Platelet Surface Markers Research Articles

Platelet Desialylation As a Predictive Marker in Childhood Immune Thrombocytopenia (ITP)

Background and aim: Immune thrombocytopenia (ITP) is the most common bleeding condition in children. Its prognosis is mostly superior, however, severe refractory disease remains diagnostic and therapeutic challenge. Low platelet counts (<100× 109/L) are associated with increased platelet clearance by two parallel mechanisms: classical antibody-mediated pathway and a novel lectin-carbohydrate mediated pathway. The latter is based on platelet desialylation, where terminal sialic acids are cleaved from glycoconjugates, mainly glycoproteins (GPs), on the platelet surface. The loss of sialic acid enhances bond of the penultimate β-galactose to asialoglycoprotein receptors (ASGPRs, also called Ashwell-Morell receptors) on hepatocytes. Desialylated platelets are then captured and phagocytosed by ASGPR-expressing hepatocytes. Desialylation has been shown to be responsible for platelet destruction in many contexts, e.g., infection-related thrombocytopenia or clearance of senescent platelets. Loss of T-cell tolerance is another underlying mechanism in ITP; CD8+ regulatory T cells (Tregs) are able to inhibit overactive immune response and maintain immune homeostasis. Forkhead box P3 (FOXP3) and GATA3 are transcription factors crucial for development and proper function of Tregs limiting the Th2-type inflammatory response. Our aims were to distinguish contribution of the mentioned processes to ITP development and to characterize immune response in children with ITP during the course of disease (diagnosis, ongoing therapy, remission, refractory/persistent ITP). Patients and Methods: We examined 30 samples from 20 children with ITP (12 males, 8 females, age 3-17 years; 3 acute ITP, 17 chronic ITP) and 10 healthy controls (age 4-15). The degree of desialylation was determined by flow cytometry using FITC-labeled Ricinus communis agglutinin (RCA-I) specific for terminal galactose or N-acetylgalactosamine. Expression of platelet surface markers was given quantitatively as mean fluorescence intensity (MFI). Presence of platelet surface-bond antibodies (IgG, IgA and IgM) was examined by flow cytometry. Subpopulations of CD4+ and CD8+ T-cells were characterized based on intracellular expression of transcription factors T-bet (Th1 cells), GATA3 (Th2 cells), ROR gamma T (Th17 cells) and FOXP3 (for Tregs) using multicolor flow cytometry. Results: Patients with ITP showed significant increase in RCA-I reactivity in comparison with healthy controls (p<0.001). Patients with newly diagnosed ITP showed the most aberrant sialylation (i.e., maximum desialylation) of platelet surface proteins. A decrease in desialylation intensity was noticeable as soon as at three days after therapy initiation. Sialylation levels returned to normal after one month of successful treatment and were similar to healthy controls in children with ITP remission. Platelet surface-bond immunoglobulins were increased in 10 (50%) patients independently on their sialylation level. We observed significant changes in T-cell subpopulations in ITP: T lymphocytes producing T-bet were decreased within both CD4+ and CD8+ populations. Percentage of CD4+ cells expressing ROR gamma T was also reduced. Proportions of cells expressing FOXP3 and GATA3 were decreased within the CD8+ but not within the CD4+ population. Conclusion: Our results highlight the importance of Fc-independent hepatic platelet clearance in ITP. Interindividual differences in ITP pathophysiology are reflected by treatment response and may improve therapeutic management and prognostication. E.g., intravenous immunoglobulins or splenectomy will be ineffective in patients with prevalent Fc-independent mechanisms, and contrarily, possibilities for novel targeted treatment (neuraminidase inhibitors) arise. Better understanding of immune-mediated processes involved in ITP pathogenesis may reduce adverse effects of immunosuppressive therapy and considerably improve quality of life in patients with ITP. Supported by: MH CZ - DRO (FNOl, 00098892), Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and Ministry of Education, Youth and Sports OPVVV CEREBIT CZ.02.1.01/0.0/0.0/16_025/0007397. Disclosures No relevant conflicts of interest to declare.

Platelet surface marker analysis by mass cytometry

Mass cytometry is a next generation flow cytometry technology which analyzes cells one at a time (up to 1000/sec) using mass spectrometry to detect probes labeled with rare-earth metals. Rare-earth metals detected by mass spectrometry have extremely low backgrounds and can be identified with high resolution enabling the routine simultaneous detection of more than 45 probes on each cell without the need for complex compensation matrices. Here we describe a panel of 14 platelet-specific metal-conjugated antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbβ3) and methods for staining and analysis of platelets by mass cytometry. High dimensional clustering algorithms, which take into account the levels of all 14 markers detected by mass cytometry on each cell, allow identification of platelet subpopulations not previously appreciated. We previously reported that platelet heterogeneity identified by mass cytometry appears similar across healthy donors and consistent over time. High dimensional analysis revealed the presence of a platelet subpopulation with significantly higher levels of surface expression of activated GPIIb-IIIa and P-selectin suggesting this subpopulation may play a greater role in thrombus formation than other platelet subpopulations. Thus, analysis by mass cytometry of platelet heterogeneity and subpopulations may suggest distinct biological roles for different platelet subpopulations and may be useful in evaluating inherited or acquired platelet disorders and platelet function in health and disease.

