Assessment of Role of Platelet Indices in the Occurrence of Retinopathy of Prematurity.

PURPOSE: Platelets have a major role in the regulation of angiogenesis. Platelets have proangiogenic factors like vascular endothelial growth factor, which causes neovascularization of immature retina. However, there is no conclusive evidence to show that platelet indices have a role in retinopathy of prematurity (ROP). This study is aimed at assessing the role of platelet indices in the occurrence and need for treatment of ROP. METHODS: This prospective cohort study included the screening of preterm babies (<37 weeks of gestation with birth weight <2000 g). The samples of platelet indices (mean platelet volume [MPV], platelet count [PLT], plateletcrit [PCT], and platelet distribution width [PDW]) collected within 1st week of life were obtained from the electronic medical records and correlated to ROP status. Statistical analysis was done using SPSS version 22, and the Chi-square test and odds ratio were used for analysis. RESULTS: A total of 300 preterm babies were screened, of whom, 55 (18.3%) babies had ROP changes. The association of the presence of ROP changes and platelet indices was not statistically significant (P value being MPV [0.22], PLT [0.58], PCT [0.98], and PDW [0.17]). Similarly, the requirement of treatment for ROP (Type I ROP) could not be correlated with abnormal platelet indices (odds ratio at 95% confidence interval - MPV [6 (0.44-81.44)], PLT [1.7 (0.25-11.37)], PCT [3 (0.44-20.90)], and PDW [0.32 (0.33-3.05)]). CONCLUSION: Abnormal platelet indices did not show any significant risk with the occurrence or need for treatment of ROP.

"A-PePSI LIGhT" Assessment Score to Predict Pressure Sore Impaired Healing Late Recurrence, Immobility, Greater Surface, Inhibited Thrombocytes.

BACKGROUND: Complication rates of up to 46 percent are reported following pressure sore surgery. Pressure sore patients often exhibit ineffective postoperative wound healing despite tension-free flap coverage, necessitating surgical revision and prolonged hospitalization. Rather than pressure sore recurrence, such impaired healing reflects a failed progress through the physiologic stages of the normal wound-healing cascade. The principal objective of the study reported here was to elucidate potentially modifiable inherent variables that predict predisposition to impaired healing and to provide a tool for identifying cases at risk for complicated early postoperative recovery following pressure sore reconstruction. METHODS: A retrospective chart review of late-stage (stage 3 or higher) sacral and ischial pressure sore patients who underwent flap reconstruction from 2014 to 2019 was performed. A multivariable logistic regression model was used to identify key patient and operative factors predictive of impaired healing. Furthermore, the Assessment Score to Predict Pressure Sore Impaired Healing (A-PePSI) was established based on the identified risk factors. RESULTS: In a cohort of 121 patients, 36 percent exhibited impaired healing. Of these, 34 patients suffered from dehiscences, necessitating surgical revision. Statistically significant risk factors comprising late recurrence (OR, 3.8), immobility (OR, 12.4), greater surface (>5 cm diameter; OR, 7.3), and inhibited thrombocytes (aspirin monotherapy; OR, 5.7) were combined to formulate a prognostic scoring system (A-PePSI LIGhT). CONCLUSIONS: The A-PePSI LIGhT score serves as a prognostic instrument for assessing individual risk for impaired healing in pressure sore patients. Preoperative risk stratification supports rational decision-making regarding operative candidacy, allows evidence-based patient counseling, and supports the implementation of individualized treatment protocols. . CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Utility of Platelet Endothelial Cell Adhesion Molecule 1 in the Platelet Activity Assessment in Mouse and Human Blood.

