An extracellular vesicle epitope profile is associated with acute myocardial infarction.

The current standard biomarker for myocardial infarction (MI) is high-sensitive troponin. Although powerful in clinical setting, search for new markers is warranted as early diagnosis of MI is associated with improved outcomes. Extracellular vesicles (EVs) attracted considerable interest as new blood biomarkers. A training cohort used for diagnostic modelling included 30 patients with STEMI, 38 with stable angina (SA) and 30 matched-controls. Extracellular vesicle concentration was assessed by nanoparticle tracking analysis. Extracellular vesicle surface-epitopes were measured by flow cytometry. Diagnostic models were developed using machine learning algorithms and validated on an independent cohort of 80 patients. Serum EV concentration from STEMI patients was increased as compared to controls and SA. EV levels of CD62P, CD42a, CD41b, CD31 and CD40 increased in STEMI, and to a lesser extent in SA patients. An aggregate marker including EV concentration and CD62P/CD42a levels achieved non-inferiority to troponin, discriminating STEMI from controls (AUC = 0.969). A random forest model based on EV biomarkers discriminated the two groups with 100% accuracy. EV markers and RF model confirmed high diagnostic performance at validation. In conclusion, patients with acute MI or SA exhibit characteristic EV biomarker profiles. EV biomarkers hold great potential as early markers for the management of patients with MI.

L-Selectin P213S and Integrin Alpha 2 C807T Genetic Polymorphisms in Pediatric Sickle Cell Disease Patients.

Sickle cell disease (SCD) is an autosomal recessive hemoglobinopathy characterized by increased cellular adhesiveness. Vaso-occlusion (VOC) is the most prevalent disease complication of SCD that could be altered by genetic factors. L-Selectin and integrin alpha 2 (ITGA2) are 2 adhesion molecules linked to vasculopathy and inflammation. The current study aimed at detecting the prevalence of genetic variants of L-selectin and ITGA2 as possible molecular modulators and novel therapeutic targets in a cohort of pediatric SCD patients. Genotyping was performed by polymerase chain reaction restriction fragment length polymorphism technique for 100 SCD patients and 100 age and gender-matched unrelated healthy controls. The homomutant genotype of ITGA2 C807T was significantly higher in SCD patients compared with controls (P=0.001) and confirmed almost a 3-fold increased risk of moderate and severe attacks of VOC. There are significant adverse effects caused by the polymorphisms of ITGA2, and hence Egyptian SCD patients could benefit from the targeted therapies specifically against ITGA2 to ameliorate the severe course of the disease and improve the quality of life. However, further studies of genotypes and expression levels of these adhesion molecules during the attacks of VOC are recommended.

Role of Platelet Size Revisited-Function and Protein Composition of Large and Small Platelets.

Epidemiological studies found an association between increased platelet size and the risk for thrombotic complications, but functional differences of large and small platelets remain poorly understood due to a lack of standardized protocols separating platelets with different size. We designed a protocol to separate large and small platelets from 15 mL whole blood. Separated large and small platelet fractions differed in mean platelet volume: 12.1 fl (10.3-13.8 fl) versus 7.7 fl (6.8-9.5 fl, p < 0.01), and forward scatter mean fluorescence intensity: 24.75 (19.9-30.9) versus 16.85 (11.3-20.6; p < 0.01). Similar fold differences were observed in cell diameter and plateletcrit. Large platelets express 30 to 50% more glycoprotein (GP) Ia, GPIb, GPIIIa, GPVI and P2Y12 on their membranes compared with small ones. Single large platelets covered a 50% larger area on a collagen surface. Adhesion to collagen was faster in large platelets compared with small ones indicating enhanced outside-in signal transduction in large platelets via collagen receptors. In contrast, integrin activation was more pronounced in small platelets after ADP stimulation. Proteome analysis revealed that 80 of the 894 proteins quantified differed in abundance: ADP-ribosylation factor 1/3, guanosine triphosphate-binding protein SAR1a, Voltage-dependent anion-selective channel protein 3 and guanylate cyclase soluble sub-unit α-3 were higher abundant in large, whereas immunoglobulins, haptoglobin, hemopexin, α-1-antitrypsin, serotransferrin and vitronectin were more abundant in small platelets. We conclude that some functions and the protein composition of large and small platelets differ, which cannot only be explained by the size difference. Our data suggest different functional roles of large and small platelets.