Hemocompatibility study of surface-attached antibiofouling polymer monolayers

The interface between biomaterials and body fluid such as blood is a critical concern, because biomaterial-centered protein fouling and infection adversely affects the quality of life of many patients and cause major health care costs. Our research focused on polymer functionalization on surfaces to create antibiofouling interfaces (i.e. interfaces that repel the adhesion of proteins and microorganisms) in biomaterial-related applications, such as surgical instruments and blood-contacting biomedical devices. To this end, we have studied two hydrophilic polymers, namely poly(2-oxazoline) and poly(ethylene glycol), attached on metal oxide surfaces in brush configuration through a polyelectrolyte surface anchor, poly(L-lysine). Apart from antibiofouling properties, blood-contacting surfaces must also serve hemocompatibility. Here the hemocompatibility of antibiofouling monolayer on silicon oxide surfaces prepared from either poly(2-oxazoline) or poly(ethylene glycol) grafted on a main backbone poly(L-lysine), has been studied. The activation of C5a (complement system), TAT and kallikrein (coagulation cascade), PF4 and sP-selectin, (platelet activation) after incubation of the polymer-modified surfaces in whole blood was measured by means of ELISA kit. The results showed that in general, the contact between blood and polymer monolayer activated the complement system, but relatively did not activate the coagulation cascade and the platelet surface marker.

Comparison of biocompatibility in polysulfone dialysis membranes with different sterilization

Polysulfone (PSf) membrane has been widely used for hemodialysis. A few studies have been reported in which a method of sterilization may affect biocompatibility. In this study, the comparison was made between two PSf membranes in order to evaluate the effect of sterilization from the biocompatibility point of view. We investigated the biocompatibility of the following two dialyzers, that is, APS-11SA (Asahi Kasei medical Co., Tokyo, Japan), sterilized by gamma-ray irradiation, and RENAK PS-1.0 (Kawasumi laboratories, Tokyo, Japan), sterilized by autoclave. Heparin of 40 units/mL was put in a syringe, and test blood was collected from healthy volunteers. Then, the dialyzer and blood circuit were filled with the test blood. Subsequently, the blood was circulated by a roller pump at the rate of 200 mL/min. We measured the platelet counts, CD41 and CD42b platelet surface markers, beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) at 30, 120, and 240 minutes, respectively. The platelet counts at 30, 120, and 240 minutes decreased significantly from the initiation of blood circulation in both dialyzers. The average beta-TG and PF4 increased from 61.3 ± 22.1 and 17.0 ± 6.4 ng/mL to 680.7 ± 217.1 and 550.7 ± 116.7 ng/mL with APS-11SA and to 454.3 ± 85.6 and 402.0 ± 58.0 ng/mL with RENAK PS-1.0, respectively. The average expression of CD41 and CD42b in APS-11SA and RENAK PS-1.0 was similar. There are some reports that the gamma-ray irradiation changes the membrane structure of the PSf membrane, crosslinking the polyvinylpyrrolidone (PVP), a hydrophilic agent, on to the membrane. On the other hand, excess amount of PVP may have been eluted during the rinsing procedure in RENAK PS-1.0 because it was sterilized with autoclave. Because both these factors influenced on our results, APS-11SA and RENAK PS-1.0 dialyzers showed excellent blood compatibility.

Circulating H3Cit is elevated in a human model of endotoxemia and can be detected bound to microvesicles

Early diagnosis of sepsis is crucial since prompt interventions decrease mortality. Citrullinated histone H3 (H3Cit), released from neutrophil extracellular traps (NETs) upon binding of platelets to neutrophils following endotoxin stimulation, has recently been proposed a promising blood biomarker in sepsis. Moreover, microvesicles (MVs), which are released during cell activation and apoptosis and carry a variety of proteins from their parental cells, have also been shown to be elevated in sepsis. In a randomized and placebo-controlled human model of endotoxemia (lipopolysaccharide injection; LPS), we now report significant LPS-induced elevations of circulating H3Cit in 22 healthy individuals. We detected elevations of circulating H3Cit by enzyme-linked immunosorbent assay (ELISA), as well as bound to MVs quantified by flow cytometry. H3Cit-bearing MVs expressed neutrophil and/or platelet surface markers, indicating platelet-neutrophil interactions. In addition, in vitro experiments revealed that H3Cit can bind to phosphatidylserine exposed on platelet derived MVs. Taken together; our results demonstrate that NETs can be detected in peripheral blood during endotoxemia by two distinct H3Cit-specific methods. Furthermore, we propose a previously unrecognized mechanism by which H3Cit may be disseminated throughout the vasculature by the binding to MVs.

