crucial role in the pathogenesis of early diabetic nephropathy, might be caused by circulating metabolic abnormalities. Platelet microparticles, extracellular vesicles released from activated platelets, have recently emerged as a novel regulator of vascular dysfunction. METHODS: We studied the effects of platelet microparticles on glomerular endothelial injury in early diabetic nephropathy in rats with streptozotocin-induced diabetes and primary rat glomerular endothelial cells. Isolated platelet microparticles were measured by flow cytometry. RESULTS: Plasma platelet microparticles were significantly increased in diabetic rats, an effect inhibited in aspirin-treated animals. In cultured glomerular endothelial cells, platelet microparticles induced production of reactive oxygen species, decreased nitric oxide levels, inhibited activities of endothelial nitric oxide synthase and SOD, increased permeability of the glomerular endothelium barrier, and reduced thickness of the endothelial surface layer. Conversely, inhibition of platelet microparticles in vivo by aspirin improved glomerular endothelial injury. Further analysis showed that platelet microparticles activated the mammalian target of rapamycin complex 1 (mTORC1) pathway in glomerular endothelial cells; inhibition of the mTORC1 pathway by rapamycin or raptor siRNA significantly protected against microparticle-induced glomerular endothelial injury in vivo and in vitro. Moreover, platelet microparticle-derived chemokine ligand 7 (CXCL7) contributed to glomerular endothelial injury, and antagonizing CXCL7 using CXCL7-neutralizing antibody or blocking CXCL7 receptors with a competitive inhibitor of CXCR1 and CXCR2 dramatically attenuated such injury. CONCLUSIONS: These findings demonstrate a pathogenic role of platelet microparticles in glomerular endothelium dysfunction, and suggest a potential therapeutic target, CXCL7, for treatment of early diabetic nephropathy.

Polyphenols: Modulators of Platelet Function and Platelet Microparticle Generation?

Platelets and platelet microparticles (PMPs) play a key role in the pathophysiology of vascular disorders such as coronary artery disease and stroke. In atherosclerosis, for example, the disruption of the plaque exposes endogenous agonists such as collagen, which activates platelets. Platelet hyper-activation and the high levels of PMPs generated in such situations pose a thrombotic risk that can lead to strokes or myocardial infarctions. Interestingly, dietary polyphenols are gaining much attention due to their potential to mimic the antiplatelet activity of treatment drugs such as aspirin and clopidogrel that target the glycoprotein VI (GPVI)-collagen and cyclooxygenease-1 (COX-1)-thromboxane platelet activation pathways respectively. Platelet function tests such as aggregometry and flow cytometry used to monitor the efficacy of antiplatelet drugs can also be used to assess the antiplatelet potential of dietary polyphenols. Despite the low bioavailability of polyphenols, several in vitro and dietary intervention studies have reported antiplatelet effects of polyphenols. This review presents a summary of platelet function in terms of aggregation, secretion, activation marker expression, and PMP release. Furthermore, the review will critically evaluate studies demonstrating the impact of polyphenols on aggregation and PMP release.

A Case of Multiple Cerebral Infarction Preceding Acute Exacerbation of Idiopathic Thrombocytopenic Purpura.

BACKGROUND: Although it was suggested that idiopathic thromobocytopenic purpura (ITP) can be a paradoxical cause of cerebral infarction, previous reports indicate that cerebral infarction associated with ITP occurs when thrombocytopenia is already evident at the onset of cerebral infarction. CASE REPORT: We report a case of multiple cerebral infarction that preceded acute exacerbation of ITP. An 80-year-old woman with a history of ITP presented with tetraplegia, and brain magnetic resonance imaging revealed multiple infarction in bilateral cerebral and cerebellar hemispheres. For ITP, she was treated with oral prednisolone and subcutaneous injection of thrombopoietin receptor agonists. Her platelet count was within the normal range at the onset of cerebral infarction. Medical work-up did not reveal the obvious causes of her multiple cerebral infarction. On day 10 of hospitalization, she showed melena and oral hemorrhage and her platelet count markedly decreased. Her platelet-associated IgG level was elevated and a diagnosis of acute exacerbation of ITP was made. She was treated with intravenous immunoglobulin and her platelet count increased moderately. However, her neurological symptoms and cerebral infarction on magnetic resonance imaging deteriorated accompanied by hemorrhagic transformation. Finally, she died of respiratory failure. CONCLUSIONS: Our case suggests that thrombophilia accompanied by ITP can precede actual exacerbation of ITP and we have to consider ITP as a possible cause of multiple cerebral infarction, even when the platelet count is within the normal range at the onset of cerebral infarction.