In our previous study, we introduced the platelet endothelial cell adhesion molecule 1 (PECAM-1)/thrombus ratio, which is a parameter indicating the proportion of PECAM-1 in laser-induced thrombi in mice. Because PECAM-1 is an antithrombotic molecule, the higher the PECAM-1/thrombus ratio, the less activated the platelets. In this study, we used an extracorporeal model of thrombosis (flow chamber model) to verify its usefulness in the assessment of the PECAM-1/thrombus ratio in animal and human studies. Using the lipopolysaccharide (LPS)-induced inflammation model, we also evaluated whether the PECAM-1/thrombus ratio determined in the flow chamber (without endothelium) differed from that calculated in laser-induced thrombosis (with endothelium). We observed that acetylsalicylic acid (ASA) decreased the area of the thrombus while increasing the PECAM-1/thrombus ratio in healthy mice and humans in a dose-dependent manner. In LPS-treated mice, the PECAM-1/thrombus ratio decreased as the dose of ASA increased in both thrombosis models, but the direction of change in the thrombus area was inconsistent. Our study demonstrates that the PECAM-1/thrombus ratio can more accurately describe the platelet activation status than commonly used parameters such as the thrombus area, and, hence, it can be used in both human and animal studies.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

Functional Assessment of Platelet Dense Granule ATP Release.

OBJECTIVES: This study was undertaken to explore the feasibility of assessing platelet dense granule release in response to platelet stimuli, using less than 1 mL of whole blood (WB). METHODS: Optimization of the luciferin-luciferase (LL) assay for ATP release, together with additional modifications, was applied to 1:10 diluted WB. RESULTS: LL assay optimization using nonstirred 1:10 diluted WB resulted in dense granule ATP release in response to thrombin receptor-activating peptide (TRAP) of similar magnitude to that observed using stirred platelet-rich plasma. Stirring of the 1:10 diluted WB restored collagen-induced dense granule secretion. Addition of lyophilized, formalin-fixed platelets, together with stirring, restored dense granule secretion responsiveness to ADP. TRAP, ADP, and collagen all stimulated ATP release in 1:10 diluted WB under the optimized conditions of this study at levels close to those observed using platelet-rich plasma. Blood sample reconstitution experiments offer hope that this assay may prove robust down to WB platelet counts as low as 50 × 103/µL. CONCLUSIONS: Platelet dense granule release in response to a number of classic stimuli, including ADP, was accomplished from less than 1 mL WB with minimal specimen processing, using widely available reagents and instrumentation.

Assessment of platelet biology in equine patients with systemic inflammatory response syndrome.

In addition to maintaining hemostasis, platelets have an important role in modulating innate and adaptive immune responses. A low platelet count has been found to be a negative prognostic factor for survival in humans and horses with critical illnesses, such as sepsis or systemic inflammatory response syndrome (SIRS). Decreased platelet aggregation, caused by in vivo activation, has been found in human patients with severe sepsis. In our prospective controlled study, we assessed platelet biology in blood samples from 20 equine SIRS cases and 120 healthy control horses. Platelet variables such as platelet count, large platelet count, clumps, plateletcrit, mean platelet volume, and mean platelet component concentration were analyzed by laser flow cytometry (Advia 2120) from K3EDTA blood and from citrate blood. Hirudin blood samples were analyzed by impedance aggregometry (Multiplate analyzer; Roche) for platelet aggregation, including spontaneous aggregation and aggregation by 4 different agonists: adenosine diphosphate (ADPtest), ADP + prostaglandin E1 (ADPtestHS), arachidonic acid (ASPItest), and collagen (COLtest). SIRS cases had significantly lower platelet counts in K3EDTA blood (p < 0.0001) compared to control horses. There were no significant differences in aggregation values between SIRS cases and controls. Non-surviving SIRS horses did not have statistically significant lower platelet counts or lower aggregation values for COLtest, ADPtest, or ADPtestHS compared to surviving SIRS horses, although 5 non-survivors were thrombocytopenic.

Intravital Assessment of Blood Platelet Function. A Review of the Methodological Approaches with Examples of Studies of Selected Aspects of Blood Platelet Function.