Comparison of biocompatibility in cellulose triacetate dialysis membranes with homogeneous and asymmetric structures

BackgroundHistorically, cellulose-based materials have been used for manufacturing hemodialysis membranes and remain to date as the commonly used materials. Though cellulose triacetate (CTA) membrane originally has a homogeneous structure, recently, CTA membrane with asymmetric structure (ATA®) was developed. By a new spinning technology, irregularities or roughness in the inner surface of ATA have become less than those of the conventional CTA membrane. In this study, a comparison was made between CTA and ATA® membranes in order to evaluate the effect of physical structure of the membrane on the biocompatibility.MethodsWe compared the biocompatibility of the following two dialyzers with different triacetate membrane: FB-190UHβ with CTA, and FA-190F with ATA® (both from Nipro, Osaka, Japan). Heparin of 40 units/mL was put in a syringe, and test blood was collected from healthy volunteers. Then, the dialyzer and blood circuit were filled with the test blood. Subsequently, the blood was circulated by a roller pump at the rate of 200 mL/min. We measured the platelet counts, platelet factor 4 (PF4), β-thromboglobulin (β-TG), and CD41 and CD42b platelet surface markers at 30, 120, and 240 min.ResultsThe average platelet counts at 30, 120, 240 min in CTA and ATA® were similar. The average β-TG and PF4 increased from 87.0 ± 49.0 ng/mL and 30.7 ± 17.3 ng/mL to 1198.5 ± 1017.2 ng/mL and 698.2 ± 574.6 ng/mL with CTA and to 774.3 ± 811.6 ng/mL and 509.0 ± 417.3 ng/mL with ATA®, respectively. The average expression of CD41 and CD42b decreased to 82.8 ± 9.7% and 80.1 ± 11.6% with CTA and 81.9 ± 14.3% and 80.2 ± 13.6% with ATA®, respectively.ConclusionsObserved dialyzers showed less platelet activation as commercial products, and differences between CTA and ATA® were not significant in terms of PF4, β-TG, CD41, and CD42b.

Targeted repair of heart injury by stem cells fused with platelet nanovesicles.

Stem cell transplantation, as used clinically, suffers from low retention and engraftment of the transplanted cells. Inspired by the ability of platelets to recruit stem cells to sites of injury on blood vessels, we hypothesized that platelets might enhance the vascular delivery of cardiac stem cells (CSCs) to sites of myocardial infarction injury. Here, we show that CSCs with platelet nanovesicles fused onto their surface membranes express platelet surface markers that are associated with platelet adhesion to injury sites. We also find that the modified CSCs selectively bind collagen-coated surfaces and endothelium-denuded rat aortas, and that in rat and porcine models of acute myocardial infarction the modified CSCs increase retention in the heart and reduce infarct size. Platelet-nanovesicle-fused CSCs thus possess the natural targeting and repairing ability of their parental cell types. This stem cell manipulation approach is fast, straightforward and safe, does not require genetic alteration of the cells, and should be generalizable to multiple cell types.

Mildly elevated unconjugated bilirubin is associated with reduced platelet activation-related thrombogenesis and inflammation in Gilbert’s syndrome

Gilbert’s syndrome (GS) is associated with a mild unconjugated hyperbilirubinemia, increased circulating antioxidant capacity, and reduced cardiovascular disease (CVD) risk. The current study investigated whether mildly elevated circulating unconjugated bilirubin (UCB) is negatively associated with multiple thrombotic risk factors including platelet activity, hemostatic function, and inflammation in individuals with GS. Blood samples were collected from matched GS and control subjects (14 per group). Activation-dependent platelet surface marker expression of PAC-1 (binds to GPIIb/IIIa surface receptors on activated platelets) and CD62P (marker for P-selectin released from activated degranulated platelets) was assessed in adenosine diphosphate (ADP)-stimulated platelets using flow cytometry. Exogenous agonists, ADP, collagen, and arachidonic acid (AA), were used to stimulate platelet aggregation. A statistically significant decrease in the expression of P-selectin (P = 0.030) on activated platelets was observed in GS subjects. Collagen and AA-induced platelet aggregation were significantly (P = 0.018; P = 0.032 for respective agonists) reduced in GS versus control group. Elevated UCB (P = 0.001) and high density lipoprotein (P = 0.033) in addition to reduced low density lipoprotein (P = 0.024) and high sensitive C-reactive protein (P = 0.043) were also observed in GS when compared to the control group. Reduced P-selectin expression suggests decreased platelet activation-dependent degranulation, while reduced platelet aggregation by AA and collagen indicates a quantitative decrease in platelet aggregation consequently targeting the cyclooxygenase-1 and GP VI pathways, respectively. These findings are the first to demonstrate that the activation of platelets is mildly inhibited in individuals with GS, an effect that might contribute to protection from platelet hyperactivation-induced thrombosis and thus cardiovascular mortality in individuals with benign hyperbilirubinemia.