A unique phenotype of acquired Glanzmann thrombasthenia due to non-function-blocking anti-αIIbß3 autoantibodies.

Essentials Acquired Glanzmann thrombasthenia (aGT) is generally caused by function-blocking antibodies (Abs). We demonstrated a unique aGT case due to marked reduction of αIIbß3 with anti-αIIbß3 Abs. The anti-αIIbß3 Abs of the patient did not inhibit platelet function but reduced surface αIIbß3. Internalization of αIIbß3 induced by the Abs binding may be responsible for the phenotype. SUMMARY: Background Acquired Glanzmann thrombasthenia (aGT) is a bleeding disorder generally caused by function-blocking anti-αIIbß3 autoantibodies. Aim We characterize an unusual case of aGT caused by marked reduction of surface αIIbß3 with non-function-blocking anti-αIIbß3 antibodies (Abs). Methods A 72-year-old male suffering from immune thrombocytopenia since his 50s showed exacerbation of bleeding symptom despite mild thrombocytopenia. Platelet aggregation was absent with all agonists but ristocetin. Analysis of αIIbß3 expression and genetic analysis were performed. We also analyzed effects of anti-αIIbß3 Abs of the patient on platelet function and αIIbß3 expression. Results Surface αIIbß3 expression was markedly reduced to around 5% of normal, whereas his platelets contained αIIbß3 to the amount of 40-50% of normal. A substantial amount of fibrinogen was also detected in his platelets. There were no abnormalities in ITGA2B and ITGB3 cDNA. These results indicated that reduced surface αIIbß3 expression caused a GT phenotype, and active internalization of αIIbß3 was suggested. Anti-αIIbß3 IgG Abs were detected in platelet eluate and plasma. These Abs did not inhibit PAC-1 binding, indicating that the Abs were non-function-blocking. Surface αIIbß3 expression of a megakaryocytic cell line and cultured megakaryocytes tended to be impaired by incubation with the patient's Abs. After 2 years of aGT diagnosis, his bleeding symptom improved and surface αIIbß3 expression was recovered to 20% of normal with reduction of anti-αIIbß3 Abs. Conclusion We demonstrated a unique aGT phenotype due to marked reduction of surface αIIbß3. Internalization induced by anti-αIIbß3 Abs may be responsible in part for the phenotype.

Associations of combined polymorphisms of the platelet membrane glycoproteins Ia and IIIa and the platelet-endothelial cell adhesion molecule-1 and P-Selectin genes with IVF implantation failures.

The aim of the study was to investigate the combined impact of the genetic heterogeneity of the glycoproteins Ia (GpIa) and IIIa (GpIIIa) and the platelet-endothelial cell adhesion molecule-1 (PECAM-1) and P-Selectin genes on IVF embryo transfer implantation failures (IVF-ET failures). Sixty nulligravida women with previous IVF-ET failures and 60 fertile controls were genotyped for the GpIa-C807T, GpIIIa-PlA1/PA2, PECAM-1-C373G (Leu125Val) and P-Selectin-A37674C (Thr715Pro) polymorphisms by pyrosequencing. Compared with wild-type combined homozygotes, carriers of combinations of risk alleles in two gene loci were at significantly increased risk for IVF-ET failure, whereas carriers of the combination of GpIa-807T, GpIIIa-PlA2 and PECAM-1-373G alleles had OR = 52.50 (95%CI: 4.05-680.95, p < .001). The area under the receiver-operating characteristic curve (AUC) based on the number of polymorphisms and the number of risk alleles per subject was 75.4% (95%CI: 66.7%-82.8%, p < .001) and 72.5% (95%CI: 63.6%-80.3%, p < .001), respectively. The OR per polymorphism and risk allele increase was 4.26 (95%CI: 2.15-8.41, p < .001) and 2.85 (95%CI: 1.71-4.76, p < .001), respectively. The above associations were more robust among younger women. The combined analysis of these polymorphisms revealed strong association of combined carriers with IVF-ET failures especially for younger women and provided a genetic risk score with good diagnostic accuracy in the prediction of IVF-ET failures.

Gain-of-Function Mutation in Filamin A Potentiates Platelet Integrin αIIbß3 Activation.