Platelet biology owes to intravital studies not only a better understanding of platelets' role in primary hemostasis but also findings that platelets are important factors in inflammation and atherosclerosis. Researchers who enter the field of intravital platelet studies may be confused by the heterogeneity of experimental protocols utilized. On the one hand, there are a variety of stimuli used to activate platelet response, and on the other hand there are several approaches to measure the outcome of the activation. A number of possible combinations of activation factors with measurement approaches result in the aforementioned heterogeneity. The aim of this review is to present the most often used protocols in a systematic way depending on the stimulus used to activate platelets. By providing examples of studies performed with each of the protocols, we attempt to explain why a particular combination of stimuli and measurement method was applied to study a given aspect of platelet biology.

Assessment of hemostatic profile in patients with mild to advanced liver cirrhosis.

BACKGROUND: Hemostasis of patients suffering from liver cirrhosis is challenging due to both, pro- and anticoagulatory disorders leading to hemostatic alterations with distinct abnormalities of coagulation. Pathological changes in conventional coagulation analysis and platelet count are common manifestations of decreased liver synthesis of coagulation factors and reduced platelet count in these patients. However, conventional coagulation analysis and platelet count do not reflect in-vivo coagulation status or platelet function. The purpose of this present observational study was therefore to assess the haemostatic profile including plasmatic coagulation using thrombelastometry and impedance aggregometry for platelet function in patients suffering from liver cirrhosis. AIM: To assess the hemostatic profile of cirrhotic patients according to model for end-stage liver disease (MELD) score. METHODS: Our study included both in- and outpatients suffering from liver cirrhosis attending the out- and inpatient care of the department of hepatology. Demographic and biochemical data as well as medical history including cause of liver cirrhosis, end stage kidney failure and medication with anticoagulants were recorded. To assess the hemostatic profile, platelet function was analyzed by multiple electrode aggregometry (MEA) using Multiplate® (ADP-, ASPI- and TRAP-test) and thrombelastometry using ROTEM® (EXTEM, INTEM, FIBTEM). Data were compared using Mann-Whitney U- or χ 2-test. Spearman correlation was performed to analyze the association between MELD Score and results of thrombelastometry and MEA. RESULTS: A total of 68 patients attending the out- and inpatient care suffering from liver cirrhosis were screened. Of these, 50 patients were included and assigned to groups according to MELD score 6 to 11 (n = 25) or ≥ 17 (n = 25). Baseline patient characteristics revealed significant differences for MELD score (8 vs 22, P < 0.0001) and underlying laboratory parameters (international normalized ratio, bilirubine, creatinine) as well as fibrinogen level (275 mg/dL vs 209 mg/dL, P = 0.006) and aPTT (30 s vs 35 s, P = 0.047). MEA showed a moderately impaired platelet function (medians: AUCADP = 43U, AUCASPI = 71U, AUCTRAP = 92U) but no significant differences between both groups. Thrombelastometry using ROTEM® (EXTEM, INTEM, FIBTEM) revealed values within normal range in both groups. No significant correlation was observed between MELD score and results of MEA/thrombelastometry. CONCLUSION: Our data demonstrate a partially impaired hemostatic profile in liver cirrhosis patients unrelated to MELD score. An individual assessment of a potential coagulopathy should therefore be considered.

Assessment of platelet indices and platelet activation markers in children with Plasmodium falciparum malaria.