Refrigerated storage of platelets initiates changes in platelet surface marker expression and localization of intracellular proteins

Platelets (PLTs) are currently stored at room temperature (22°C), which limits their shelf life, primarily due to the risk of bacterial growth. Alternatives to room temperature storage include PLT refrigeration (2-6°C), which inhibits bacterial growth, thus potentially allowing an extension of shelf life. Additionally, refrigerated PLTs appear more hemostatically active than conventional PLTs, which may be beneficial in certain clinical situations. However, the mechanisms responsible for this hemostatic function are not well characterized. The aim of this study was to assess the protein profile of refrigerated PLTs in an effort to understand these functional consequences. Buffy coat PLTs were pooled, split, and stored either at room temperature (20-24°C) or under refrigerated (2-6°C) conditions (n = 8 in each group). PLTs were assessed for changes in external receptor expression and actin filamentation using flow cytometry. Intracellular proteomic changes were assessed using two-dimensional gel electrophoresis and Western blotting. PLT refrigeration significantly reduced the abundance of glycoproteins (GPIb, GPIX, GPIIb, and GPIV) on the external membrane. However, refrigeration resulted in the increased expression of high-affinity integrins (αIIbβ3 and β1) and activation and apoptosis markers (CD62P, CD63, and phosphatidylserine). PLT refrigeration substantially altered the abundance and localization of several cytoskeletal proteins and resulted in an increase in actin filamentation, as measured by phalloidin staining. Refrigerated storage of PLTs induces significant changes in the expression and localization of both surface-expressed and intracellular proteins. Understanding these proteomic changes may help to identify the mechanisms resulting in the refrigeration-associated alterations in PLT function and clearance.

Treatment of platelet concentrates with ultraviolet C light for pathogen reduction increases cytokine accumulation.

Pathogen reduction technologies use photoactive substances in combination with ultraviolet (UV) light to inactivate pathogens. A new method uses only UVC light for pathogen reduction. This study assesses the effects of UVC light treatment on cytokine release in platelet (PLT) concentrates (PCs). A PC with 35% plasma and 65% PLT additive solution (SSP+) was prepared from five buffy coats. Three such PCs were pooled and divided into 3 units. One unit was used as a nonirradiated control, the second was a gamma-irradiated control, and the third unit was treated with UVC light technology. Ten units of each type were investigated. Cytokine release was analyzed on Days 1, 5, and 7 of storage. Correlation between cytokines, PLT surface markers, and hemostatic properties was investigated. Swirling was well preserved and pH was above the reference limit of 6.4 during storage of PLTs in all groups. Cytokine levels increased during storage in all groups but to a larger degree in PCs treated with UVC light. Only weak correlation was found between cytokines and PLT surface markers (r < 0.5). However, several cytokines showed strong correlation (r > 0.6) with the PLTs' ability to promote clot retraction. UVC treatment resulted in increased release from PLT alpha granules as evident by a higher cytokine release compared to nonirradiated and gamma-irradiated PCs. The clinical relevance of these findings needs to be further evaluated.

A Dominant Negative Mutation (p.P214L) in ETV6 is Associated with Megakaryocyte and Erythroid Transcript Misregulation