OBJECTIVE: Dominant mutations of the X-linked filamin A (FLNA) gene are responsible for filaminopathies A, which are rare disorders including brain periventricular nodular heterotopia, congenital intestinal pseudo-obstruction, cardiac valves or skeleton malformations, and often macrothrombocytopenia. APPROACH AND RESULTS: We studied a male patient with periventricular nodular heterotopia and congenital intestinal pseudo-obstruction, his unique X-linked FLNA allele carrying a stop codon mutation resulting in a 100-amino acid-long FLNa C-terminal extension (NP_001447.2: p.Ter2648SerextTer101). Platelet counts were normal, with few enlarged platelets. FLNa was detectable in all platelets but at 30% of control levels. Surprisingly, all platelet functions were significantly upregulated, including platelet aggregation and secretion, as induced by ADP, collagen, or von Willebrand factor in the presence of ristocetin, as well as thrombus formation in blood flow on a collagen or on a von Willebrand factor matrix. Most importantly, patient platelets stimulated with ADP exhibited a marked increase in αIIbß3 integrin activation and a parallel increase in talin recruitment to ß3, contrasting with normal Rap1 activation. These results are consistent with the mutant FLNa affecting the last step of αIIbß3 activation. Overexpression of mutant FLNa in the HEL megakaryocytic cell line correlated with an increase (compared with wild-type FLNa) in PMA-induced fibrinogen binding to and in talin and kindlin-3 recruitment by αIIbß3. CONCLUSIONS: Altogether, our results are consistent with a less binding of mutant FLNa to ß3 and the facilitated recruitment of talin by ß3 on platelet stimulation, explaining the increased αIIbß3 activation and the ensuing gain-of-platelet functions.

Cardiac rehabilitation programme as a non-pharmacological platelet inhibitory tool in acute coronary syndrome survivors.

Background Acute coronary syndrome is associated with platelet hyperactivity, which in its persistent form, promotes recurrent thrombotic events. Complex cardiac rehabilitation after acute coronary syndrome improves clinical outcome; however, its effect on platelet hyperactivity is unknown. Design and methods We enrolled 84 acute coronary syndrome patients on dual antiplatelet therapy, who underwent a new complex cardiac rehabilitation programme (NovaCord physiotherapy, lifestyle counselling, strict diet, stress management and regular coaching) and 51 control acute coronary syndrome patients with traditional cardiac rehabilitation. Platelet functionality was determined at enrolment and at three months follow-up by aggregometry, serum platelet-derived growth factor levels, total- and platelet-derived microvesicle counts (PMV; CD41a+/CD61+, CD62P+). Results Platelet aggregation parameters and platelet-derived growth factor levels were significantly decreased in the complex cardiac rehabilitation group at three months (1 µg/ml collagen, median (interquartile range): 22 (10-45) vs 14 (7.5-25.5)%, p = 0.0015; 2 µg/ml collagen: 36 (22-60) vs 26.5 (16-37)%, p = 0.0019; 1.25 µM adenosine-diphosphate: 4.5 (1-10) vs 1 (0-3)%, p = 0.0006; 5 µM adenosine-diphosphate: 27 (16-38) vs 22 (12-31)%, p = 0.0078; epinephrine: 33 (15-57) vs 27 (12-43)%, p = 0.01; platelet-derived growth factor: 434.6 (256.0-622.7) vs 224.8 (148.5-374.1) pg/ml, p = 0.0001). In contrast, these changes were absent or did not reach statistical significance in the traditional cardiac rehabilitation group. Platelet-derived microvesicle counts were significantly decreased in both groups, while total microvesicle count was significantly reduced only in the complex cardiac rehabilitation group (median (interquartile range): 3945.5 (2138-5661) vs 1739 (780-2303) count/µl; p = 0.0001). Conclusions Platelet hyperactivity three months after acute coronary syndrome significantly decreased in patients undergoing complex cardiac rehabilitation. Besides dual antiplatelet therapy, effective management and comprehensive control of cardiovascular risk factors might represent a new, non-pharmacological approach to influence platelet functionality.

Oscillatory shear stress, flow-mediated dilatation, and circulating microparticles at sea level and high altitude.