BACKGROUND: Plasmodium falciparum malaria remains one of the world's major infectious diseases that cause most morbidity and mortality, particularly in children. In Ghana, most children below the ages of 5 years depending on the severity of the infection often lose their lives. However, it is still debatable why infection with falciparum malaria contributes to thrombocytopenia. METHODS: This study sought to investigate the expression of the various platelet indices and activation markers in children with falciparum malaria. Platelet indices (Platelet count [PLT], Plateletcrite [PCT], Mean Platelet Volume [MPV], Platelet Distribution Width [PDW] and Platelet-Large Cell Ratio [P-LCR]) and platelet surface membrane glycoproteins (GPIIb/IIIa [PAC-1], P-selectin [CD62p] and GPIV [CD36]) expressions were determined in children with falciparum malaria (cases) and healthy children (controls) using automated blood cell analysis and flow cytometry techniques, respectively. RESULTS: Except for P-LCR, all the other platelet indices (PLT, MPV, PDW, and PCT) were significantly lower in the cases than the controls (P < 0.05). Also, it was observed that the level of expression of the activation markers; PAC 1 and CD62p showed a significant (P < 0.05) decreased before and after activation in falciparum malaria cases than in the controls. On the contrary, CD36 expression in the controls did not differ significantly (p > 0.05) from the malaria cases. Platelet activation markers were known to be associated with increased risk of falciparum malaria with the mean fluorescence intensity of PAC1 (Odds Ratio [OR] 34.0, Relative Risk [RR] 4.47, 95% Confidence Interval [CI] 4.904-235.7; p < 0.0001) and CD36 (OR 4.2, RR 1.82, 95% CI 0.9824-17.96; p = 0.04). Moreover, the percentage expression of CD62p (OR 4.0, RR 1.80, 95% CI 0.59-27.24; p = 0.19) was also observed to be probably associated with increased risk of falciparum malaria although not statistically significant (p > 0.05). CONCLUSION: Plasmodium falciparum malaria has been known to be associated with platelet activation markers, which probably contributes to thrombocytopenia.

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling.

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

The key events of thrombus formation: platelet adhesion and aggregation.

Thrombus formation is a complex, dynamic and multistep process, involving biochemical reactions, mechanical stimulation, hemodynamics, and so on. In this study, we concentrate on its two crucial steps: (i) platelets adhered to a vessel wall, or simply platelet adhesion, and (ii) platelets clumping and arrested to the adherent platelets, named platelet aggregation. We report the first direct simulation of three modes of platelet adhesion, detachment, rolling adhesion and firm adhesion, as well as the formation, disintegration, arrestment and consolidation of platelet plugs. The results show that the bond dissociation in the detachment mode is mainly attributed to a high probability of rupturing bonds, such that any existing bond can be quickly ruptured and all bonds would be completely broken. In the rolling adhesion, however, it is mainly attributed to the strong traction from the shear flow or erythrocytes, causing that the bonds are ruptured at the trailing edge of the platelet. The erythrocytes play an important role in platelet activities, such as the formation, disintegration, arrestment and consolidation of platelet plugs. They exert an aggregate force on platelets, a repulsion at a near distance but an attraction at a far distance to the platelets. This aggregate force can promote platelets to form a plug and/or bring along a part of a platelet plug causing its disintegration. It also greatly influences the arrestment and consolidation of platelet plugs, together with the adhesive force from the thrombus.

Modulation of Platelet Functions Assessment during Menstruation and Ovulatory Phases.

During menstruation, endometrial hemostasis is achieved by platelet aggregation, fibrin deposition, and thrombus formation that interact with local endocrine and immunological factors which cause termination of menstrual bleeding. Interactions between steroidal sex hormones and platelet functions are not well understood. The aim of this study was to evaluate the effect of platelet function during the menstrual cycle and luteal phase in women of reproductive age. The cross-sectional study on women of reproductive age included 44 healthy women. Platelet function was assessed by PFA-100TM analyzer with collagen/epinephrine and collagen/ADP cartridges during the menstrual cycle and luteal phase. There were no significant differences in platelet function between menstruation and ovulatory phase. Platelet activity in Arab collagen/epinephrine cartridge increased during menstruation compared to non-Arab ethnic subjects and no significant differences in platelet function were found when using collagen/ADP cartridge. This study suggested modulation in platelet functions during menstruation and luteal phase in women of reproductive age. Further studies, including a large number of subjects, platelet genetic and progesterone factors change in platelet clotting associated to menstrual cycle should be conducted.

Standardization of Light Transmission Aggregometry for Diagnosis of Platelet Disorders: An Inter-Laboratory External Quality Assessment.