Background: Our group has recently described a family with autosomal dominant thrombocytopenia, high mean corpuscular volume (MCV) of red cells, and 2 occurrences of pre B-cell acute lymphoblastic leukemia (ALL) associated with a mutation in the transcription factor ETV6. ETV6 is a transcriptional repressor which functions through dimerization with itself or with other transcription factors such as FLI1, mutations in which cause a familial form of thrombocytopenia called Paris-Trousseau syndrome. The missense mutation found in this family (p.P214L) has a dominant negative effect by dimerizing with and sequestering WT ETV6 from the nucleus. This dominant negative mutation causes a phenotype in both platelets and red cells, suggesting a disruption in megakaryocyte-erythroid differentiation. Therefore, we aimed to study the effect of p.P214L ETV6 on megakaryocyte-erythroid differentiation by studying the platelet transcriptome, protein from platelet lysates, and platelet surface markers of patients with the ETV6 mutation. To investigate whether this platelet and red cell phenotype is due to interactions with FLI1, which is essential for megakaryocyte-erythroid lineage determination, we also aimed to study the interaction of p.P214L ETV6 with FLI1.Methods: RNA was isolated from leukoreduced platelets of 2 patients with the p.P214L mutation (affected), 3 unaffected family members, and 2 unrelated controls for whole transcriptome analysis. GPIX quantification from platelet lysates of 2 affected patients and 3 controls was done by Western blot. Analysis of protein dimerization was done by expression of differentially tagged ETV6 and FLI1 constructs (His or c-myc) in HEK293T cells, pull down with anti-c-myc beads, and quantification of both c-myc and His by Western blot. To determine platelet receptor levels, flow cytometry was done with antibodies against GPIIb, GPIIIa, GPIX, and GPIb on peripheral blood from one affected patient and one control.Results: Platelet transcriptome analysis of patients with the p.P214L mutation revealed significant differential expression of more than 200 transcripts between patients and controls. Seven of the significant transcripts were key erythrocyte transcripts, which were upregulated in affected patients; and 9 transcripts were key platelet transcripts, which were downregulated. Of these, the red cell marker GYPA was upregulated 25 fold in affected patients (p < 5x10-5), and the platelet markers VWF and GPIX, a target of FLI1, were downregulated 6.4 and 5.6 fold in affected patients (p < 0.05 and p < 0.07), respectively. Platelet GPIX protein expression was also downregulated in 2 affected patients as determined by Western blot. Furthermore, flow cytometry of platelets from one patient with the p.P214L mutation indicated a marginally decreased Ib-IX:IIb-IIIa ratio (0.321) when compared to a normal control (0.500). In vitro studies confirmed that the p.P214L mutation did not disrupt dimerization with WT ETV6 or FLI1, indicating that this defect could be due to dominant negative interactions with both ETV6 and FLI1.Conclusions: Several transcription factors, including FLI1, have been described to play a role in the fate determination between erythroid and megakaryocyte lineages. We report that a dominant negative mutation in ETV6, found in a family with both platelet and red blood cell abnormalities, is associated with differentially regulated red blood cell and platelet transcripts, decreased GPIX protein in platelet lysates, decreased Ib-IX:IIb-IIIa ratio, and dimerization with WT ETV6 and FLI1 in vitro. Our data indicate an important role for ETV6, either alone or through interaction with FLI1, in megakaryocyte-erythroid differentiation. DisclosuresNo relevant conflicts of interest to declare.

Platelet Activation and Aggregation in Patients with Advanced Adenocarcinoma Undergoing Chemotherapy: Correlation with a Validated Venous Thromboembolism Risk Score

Background: Cancer is known to increase risk of venous thromboembolism (VTE), which is associated with considerable mortality and morbidity. Chemotherapy is an independent risk factor for thrombosis in cancer patients. We examined platelet activation and reactivity based on the Khorana Score, a validated scoring model for VTE risk in patients receiving chemotherapy.Materials and Methods: Patients (n=25) with advanced stage adenocarcinomas (TNM stage III or greater), were enrolled in this study. Patients on antiplatelet therapy or on anticoagulation were excluded. Approximately 10 ml of Citrated blood samples were collected from central access catheters after discarding initial 5 ml to minimize iatrogenic platelet activation. 1μMol, 2μMol, 5μMol of ADP-induced, 2μMol Arachidonic Acid (AA)-induced and 4μMol Collagen-induced platelet aggregation in Platelet Rich plasma (PRP) was assessed with a light transmission aggregometry assay. Maximal aggregation and aggregation velocity were recorded. Concurrent flowcytometry analysis was done to assess the expressions of CD41 (Glycoprotein GpIIb), CD62p (platelet surface p-Selectin), and PAC-1(Activated GP IIb/IIIa).Thromboembolic risk scores (Khorana Scores, KS) were calculated based on a validated scoring system (Khorana AA, et al. Blood. 2008;111:4902-7) as follows: Table 1Very high risk cancers (pancreatic or gastric)+2 pointsHigh risk cancers (lung, ovarian, or bladder cancer)+1 pointPlatelet count ≥350 x 109/L+1 pointHemoglobin < 10 gm/dL or use of erythropoietin+1 pointLeukocytosis >11 x 109/L+1 pointBMI > 35+1 pointResults: Mean values for maximum platelet aggregation were calculated by groups and compared between patient with KS < 3 and KS > = 3 by two sample t-tests. Differences were statistically significant for all concentrations of ADP and collagen, with a positive trend for Arachidonic acid (Table). A significant linear relationship between maximal platelet aggregation and higher KS was observed. However there were no significant differences observed in the expression of platelet surface p-selectin, CD41 or PAC-1 when comparing patients at Khorana scores 0, 3 or more than 3 (not shown). Table 2Agonist ConcentrationKhorana Score <3Khorana Score >= 3P valueADP 1μMol20.8%28.14%0.02ADP 2μMol38.6%67%0.002ADP 5μMol66.5%79.85%0.006Arachidonic acid 2μMol61.6%73.85%0.12Collagen 4μMol78.6%90.14%0.01Discussion: The mechanism of cancer-related thromboembolism is not well understood. Chemotherapy is an added risk factor for the development of VTEs. Several new markers such as soluble p-selectin and mean platelet volume have been investigated as adjunct factors to improve predictive ability. Although our study did not find a correlation of some common platelet surface markers for activation with thrombotic risk scores; we find a strong positive correlation of future thrombotic risk derived via Khorana scores with heightened platelet reactivity. Platelet reactivity may mediate a final common pathway for venous thromboembolism in cancer patients and should be validated further as an adjunctive marker in a large scale study. DisclosuresNo relevant conflicts of interest to declare.