Platelet microparticle delivered microRNA-Let-7a promotes the angiogenic switch.

Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3'UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

Interaction of Platelet-Derived Microparticles with a Human B-Lymphoblast Cell Line: A Clue for the Immunologic Function of the Microparticles.

BACKGROUND: Platelets are blood cells with extensive capabilities in hemostasis. They also play a central role in the development of innate and adaptive immune responses. Little information exists about the immunostimulatory role of platelet-derived microparticles (Plt-MPs). To further elucidate this issue, we conducted this study using the B-lymphoblast cell line 'Daudi' as an available surrogate cell line for peripheral blood B lymphocytes. This cell line does not produce immunoglobulins (Igs) and has low expression of activation markers. METHODS: Plt-MPs were isolated from platelet concentrate (PC) using a multi-step centrifugation method. Daudi cells were treated with Plt-MPs in the culture medium while no treatment was given to the control cells. During 5-day co-culture, Daudi cells were evaluated for the Ig production and the expression of the cell surface markers CD86, CD27, and IgD. RESULTS: An increase was observed for the production of IgG and the expression of CD27 and CD86 on Daudi cells in response to Plt-MPs, whereas the IgD level was decreased. The response of Daudi cells was dependent on the concentration of Plt-MPs and the time of their isolation from PCs during storage. The differences of the variables were significant between the treatment and control groups. CONCLUSION: Plt-MPs could induce the activation and differentiation of immortalized cells of B-cell origin. Thus it is conceivable that Plt-MPs may play a significant role as immortalized cell activators in human monoclonal antibody technology in near future.

Effects of transplanted circulating endothelial progenitor cells and platelet microparticles in atherosclerosis development.

BACKGROUND INFORMATION: Atherosclerosis is an inflammatory disease, in which risk factors such as hyperlipidemia and hypertension affect the arterial endothelium, resulting in dysfunction, cell damage or both. The number of circulating endothelial progenitor cells and microparticles provides invaluable outcome prediction for atherosclerosis disease. However, evidence for the therapeutic potential of endothelial progenitor cells and microparticles in atherosclerosis development is limited. Our study was designed to investigate the possible protective role of a cell therapy-based approach, using endothelial progenitor cells and the dual behaviour of circulating platelet microparticles, on atherosclerosis development in hypertensive-hypercholesterolemic hamster model. Consequently, control hamsters received four intravenous inoculations of: (1) 1×10(5) endothelial progenitor cells of healthy origins in one dose per month, during four months of diet-induced atherosclerosis, and after hypertensive-hypercholesterolemic diet for further four months; (2) in a second set of experiments, 1×10(5) endothelial progenitor cells of healthy origins or/and 1×10(5) platelet microparticles of atherosclerotic origins were inoculated every other month during hypertensive-hypercholesterolemic diet. RESULTS: Endothelial progenitor cell treatment had the following effects: (1) re-established plasmatic parameters: cholesterol and triglyceride concentrations, blood pressure, heart rate, cytokine and chemokine profiles, platelet microparticle pro-thrombotic activity and endothelial progenitor cell paracrine activity reflected by cytokine/chemokine detection; (2) reduced lipid, macrophage and microparticle accumulation in liver; (3) reduced atherosclerosis development, revealed by decreased lipid, macrophage and microparticle content of arterial wall; (4) induced the recruitment and incorporation of endothelial progenitor cells into liver and arterial wall; (5) improved arterial dysfunction by increasing contraction and relaxation; (6) reduced the protein expression of specific pro-inflammatory molecules in liver and arterial wall. Platelet microparticle transplantation aggravated the above-mentioned biomarkers and atherosclerosis process, which were partially reverted with co-inoculation of platelet microparticles and endothelial progenitor cells. CONCLUSIONS: With this study, we demonstrate in a hypertensive-hypercholesterolemic hamster model, that the endothelial progenitor cell-based therapy suppresses the development of atherosclerosis and reduces hepatic lipid and macrophage accumulation with the consequent alleviation of dyslipidaemia and hypertension. SIGNIFICANCE: Our results support the notion that increasing the number of circulating endothelial progenitor cells by different ways could be a promising therapeutic tool for atherosclerosis.

Platelet microvesicles in health and disease.