Several in vitro platelet function tests are available for the diagnosis of inherited platelet function disorders. Currently, the light transmission aggregometry (LTA) is recommended as one of the first-step tests. LTA is available in most specialized hemostasis laboratories. Although the LTA is accepted as a 'gold standard' assay for the evaluation of platelet function, its standardization in the clinical practice is still challenging. The GTH-based THROMKID-Plus Study Group has performed an inter-laboratory trial in Germany and Austria. Five different agonists were selected according to the Scientific and Standardization Committee/International Society on Thrombosis and Haemostasis recommendations and shipped in 3 different sets (one should represent a healthy control and two should simulate platelet function disorders) to 15 specialized laboratories in Germany and Austria. Agonists were analyzed by APACT or PAP4/8 aggregometer using platelet-rich plasma from healthy donors. In addition, laboratory-internal platelet agonists were tested in platelet-rich plasma from a healthy donor. All laboratories (9 used APACT, 6 used PAP4/PAP8) showed very consistent data regarding the maximum percentage of aggregation induced by the tested agonists and identified the differential diagnosis of the simulated platelet function disorders with one exception, which was due to technical problems. In contrast, there was a high variability of the laboratory-internal inductors regarding reagent type, concentrations and pathological cut-off values. Our study showed that the shipment of agonists is suitable for an inter-laboratory survey of LTA. However, there is still a remarkable need for standardization of agonist reagents and their concentration as well as for definition of reference ranges.

Adverse events in patients with high platelet reactivity following successful chronic total occlusion PCI: The Assessment of Dual AntiPlatelet Therapy with Drug-Eluting Stents (ADAPT-DES) study.

BACKGROUND: Chronic total occlusion (CTO) percutaneous coronary intervention (PCI) typically requires a greater number of stents and longer stent length than non-CTO PCI, placing these patients at greater risk for adverse ischemic events. We sought to determine whether the association between high platelet reactivity (HPR) and the risk of ischemic events is stronger after CTO than non-CTO PCI. METHODS: Patients undergoing successful PCI in the multicenter ADAPT-DES study were stratified according to whether they underwent PCI of a CTO. HPR was defined as VerifyNow platelet reaction units >208. The study primary endpoint was the 2-year risk target vessel failure ([TVF] defined as cardiac death, myocardial infarction, or target lesion revascularization). RESULTS: CTO PCI was performed in 400 of 8448 patients. HPR was present in 34.5% of CTO PCI patients and 43.1% of non-CTO PCI patients (P = .0007). Patients undergoing CTO PCI with versus without HPR had significantly higher 2-year rates of TVF (15.0% versus 8.3%, P = .04) without significant differences in bleeding. HPR was an independent predictor of 2-year TVF (adjusted HR 1.16, 95% CI 1.02-1.34, P = .03) whereas CTO PCI was not (adjusted HR 0.89, 95% CI 0.65-1.22, P = .48). There was a significant interaction between CTO versus non-CTO PCI and PRU as a continuous variable for 2-year TVF (Pinteraction = 0.02). CONCLUSIONS: In ADAPT-DES, HPR was associated with an increased 2-year risk of TVF after PCI, an association that was at least as strong after CTO PCI compared with non-CTO PCI.

Platelets function assessment in patients qualified for cardiac surgery - clinical problems and a newer diagnostic possibilities.