Would mean platelet volume/platelet count ratio be used as a novel formula to predict 22q11.2 deletion syndrome?

The diagnosis of 22q11.2 deletion syndrome depends on a time-consuming and expensive method, fluorescence in situ hybridisation (FISH). We aimed to determine new parameters which can aid for in the diagnosis of 22q11.2 deletion syndrome. Twenty two patients with 22q11.2 or 10p13 deletion were evaluated retrospectively. Facial-dysmorphism and mental-motor retardation were detected in 100% of patients. Mean platelet (PLT) counts were lower (224,980 versus 354,000, p = 0.001), mean PLT volume (MPV) (9.95 versus 7.07, p = 0.002), and MPV/PLTx105 ratios (5.36 versus 2.08, p < 0.001) were higher in patients with 22q11.2 deletion compared with the control group. Area under the receiver-operator characteristic (ROC) curve was 0.864, sensitivity was 84.6%, specificity was 90.9%, positive predictive value (PPV) was 91.7%, and negative predictive value (NPV) was 83.3% when MPV was 8.6. Area under ROC curve was 0.864, sensitivity was 76.9%, specificity was 90.1%, PPV was 90.1%, and NPV was 76.3% when PLT was 265,500. Area under ROC curve was 0.906, sensitivity was 84.6%, specificity was 100%, PPV was 100%, and NPV was 84.6% when MPV/PLTx105 was 3.3. Expression of PLT surface markers which were not in the GPIb-V-IX receptor complex (CD61, CD41a) increased as the surface area increased, but markers which were in a complex (CD42a, CD42b) did not change. High MPV/PLT value can be a good predictor for the diagnosis of 22q11.2 deletion syndrome. We suggest that in patients with facial dysmorphism and retardation in neurodevelopmental milestones and if MPV≥8.6fl, MPV/PLTx105 ratio≥3.3 and PLT count ≤265,500/mm3, the patients should be tested by FISH analysis to confirm the 22q11.2 deletion. If there are no macrothrombocytes, the 10p13 deletion should be tested in suspected cases.

Oxaliplatin-based chemotherapy induces extravasated platelet aggregation in the liver.

Oxaliplatin-based chemotherapy plays a central role in the treatment of patients with colorectal liver metastasis (CRLM). This treatment, however, has been associated with hepatic sinusoidal obstruction syndrome (SOS), a clinically important adverse effect characterized by a bluish hue of the liver, splenomegaly and thrombocytopenia, resulting in liver dysfunction. The significant association between the sinusoidal endothelium and platelets has suggested that oxaliplatin-based chemotherapy affects platelets in the liver. This study compared platelet counts in patients who did and did not receive oxaliplatin-based neoadjuvant chemotherapy (NAC). The peripheral blood platelet count was significantly lower in the NAC group (n=17) compared to that in the non-NAC, or control group (n=15) (P<0.05). The spleen index was also higher in the NAC group, although the difference was not significant. However, the spleens of the patients in the NAC group were significantly enlarged following treatment (P<0.01). Immunostaining for the platelet surface marker CD42b (glycoprotein Ib), revealed more platelets in the liver in the NAC compared to the control group, particularly in the centrilobular zone III, adjacent to the hepatic central vein and in contact with hepatocytes (P<0.01). The platelets present in the spaces of Disse, referred to as extravasated platelet aggregation (EPA), secrete a number of growth factors, including transforming growth factor-β, vascular endothelial growth factor-A, plasminogen activator inhibitor-1 and thromboxane A2. In conclusion, EPA may play an important role in the development of hepatic SOS. Moreover, antiplatelet drugs may prevent the onset of SOS and hepatic injury in patients treated with oxaliplatin-based chemotherapy for CRLM.