BACKGROUND AND AIMS: Exposing the endothelium to acute periods of imposed oscillatory shear stress reduces endothelial function and elevates circulating microparticles (MPs). Oscillatory shear stress may be especially pathogenic when superimposed on hypoxia, an environmental stimulus that disrupts the endothelial milieu. We examined the effects of acute manipulation of oscillatory shear stress on endothelial function and circulating MPs at sea level (SL) and high altitude (HA). METHODS: Healthy adults (n = 12) participated, once at SL and once on the second or third day at HA (3800 m). Oscillatory shear stress was provoked using a 30-min distal cuff occlusion intervention (75 mmHg). Endothelial function was assessed before and immediately after the intervention in the brachial artery by reactive hyperaemia flow-mediated dilatation (FMD). Venous blood samples of MPs (flow cytometry) were obtained before and during the last five minutes of the shear intervention. RESULTS: At baseline, circulating MPs were two-fold higher at HA (p = 0.011) and brachial artery diameter was constricted (p = 0.015). Although the intervention at SL increased endothelial-derived MPs by 83 ± 39% (mean ± SEM; p = 0.021), FMD was unaltered. Conversely, at HA, the intervention elicited a 26 ± 11% reduction in FMD (p = 0.020); this reduction was inversely correlated with the change in total circulating MPs (r = -0.737, p = 0.006) and the change in endothelial-derived MPs (r = -0.614, p = 0.034). CONCLUSIONS: The vascular endothelium appears to be susceptible to periods of oscillatory shear stress at HA, where impairments in endothelium-dependent vasodilatation may be amplified by endothelial injury. These findings have important implications for understanding the early impact of clinical situations of hypoxaemia on the vascular endothelium.

Common Variant in Glycoprotein Ia Increases Long-Term Adverse Events Risk After Coronary Artery Bypass Graft Surgery.

BACKGROUND: This study was aimed to investigate the clinical relevance between glycoprotein Ia (GPIA) rs1126643C/T polymorphism and the outcome of coronary artery disease after coronary artery bypass graft (CABG) surgery and explore the involved potential mechanisms. METHODS AND RESULTS: We genotyped GPIA rs1126643 polymorphism of 1592 patients who underwent CABG and followed up for a median period of 72.8 months. Patients who are GPIA rs1126643 T-allele carriers have a higher major adverse cardiac or cerebrovascular events risk post-CABG than those who are CC homozygotes (hazard ratio [HR]=1.29; P=0.022). The clinical association between the risk allele (T) carriage and major adverse cardiac or cerebrovascular events was confirmed in another cohort study, which included 646 CABG patients from various health centers across China. Meanwhile, rs1126643 T allele was also linked with increased risk of major adverse cardiac or cerebrovascular events (HR=1.73; P=0.019). To explore the underlying mechanisms, we prospectively recruited 131 coronary artery disease patients, assessed their platelet aggregation function, and focused on detecting their GPIA mRNA level and protein expression. Results showed that patients with rs1126643 T allele have elevated platelet aggregation activity (P=0.029) when protein expression is increased (P<0.001) and not affected by glycoprotein Ia mRNA level. CONCLUSIONS: The synonymous common variant, GPIA rs1126643, increases the long-term adverse events risk of CABG by augmenting GPIa protein expression and enhancing platelet aggregation function. This finding can serve as the implication of improving secondary prevention of CABG patients.

Vesicular and Extra-Vesicular RNAs of Human Blood Plasma.

Human blood contains a great variety of membrane-covered RNA carrying vesicles which are spherical or tubular particles enclosed by a phospholipid bilayer. Circulating vesicles are thought to mediate cell-to-cell communication and their RNA cargo can act as regulatory molecules. In this work, we separated blood plasma of healthy donors by centrifugation and determined that vesicles precipitated at 16,000 g were enriched with CD41a, marker of platelets. At 160,000 g, the pellets were enriched with CD3 marker of T cells. To characterize the RNA-content of the blood plasma sub fractions, we performed high throughput sequencing of the RNA pelleted within vesicles at 16,000 g and 160,000 g as well as RNA remaining in the vesicle-free supernatant. We found that blood plasma sub fractions contain not only extensive set of microRNAs but also fragments of other cellular RNAs: rRNAs, tRNAs, mRNAs, lncRNAs, small RNAs including RNAs encoded by mtDNAs. Our data indicate that a variety of blood plasma RNAs circulating within vesicles as well as of extra-vesicular RNAs are comparable to the variety of cellular RNA species.