BACKGROUND: As the incidence of cardiovascular diseases increases, the use of antiplatelet therapy is widely recognized. This presents clinicians with the challenge of balancing the risk of thrombotic and bleeding complications. Platelet dysfunction is one of the causes of postoperative bleedings and their etiology is not fully understood. Platelets receptors point-of-care investigation is of a remarkable value in assessing patients risk of bleeding. Reliable assessment of platelet function can improve treatment. The aim of this study was to evaluate the activity of platelet receptors in patients qualified for cardiac surgery, taking into account organ dysfunctions and pharmacological therapy applied in these patients. METHODS: Seventy-one cardiac surgical patients were analyzed before surgery using multiple electrode aggregometry with the use of the ADP test and ASPI test. The cut-off values were determined based on the manufacturer's recommendations. Patients were divided into four groups: Group I (33/71 patients, without platelet dysfunctions), Group II (6/71 patients, ADP < 710 AU x min), Group III (13/71 patients, ASPI < 570 AU x min) and Group IV (19 / 71 patients, ADP < 710 AU x min and ASPI < 570 AU x min). Biochemical data defining the efficiency of the liver and kidneys, the list of preoperative drugs used and the requirement for transfusion throughout the study group were collected. RESULTS: The study group included 41 males (57.7%) and 30 females (42.3%), mean age 66 years. The majority of patients (94.4%) had platelet counts within the normal range, but platelet function was impaired in more than half of the studied patients (53.5%). No relationship was found between the biochemical markers of the kidneys and liver and the function of the ADP and ASPI receptors, while receptors activities were related (rs = 0.72, p < 0.001), and both associated with platelet count (rs = 0.55, p < 0.001 and rs = 0.42, p < 0.001, respectively). Platelet receptors activity was not related to the postoperative need for any type of transfusion as well as the applied preoperative pharmacological therapy. CONCLUSIONS: Early identification of patients at high risk of bleeding, using point-of-care platelet function assessment tests, enables a targeted therapeutic pathway. Due to the variety of factors affecting the activity of platelets, finding a specific cause of this pathology is extremely difficult. According to our study, the correlation between platelet receptor disorders and mild to moderate liver and kidney injury has not been demonstrated. However, platelet receptors dysfunction has been shown to be associated with a decreased number of platelets.

Assessment of Thrombelastography and Platelet Life Span in Ovines.

Ovines are a common animal model for the study of cardiovascular devices, where consideration of blood biocompatibility is an essential design criterion. In the ovine model, tools to assess blood biocompatibility are limited and continued investigation to identify and apply additional assays is merited. Toward this end, the thrombelastograph, clinically utilized to assess hemostasis, was used to characterize normal ovine parameters. In addition, platelet labeling with biotin was evaluated for its potential applicability to quantify ovine platelet life span. Mean ovine thrombelastograph values were reaction-time: 4.9 min, K-time: 2 min, angle: 64.1°, maximum amplitude: 68.6mm, actual clot strength: 11.9 kd/s, and coagulation index: 1.5. Reaction time was significantly shorter and maximum amplitude, actual clot strength, and coagulation index were all significantly higher when compared to normal human thrombelastograph values suggesting some hypercoagulability of sheep blood. Biotinylation and reinfusion of ovine platelets allowed temporal tracking of the labeled platelet cohort with flow cytometry. These data indicated a mean ovine platelet life span of 188h with a half-life of 84h. The collection of these parameters for normal ovines demonstrates the applicability of these techniques for subsequent studies where cardiovascular devices may be evaluated and provides an indication of normal ovine values for comparison purposes.

Programmable Multivalent DNA-Origami Tension Probes for Reporting Cellular Traction Forces.

Mechanical forces are central to most, if not all, biological processes, including cell development, immune recognition, and metastasis. Because the cellular machinery mediating mechano-sensing and force generation is dependent on the nanoscale organization and geometry of protein assemblies, a current need in the field is the development of force-sensing probes that can be customized at the nanometer-length scale. In this work, we describe a DNA origami tension sensor that maps the piconewton (pN) forces generated by living cells. As a proof-of-concept, we engineered a novel library of six-helix-bundle DNA-origami tension probes (DOTPs) with a tailorable number of tension-reporting hairpins (each with their own tunable tension response threshold) and a tunable number of cell-receptor ligands. We used single-molecule force spectroscopy to determine the probes' tension response thresholds and used computational modeling to show that hairpin unfolding is semi-cooperative and orientation-dependent. Finally, we use our DOTP library to map the forces applied by human blood platelets during initial adhesion and activation. We find that the total tension signal exhibited by platelets on DOTP-functionalized surfaces increases with the number of ligands per DOTP, likely due to increased total ligand density, and decreases exponentially with the DOTP's force-response threshold. This work opens the door to applications for understanding and regulating biophysical processes involving cooperativity and multivalency.