Signature biomarkers in Diabetes Mellitus and associated Cardiovascular diseases

Platelet signatures indicating differential dysfunction, hyperactivation, aggregation or adhesion are capable of expressing their characters during the journey of a disease process, and can be utilized as cost effective biomarkers with immense clinical value. Type 2 diabetes mellitus (T2DM) is a major lifestyle disease of contemporary world with progression to diabetes associated cardiovascular diseases (DM-CVD). We identified a few potential biomarkers in platelets of T2DM to analyze the thrombotic risk in diabetes subjects by utilizing flow cytometric quantification with different flurochrome conjugated monoclonal antibodies. Our study describes interesting correlations (p<0.0001) for different clinical parameters of concurrent threat for vessel occlusion and the status of indices like reactive oxygen species, von Willebrand factor and mitochondrial membrane potential using western blotting and fluorescence techniques. Our study involved 32 T2DM, and 31 T2DM-CVD subjects compared to 29 healthy controls without any history of T2DM or CVD. An altered expression of platelet surface markers P-selectin (CD62p) and GpIIb/IIIa (CD 41/61, PAC1) along with changes in the platelet size due to agonist induced activation contributed to the enhanced thrombotic potential in the patients. This work elucidates the prospect of platelet biomarkers as diagnostic tool to predict cardiovascular risk in DM subjects.

High‐density lipoprotein 3 and apolipoprotein A‐I alleviate platelet storage lesion and release of platelet extracellular vesicles

Stored platelet (PLT) concentrates (PLCs) for transfusion develop a PLT storage lesion (PSL), decreasing PLT viability and function with profound lipidomic changes and PLT extracellular vesicle (PL-EV) release. High-density lipoprotein 3 (HDL3 ) improves PLT homeostasis through silencing effects on PLT activation in vivo. This prompted us to investigate HDL3 and apolipoprotein A-I (apoA-I) as PSL-antagonizing agents. Healthy donor PLCs were split into low-volume standard PLC storage bags and incubated with native (n)HDL3 or apoA-I from plasma ethanol fractionation (precipitate IV) for 5 days under standard blood banking conditions. Flow cytometry, Born aggregometry, and lipid mass spectrometry were carried out to analyze PL-EV release, PLT aggregation, agonist-induced PLT surface marker expression, and PLT and plasma lipid compositions. Compared to control, added nHDL3 and apoA-I significantly reduced PL-EV release by up to -62% during 5 days, correlating with the added apoA-I concentration. At the lipid level, nHDL3 and apoA-I antagonized PLT lipid loss (+12%) and decreased cholesteryl ester (CE)/free cholesterol (FC) ratios (-69%), whereas in plasma polyunsaturated/saturated CE ratios increased (+3%) and CE 16:0/20:4 ratios decreased (-5%). Administration of nHDL3 increased PLT bis(monoacylglycero)phosphate/phosphatidylglycerol (+102%) and phosphatidic acid/lysophosphatidic acid (+255%) ratios and improved thrombin receptor-activating peptide 6-induced PLT aggregation (+5%). nHDL3 and apoA-I improve PLT membrane homeostasis and intracellular lipid processing and increase CE efflux, antagonizing PSL-related reduction in PLT viability and function and PL-EV release. We suggest uptake and catabolism of nHDL3 into the PLT open canalicular system. As supplement in PLCs, nHDL3 or apoA-I from Fraction IV of plasma ethanol fractionation have the potential to improve PLC quality to prolong storage.

Pathogen reduction treatment alters the immunomodulatory capacity of buffy coat–derived platelet concentrates

Storage of platelet concentrates (PCs) after Mirasol pathogen reduction technology (PRT) treatment changes platelet (PLT) surface marker expression and secretion of immunomodulatory factors. Given that PLTs are known to participate in immune function, PRT may alter the way PLTs interact with the immune cells of a recipient upon transfusion. As such, the aim of this study was to assess the effects of PRT treatment on the functional ability of PLTs to interact with peripheral blood mononuclear cells (PBMNCs). Buffy coat-derived PCs were pooled and split to obtain matched pairs. One unit was treated using the Mirasol PRT system, while the control PC remained untreated. After 5 days of storage, either the PLTs or the PLT supernatants from the PCs were cocultured with PBMNCs, with or without lipopolysaccharide (LPS). The immunomodulatory factors secreted into culture medium after coculture were examined. PRT-treated PLTs and PLT supernatant significantly increased the interleukin (IL)-8 concentration, which was manifested only in the presence of LPS. Conversely, PRT-treated PLTs secreted less soluble P-selectin (sCD62P) upon coculture with PBMNCs. PRT-treatment induced differential secretion of IL-8 and sCD62P during coculture, which may be attributed to either bioactive substances present in PLT supernatant or as a result of cell-cell interactions.