Interest in cell-derived extracellular vesicles and their physiological and pathological implications is constantly growing. Microvesicles, also known as microparticles, are small extracellular vesicles released by cells in response to activation or apoptosis. Among the different microvesicles present in the blood of healthy individuals, platelet-derived microvesicles (PMVs) are the most abundant. Their characterization has revealed a heterogeneous cargo that includes a set of adhesion molecules. Similarly to platelets, PMVs are also involved in thrombosis through support of the coagulation cascade. The levels of circulatory PMVs are altered during several disease manifestations such as coagulation disorders, rheumatoid arthritis, systemic lupus erythematosus, cancers, cardiovascular diseases, and infections, pointing to their potential contribution to disease and their development as a biomarker. This review highlights recent findings in the field of PMV research and addresses their contribution to both healthy and diseased states.

Microparticles in patients undergoing transcatheter aortic valve implantation (TAVI).

Degenerative aortic stenosis (AS) is the most frequent form of acquired valvular heart disease. AS is known to entail endothelial dysfunction caused by increased mechanical shear stress leading to elevated circulatory levels of microparticles. Endothelial and platelet microparticles (EMP and PMP) are small vesicles that originate from activated cells and thrombocytes. We sought to evaluate whether transcatheter aortic valve implantation (TAVI) procedure would elicit effects on circulating EMP and PMP. 92 patients undergoing TAVI procedure for severe AS were included in this study. Samples were obtained at each visit before TAVI, 1 week post-procedure and at 1, 3 and after 6 months after TAVI and were evaluated using flow cytometry. A 12 month clinical follow-up was also performed. CD62E+ EMP concentration before TAVI was 21.11 % (±6.6 % SD) and declined to 20.99 % (±6.8 % SD) after 1 week, to 16.63 % (±5.4 % SD, p < 0.0001) after 1 month, to 17.08 % (±4.6 % SD, p < 0.0001) after 3 months and to 15.94 % (±5.4 % SD, p < 0.0001) after 6 months. CD31+/CD42b-, CD31+/Annexin+/- EMP remained unchanged. CD31+/CD41b+ PMP evidenced a slight, but statistically significant increase after TAVI and remained elevated during the entire follow-up. Apart from a procedure-related improvement in echocardiographic parameters, TAVI procedure led also to a decline in CD62E+ EMP. The reduction in pressure gradients with less hemodynamic shear stress seems also to have beneficially affected endothelial homeostasis.

[Real-time investigation of dynamic morphology of live platelets and generation of platelet microparticles using hopping probe ion conductance microscopy].

Platelets are rapidly activated by activators and produce a large number of platelet microparticles (PMPs) with high coagulation activity, resulting in coagulation dysfunction. However, the generation mechanism of PMPs is still not clear. Hopping probe ion conductance microscopy (HPICM) has special technical advantages in non-contact, real-time, high-resolution imaging of living cells under physiological conditions. Using HPICM, this study monitored the processes of platelet activation and generation of PMPs in real time in the presence of calcium ionophore A23187 and cytochalasin D (CD), respectively. The results proved that the intracellular calcium concentration and the cytoskeletal proteins played important roles in the platelet activation and the generation of PMPs. Compared with the low density spread shape platelets (LDSS), the high density bubble shape platelets (HDBS) were more sensitive to the calcium ionophore A23187 and cytochalasin D. This research has a guiding significance for the further study on the relationship between platelet activation and coagulation function using HPICM.

The biology of extracellular vesicles with focus on platelet microparticles and their role in cancer development and progression.

Extracellular vesicles (EVs) are a heterogeneous group of structures which can be classified into smaller in size and relatively homogenous exosomes (EXSMs)-spherical fragments of lipid bilayers from inner cell compartments-and bigger in size ectosomes (ECSMs)-a direct consequence of cell-membrane blebbing. EVs can be found in body fluids of healthy individuals. Their number increases in cancer and other pathological conditions. EVs can originate from various cell types, including leukocytes, erythrocytes, thrombocytes, and neoplastic cells. Platelet microparticles (PMPs) are the most abundant population of EVs in blood. It is well documented that PMPs, being a crucial element of EVs signaling, are involved in tumor growth, metastasis, and angiogenesis and may participate in the development of multidrug resistance by tumor cells. The aim of this review is to present the role of PMPs in carcinogenesis. The biology and functions of PMPs with a particular emphasis on the most recent scientific reports on EV properties are also characterized.

Platelets are relevant mediators of renal injury induced by primary endothelial lesions.