[Decreased expression levels of LAG-3 and CD49b on CD14+ cells in peripheral blood of patients with recurrent spontaneous abortion].

OBJECTIVE: To investigate the expression levels of adhesion molecule CD49b and negative regulation molecule LAG-3 (CD223) on peripheral blood CD14(+) cells in patients with recurrent spontaneous abortion (RSA). METHODS: Peripheral blood samples were collected from 7 normal female individuals and 12 patients with RSA, and then peripheral blood mononuclear cells (PBMCs) and plasma were separated from the peripheral blood via centrifugation. The expression levels of CD49b and LAG-3 on CD14(+) cells in PBMCs were detected by flow cytometry and the levels of interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß) in plasma were detected by ELISA. RESULTS: In the RSA patient group, there was no significant difference in the percentage of monocytes compared with that of the normal female group. The numbers of CD14(+) CD49b(+), CD14(+) LAG-3(+) and CD14(+) CD49b(+) LAG-3(+) cells in the RSA patient group were lower than those of the normal female group. The plasma level of TGF-ß in the RSA patient group was lower than that of the normal female group. However, there was no significant difference in the plasma level of IL-10 between the two groups. CONCLUSION: In RSA patients, the expression levels of CD49b and LAG-3 on CD14(+) monocytes and the plasma level of TGF-ß decreased obviously compared with that of the normal females.

CEACAM1 regulates integrin αIIbß3-mediated functions in platelets.

Previous studies have implicated that the Ig-ITIM superfamily member, CEACAM1 may regulate integrin function. While CEACAM1 has been demonstrated to play a role as an inhibitory co-receptor of ITAM-associated GPVI/FcR γ-chain signaling pathways in platelets, its physiologic role in integrin αIIbß3-mediated platelet function is unclear. In this study, we investigate the functional importance of Ceacam1 in murine platelets. We show that CEACAM1 is constitutively associated with integrin αIIbß3 in resting human and mouse platelets as demonstrated by co-immunoprecipitation studies. Using Ceacam1-deficient mice, we show that they have prolonged tail bleeding times and volume of blood lost that is corrected by reconstitution with platelet Ceacam1. Ceacam1(-/-) platelets have moderate integrin αIIbß3 mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and failure to retract fibrin clots in vitro. This functional integrin αIIbß3 defect could not be attributed to altered integrin αIIbß3 expression. Ceacam1(-/-) platelets displayed normal "inside-out" signaling properties as demonstrated by normal agonist-induced binding of soluble (fluorescein isothiocyanate) FITC-fibrinogen, JON/A antibody binding, and increases in cytosolic free calcium levels. This study provides direct evidence that Ceacam1 is essential for normal integrin αIIbß3-mediated platelet function and that disruption of mouse Ceacam1 induced moderate integrin αIIbß3-mediated functional defects.

CD49b, a major marker of regulatory T-cells type 1, predicts the response to antiviral therapy of recurrent hepatitis C after liver transplantation.

The TRANSPEG study was a prospective study to assess the efficacy of antiviral therapy in patients with a recurrent hepatitis C virus (HCV) after liver transplantation. The influence of regulatory T-cells (Tregs) on the response to antiviral therapy was analyzed. Patients were considered as a function of their sustained virological response (SVR) at 18 months after treatment initiation. A transcriptomic analysis was performed to assess Treg markers (Tr1 and FoxP3(+)) in serum, PBMC, and liver biopsies. 100 patients had been included in the TRANSPEG study. Data from 27 of these patients were available. The results showed that the expression of CD49b (a predominant marker of Tr1) before the introduction of antiviral therapy was significantly associated with SVR. Responders displayed lower serum levels of CD49b than nonresponders (P < 0.02). These findings were confirmed in PBMC and liver biopsies even if in a nonsignificant manner for the limited number of samples. The assessment of CD49b levels is thus predictive of the response to antiviral therapy. This data suggests that CD49b may be a marker of the failure of the immune response and antiviral therapy during HCV recurrence. The assessment of CD49b could help to select patients who require earlier and more intensive antiviral therapy.

[Modern methods of diagnosis of thrombophylic states and complex treatment of patients with thrombotic complications of severe forms of varicose disease].