Effects of outdoor air pollutants on platelet activation in people with type 2 diabetes

Exposure to air pollution is associated with increased morbidity and mortality from cardiovascular disease. We hypothesized that increases in exposure to ambient air pollution are associated with platelet activation and formation of circulating tissue factor-expressing microparticles. We studied 19 subjects with type 2 diabetes, without clinical evidence of cardiovascular disease, who had previously participated in a human clinical study of exposure to ultrafine particles (UFP). Blood was obtained for measurements of platelet activation following an overnight stay in the Clinical Research Center, prior to each of their two pre-exposure visits. Air pollution and meteorological data, including UFP counts, were analyzed for the 5 days prior to the subjects’ arrival at the Clinical Research Center. Contrary to expectations, increases in UFP were associated with decreases in surface expression of platelet activation markers. The number of platelet-leukocyte conjugates decreased by −80 (95% confidence interval (CI) −123 to −37, p = 0.001) on the first lag day (20–44 h prior to the blood draw) and by −85 (CI −139 to −31, p = 0.005) on combined lag days 1 to 5, per interquartile range (IQR) increase in UFP particle number (2482). However, levels of soluble CD40L increased 104 (CI 3 to 205, p = 0.04) pg/ml per IQR increase in UFP on lag day 1, a finding consistent with prior platelet activation. We speculate that, in people with diabetes, exposure to UFP activates circulating platelets within hours of exposure, followed by an increase in soluble CD40L and a rebound reduction in circulating platelet surface markers.

Blood Rheology and Platelet Function in Untreated Early-Stage Essential Hypertensives Complicated with Metabolic Syndrome

We examined whether hemorheology and platelet function are affected in essential hypertensives (EHTs) of the World Health Organization stage I when complicated with metabolic syndrome (Mets). In 156 untreated EHTs, blood viscosity and platelet surface markers were determined. Blood viscosity was significantly elevated in 54 subjects with Mets compared with 102 subjects without Mets. Hematocrit and plasma viscosity increased in the group with Mets, although red blood cell rigidity index “k” did not differ between groups. As a whole group, blood viscosity correlated positively with hematocrit and plasma viscosity. Additionally, plasma viscosity correlated positively with plasma leptin, triglyceride, homeostasis model assessment index, C-reactive protein, and plasma fibrinogen, but negatively with high-density lipoprotein cholesterol. In contrast, no differences were seen in platelet surface markers between groups. In conclusion, EHTs of the early stage complicated with Mets are characterized by increased blood viscosity due to hemoconcentration and increased plasma viscosity.

The Human Endogenous Circadian System Causes Greatest Platelet Activation during the Biological Morning Independent of Behaviors

BackgroundPlatelets are involved in the thromboses that are central to myocardial infarctions and ischemic strokes. Such adverse cardiovascular events have day/night patterns with peaks in the morning (∼9AM), potentially related to endogenous circadian clock control of platelet activation. The objective was to test if the human endogenous circadian system influences (1) platelet function and (2) platelet response to standardized behavioral stressors. We also aimed to compare the magnitude of any effects on platelet function caused by the circadian system with that caused by varied standardized behavioral stressors, including mental arithmetic, passive postural tilt and mild cycling exercise.Methodology/Principal FindingsWe studied 12 healthy adults (6 female) who lived in individual laboratory suites in dim light for 240 h, with all behaviors scheduled on a 20-h recurring cycle to permit assessment of endogenous circadian function independent from environmental and behavioral effects including the sleep/wake cycle. Circadian phase was assessed from core body temperature. There were highly significant endogenous circadian rhythms in platelet surface activated glycoprotein (GP) IIb-IIIa, GPIb and P-selectin (6–17% peak-trough amplitudes; p≤0.01). These circadian peaks occurred at a circadian phase corresponding to 8–9AM. Platelet count, ATP release, aggregability, and plasma epinephrine also had significant circadian rhythms but with later peaks (corresponding to 3–8PM). The circadian effects on the platelet activation markers were always larger than that of any of the three behavioral stressors.Conclusions/SignificanceThese data demonstrate robust effects of the endogenous circadian system on platelet activation in humans—independent of the sleep/wake cycle, other behavioral influences and the environment. The ∼9AM timing of the circadian peaks of the three platelet surface markers, including platelet surface activated GPIIb-IIIa, the final common pathway of platelet aggregation, suggests that endogenous circadian influences on platelet function could contribute to the morning peak in adverse cardiovascular events as seen in many epidemiological studies.