Several studies have suggested a prominent (pro)inflammatory and harmful role of platelets in renal disease, and newer work has also demonstrated platelet release of proangiogenic factors. In the present study, we investigated the role of platelets in a mouse model of selective endothelial cell injury using either platelet depletion or the pharmacological P2Y12 receptor blocker clopidogrel as an interventional strategy. The concanavalin A/anti-concanavalin A model was induced in left kidneys of C57bl/6J wild-type mice after initial platelet depletion or platelet-inhibiting therapy using clopidogrel. FACS analysis of glycoprotein IIb/IIIa/P-selectin double-positive platelets and platelet-derived microparticles demonstrated relevant platelet activation after the induction of selective endothelial injury in mice. Enhanced platelet activation persisted for 5 days after disease induction and was accompanied by increased amounts of circulating platelet-derived microparticles as potential mediators of a prolonged procoagulant state. By immunohistochemistry, we detected significantly reduced glomerular injury in platelet-depleted mice compared with control mice. In parallel, we also saw reduced endothelial loss and a consequently reduced repair response as indicated by diminished proliferative activity. The P2Y12 receptor blocker clopidogrel demonstrated efficacy in limiting platelet activation and subsequent endothelial injury in this mouse model of renal microvascular injury. In conclusion, platelets are relevant mediators of renal injury induced by primary endothelial lesions early on, as demonstrated by platelet depletion as well as platelet inhibition via the P2Y12 receptor. While strategies to prevent platelet-endothelial interactions have shown protective effects, the contribution of platelets during renal regeneration remains unknown.

Platelet-derived microparticles trigger THP-1 monocytic cell aggregation and release of pro-coagulant tissue factor-expressing microparticles in vitro.

Microparticles (MPs) released by blood or endothelial cells are present in plasma for transfusion. They originate from the collected donor blood or are triggered by the variable steps taking place during collection and production/storage processes of blood components. While MPs may contribute to hemostasis, their presence in transfused plasma may lead to uncontrolled thrombin generation when transfused to susceptible cancer or hypercoagulable patients. Understanding the biochemical and cellular triggers of MP-mediated thrombogenesis is therefore crucial. We isolated platelet MPs (PMPs) present in platelet concentrate supernatant plasma (N-PMPs) or prepared by activation of isolated platelets using 0.1 IU/mL thrombin (T-PMPs). N-PMPs and T-PMPs were characterized by dynamic light scattering and counted by tunable resistive pulse sensing to determine population size and number. T-MPMs, but not N-PMPs, induced immediate, long-lasting, strong aggregation of THP-1 monocytic cells in vitro. In addition, co-cultures of THP-1 cells with both N-PMPs and T-PMPs triggered the generation of pro-coagulant tissue factor (TF)-bearing MPs from THP-1 cells. Therefore, some PMPs may induce THP-1 monocytic cell aggregation in vitro and trigger immune cell-mediated thrombogenicity linked to the release of pro-coagulant tissue factor-bearing MPs. Controlling the impact of the presence of PMPs in transfused blood components in certain patient population or critically ill patients deserves in-depth consideration.

The Role of Circulating Platelets Microparticles and Platelet Parameters in Acute Ischemic Stroke Patients.

BACKGROUND: Platelet activation and aggregation are critical in the pathogenesis of acute ischemic stroke (AIS). Circulating platelet microparticles (PMPs) and platelet parameters are biologic markers of platelet function in AIS patients; however, their associations with stroke subtypes and infarct volume remain unknown. METHODS: We recruited 112 AIS patients including large-artery atherosclerosis (LAA) and small-artery occlusion [SAO] subtypes and 35 controls in this study. Blood samples were collected at admission and after antiplatelet therapy. The levels of circulating PMPs and platelet parameters (mean platelet volume [MPV], platelet count, plateletocrit, and platelet distribution width) were determined by flow cytometry and hematology analysis, respectively. Infarct volume was examined at admission by magnetic resonance imaging. RESULTS: (1) The levels of circulating PMPs and MPV were significantly elevated in AIS patients compared with healthy controls; (2) the level of circulating PMPs, but not platelet parameters, was decreased after antiplatelet therapy in AIS patients; (3) the infarct volume in LAA subtype was larger than that in SAO subtype. Notably, circulating PMP level was positively correlated with the infarct volume in LAA subtype. No association with infarct volume in either AIS subtype was observed for platelet parameters; and (4) according to the regression analysis, circulating PMP was an independent risk factor for the infarct volume in pooled AIS patients after adjustments of other impact factors (hypertension and diabetes). CONCLUSIONS: Our results suggest that circulating PMP level is associated with cerebral injury of AIS, which offers a novel evaluation parameter for AIS patients.