Actual issues of surgical treatment of patients, suffering complications of severe forms of varicose disease of the lower extremities (VDLE) are discussed. The causes of unsatisfactory results of treatment in patients, suffering varicothrombophlebitis (VTHPH), the main of which--absence of the only one tactics for operative treatment and anticoagulant therapy, were analyzed. The results of patients examination, suffering thrombotic complications of severe forms of VDLE, while its recurrent course, in conjunction of VTHPH and thrombosis of deep veins of the lower extremities, using diagnostic complex "PLR genetics thrombophilia", are adduced. Differential tactics of treatment in patients, suffering severe forms of VDLE, while various localization of thrombotic process, concerning the presence of thrombophilic states, is proposed.

[Thrombotic complications of severe forms of varicose disease: modern approach to diagnosis and treatment of hereditary thrombophilia and immunohistochemical peculiarities of vascular wall].

Experience of treatment of 176 patients, suffering thrombotic complications of severe forms of the lower extremities varicose disease (VDLE), was analyzed. In 20 patients, suffering varicothrombophlebitis (VTHPH) in severe forms of VDLE, morphological and immunohistochemical changes in the venous wall and surrounding tissues were studied. There were examined 28 patients, in whom thrombotic complications of the VDLE have had occurred, using diagnostic complex "PLR genetics thrombophilia". Recurrent course of thrombotic complications and coexistence of VTHPH and thrombosis of deep veins have had constitute the main criterion of such patients selection. The groups of patients, suffering severe forms of VDLE, were delineated, depending on thrombotic process localization, differentiated tactics of their surgical treatment was proposed.

Sensitivity of assays for the detection of HPA-1a antibodies: results of an international workshop demonstrating the impact of cation chelation from integrin αIIbß3 on three widely used assays.

BACKGROUND AND OBJECTIVES: HPA-1a antibodies account for 70-80% of cases of fetal-neonatal alloimmune thrombocytopenia (FNAIT) in Caucasians. However, numerous workshops have demonstrated variability in their detection. We recently showed that exposure of αIIbß3 to ethylene diamine tetraacetic acid (EDTA) affected binding of many anti-αIIbß3 monoclonal, and HPA-1a allo-, antibodies; this adversely affected sensitivity of the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay and indirect platelet immunofluorescence test (PIFT). This study presents results from an international workshop studying the impact of cation chelation on HPA-1a antibody detection in routine diagnostic laboratories. MATERIALS AND METHODS: Serum and EDTA-anticoagulated plasma samples containing anti-HPA-1a were distributed to 39 laboratories. Participants were asked to detect and identify any HPA antibodies present. RESULTS: 2/39 (5.1%) participants were able to detect and identify anti-HPA-1a in the serum, but not in the plasma sample. EDTA plasma reduced MAIPA assay sensitivity by ≥ 20% in 17/24 (70.8%) laboratories and by ≥ 50% in 9/24 (37.5%) when using HPA-1a1a platelets (mean: 27.7%, range 0-85.1%); when using HPA-1a1b platelets 3/4 (75%), participants reported ≥ 50% loss of sensitivity (mean 65.6%, range 0-96.6%). A small but significant increase in optical densities was observed in antigen capture ELISA assays when using plasma (mean difference: 0.081, P < 0.01). Insufficient PIFT data were returned to draw firm conclusions. CONCLUSION: Use of EDTA plasma significantly affects the sensitivity of the MAIPA assay and can affect detection of even potent, FNAIT-causing examples of anti-HPA-1a. These data highlight the importance of use of αIIbß3 in an appropriate conformation for the sensitive detection of anti-HPA-1a.

Nanoparticles induce platelet activation in vitro through stimulation of canonical signalling pathways.

Nanomaterials are attracting growing interest for their potential use in several applications as nanomedicine; therefore, the analysis of their potential toxic effects on various cellular models, including circulating blood cells, is mandatory. This study aimed to investigate the effect of three unrelated nanomaterials, namely nanoscale silica, multiwalled carbon nanotubes, and carbon black, on platelet activation and aggregation. We found that these nanomaterials stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in the integrin α(IIb)ß3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2. Importantly, we found that doses of nanoparticles unable to trigger appreciable responses can synergize with subthreshold amounts of physiological agonists to mediate platelet aggregation, indicating that even small amounts of nanomaterials in the bloodstream might contribute to the development of thrombosis. FROM THE CLINICAL EDITOR: In this study, nanosized particles of three virtually unrelated materials (silica, multi-walled carbon nanotubes and carbon black) were investigated regarding their effects on platelet activation and aggregation. All were found to stimulate some of the typical biochemical pathways involved in canonical platelet activation, and were found to have synergistic effects with physiologic platelet activator agonists.