Influencing factors in quantitative measurement using activated platelet levels and platelet-activating capacity for the assessment of thrombosis in pre-metabolic syndrome and type 2 diabetes mellitus.

Activated platelet levels and platelet-activating capacity are well recognized as useful index parameters for the physiological and pharmacological prediction of thrombotic events. Recently, quantitative measurements for platelet functions using a flow cytometer have been increasing gradually. However, the relation of physiological factors, such as sex, aging, and laboratory tests, to platelet functions has not been well documented. We conducted a blood analysis of people with normal/pre-metabolic syndrome and patients with type 2 diabetes mellitus to clarify the pathological factors. The levels of basal (non-stimulated)-activated, platelet-expressed P-selectin and activated platelet stimulated by agonists were measured by a flow cytometer, and ratios of platelet-activating capacity were also calculated. Statistical analyses indicated significantly high basal-activated platelet in pre-metabolic syndrome, and basal-activated platelet was positively associated with hyperlipidemia and hepatic damage. Platelet-activating capacity was significantly low in aging and hyperlipidemia, but high in hyperglycemia, and was negatively associated with hyperlipidemia and hepatic damage. Aging and high nutrient condition impaired platelet functions. Quantitative measurements of basal-activated platelet and platelet-activating capacity are precise parameters for the prediction of thrombotic events.

Clinical and laboratory assessment of a patient with thrombocytosis.

Elevated platelet counts are frequently encountered in hospital medicine and arise from both physiological and pathological mechanisms. Thrombocytosis may be secondary, reflecting an inflammatory state, iron deficiency, recent surgery or point towards an underlying neoplasm. Thrombocytosis may be the presenting sign of solid tumours and haematological conditions. The discovery of the activating mutations affecting thrombopoiesis led to greater understanding of the pathobiology of essential thrombocythaemia and other myeloproliferative neoplasms. The investigation of suspected primary thrombocytosis has evolved to include testing for these disease-associated mutations. Therapy for patients with essential thrombocythaemia aims to reduce their risk of thrombotic complications by addressing cardiovascular risk factors, and using antiplatelet agents and, in selected patients, cytoreductive therapy. This article provides a logical approach to distinguishing reactive or secondary thrombocytosis from thrombocytosis associated with an underlying myeloproliferative neoplasm and gives an overview of the management of essential thrombocythaemia.

Genotyping of six clopidogrel-metabolizing enzyme polymorphisms has a minor role in the assessment of platelet reactivity in patients with acute coronary syndrome.

OBJECTIVE: To evaluate the contribution of six polymorphisms to the platelet reactivity in patients with acute coronary syndrome (ACS) treated with clopidogrel. METHODS: Cross-sectional study of 278 consecutive patients with ACS. Detailed clinical information for each patient was collected and genotypes (CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*17, CYP3A4*1B, and PON1-Q192R) were evaluated with TaqMan® and KASPar® assays. Platelet reactivity was measured with VerifyNow®. RESULTS: Mean age of patients was 66±11 years and 182 (65.5%) patients presented ACS without ST-segment elevation. A total of 206 (74.1%) patients presented poor response to clopidogrel (PRC). CYP2C19*2 polymorphism (p=0.038) was associated with PRC in the univariate setting. In the multiple logistic regression analysis, the risk factors for PRC were the presence of CYP3A4*1B allele (odds ratio [OR] 4.03; 95% confidence interval [CI] 1.01-16.34), age (OR 1.43; 95% CI 1.03-2.00), and body mass index (OR 4.05; 95% CI 1.21-13.43), whereas elevated hemoglobin was a protective factor. Discrimination of PRC through the model that included the six polymorphisms added modest information to the model based on clinical variables (C statistic difference 3.9%). CONCLUSION: CYP3A4*1B allele may be an independent determinant of PRC in patients with ACS, although the variability in response to clopidogrel explained by the six polymorphisms is poor when compared to clinical variables.