Platelet reactivity in diabetic patients subjected to acute exercise stress test.

BACKGROUND: Previous studies have reported ambiguous results regarding the effect of acute exercise on platelet reactivity in healthy and cardiac patients. OBJECTIVES: We aimed to assess platelet reactivity among diabetic patients before and immediately after an acute exercise stress test. METHODS: Patients (controls: mean age 53.1 ± 12.1 years; four males; body mass index 27.0 ± 5.7 kg/m(2); HbA(1c) 6.0 ± 1.1%, n = 8) and diabetic patients (52.9 ± 11.3; six males; body mass index 30.7 ± 2.2 kg/m(2); HbA(1c) 7.8 ± 1.7%, n = 8) referred for diagnostic nuclear exercise stress test were recruited. Blood samples obtained at rest and immediately post-exercise were stimulated with adenosine diphosphate (ADP), collagen and arachidonic acid. Expression of CD41 (pan-platelet marker) and CD62p (platelet stimulation marker) were measured by flow cytometry. Aspirin responsiveness was measured using VerifyNow. RESULTS: Although peak systolic blood pressure was significantly higher in the diabetics compared with nondiabetics (186.3 ± 25.4 vs. 157.1 ± 19.1, respectively, P = .028), peak exercise heart rate was similar (156.5 ± 8.3 vs. 155.5 ± 12.1 for diabetics and nondiabetics, respectively). No differences were observed between groups for aspirin resistance. Platelet stimulation with ADP exhibited significantly lower CD62p-positive cell population (%) in the diabetic patients both prior to and following the exercise stress test (P = .03). In addition, although not significant, platelet stimulation was higher post-exercise in the diabetic patients (6.3 ± 4.7% vs. 12.0 ± 5.6%, for pre- and post-exercise, respectively, P = .2) with no difference in controls (9.2 ± 5.5% vs. 8.9 ± 5.9%). CONCLUSION: Platelet stimulation in diabetic patients is blunted and might be explained by the prolonged exposure of platelets to multiple diabetic risk factors.

Phase I study of E7820, an oral inhibitor of integrin alpha-2 expression with antiangiogenic properties, in patients with advanced malignancies.

PURPOSE: This phase I study was conducted to characterize the safety profile, pharmacokinetics, pharmacodynamics, dose-limiting toxicity (DLT), and the maximum-tolerated dose of E7820, a novel oral sulfonamide derivative with antiangiogenic properties, when administered to patients with advanced solid malignancies. PATIENTS AND METHODS: Patients received single daily doses of E7820 orally for 28 days in cycle 1, followed by a 7-day no-treatment period, after which time-uninterrupted daily dosing ensued. The starting dose of E7820 was 10 mg/d, which was increased to 20, 40, 70, 100, and 200 mg/d in cohorts of new patients. RESULTS: Thirty-seven patients [21 male; median age 65 (40-82] were enrolled. At 100 mg/d, 1 patient experienced a DLT consisting of grade 3 neutropenia, thrombocytopenia, and elevated liver enzymes. At the 200-mg dose level, 2 patients experienced grade 4 thrombocytopenia and neutropenia. No partial or complete responses were observed; 8 patients had stable disease (≥ 4 months), including 5 patients with protracted stable disease exceeding 6 months. Mean time to maximum plasma concentration values ranged from 1 to 12 hours, whereas mean terminal half-life values ranged from 5.6 to 8.6 hours. Flow cytometric analysis of platelet integrin α-2 expression showed a sustained greater than 50% decrease beyond day 28 in 3 of 4 patients at 200 mg, whereas moderate (<30%) decreases were observed at 70- and 100-mg dose levels. CONCLUSIONS: The recommended phase II dose of E7820 is 100 mg/d, based on a fasting schedule. E7820 downregulates integrin α-2 expression in surrogate tissues (platelets) and is associated with stable disease in a wide variety of heavily pretreated malignancies.