Till Örebro universitet

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the ISTH SSCs, we assessed how three of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALLTM filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting the techniques are transferrable and reproducible. Moreover, all three isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ3 activation is reduced by anti-CD45 bead isolation compared to washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

We report a disposable self-powered integrated microfluidic chip that enables a rapid and simple platelet-function assay from small samples of whole blood. The chip integrates a single-cell adhesion assay with a microfluidic platform; it enables accurate quantification of platelet adhesion, and it controls whole blood flow rate, shear stress, volume of sample, and assay time.

We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates By separating platelets From whole blood using specific binding to protein spots of a defined size. iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (I) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopuladons from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated To date, we have demonstrated 1-4 of the above list Platelets were separated from whole blood using tPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 full) The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (aIlb beta 3) and, relevant to diagnostic applications, platelet adhesion correlates strongly with normal versus abnormal platelet function A critical function of platelets is to adhere to regions of damage on blood vessel walls, in contrast to conventional flow cytometry, where platelets are suspended in solution, iPC enables physiologically relevant platelet bioassays based on platelet/protein-matrix inter actions on surfaces. This technology should be inexpensive to implement in clinical assay format, is readily integrable into fluidic microdevices, and paves the way for high-throughput platelet assays from microliter volumes of whole blood.

Pentraxin-3 (PTX3) and neprilysin have been associated with increased morbidity and mortality in chronic inflammatory disease and heart failure, but these biomarkers have been studied less in patients with ST segment elevation myocardial infarction (STEMI). We investigated the dynamic changes in these biomarkers, as well as the well-known C-reactive protein (CRP), in STEMI patients. PTX3, neprilysin and CRP were measured in samples from 165 STEMI patients, collected at the acute stage, 1-3 days after and 3 months after percutaneous coronary intervention (PCI), and from 40 healthy donors. Patient survival was followed for approximately 8 years after the PCI. As compared with samples from healthy donors, plasma levels of CRP and PTX3 were significantly increased in the acute samples and 1-3 days after PCI, but not at 3 months. CRP levels peaked at 1-3 days, while PTX3 was similarly high in both acute and 1-3 days samples. For neprilysin, no significant differences were observed at the group level. We found no significant differences when comparing patients with patent versus occluded culprit vessels or between patients having a thrombus aspiration or not. However, we found a significant reduction in survival for individuals with PTX3 above the median, both for samples collected at the acute stage and 1-3 days after PCI (p = 0.0001 and p = 0.0008, respectively). For CRP, no significant differences were observed using this approach, but patients above the reference range for healthy donors in the acute samples showed significantly lower survival (p = 0.0476). Conclusions: Survival analysis suggests that PTX3 might be a promising marker to predict mortality in this patient population.

The soluble tumor necrosis factor receptors (sTNFR1 and sTNFR2) are suggested to play dual roles on physiological and pathophysiological actions of TNF-α. The aim of this study was to investigate the dynamic changes of these biomarkers in patients with ST-segment elevation myocardial infarction (STEMI). Blood was collected from 165 STEMI patients at admission, 1-3 days and 3 months after percutaneous coronary intervention (PCI) and from 40 healthy blood donors. sTNFR1 and sTNFR2 were measured with ELISA. The plasma levels of both sTNFR1 and sTNFR2 were significantly higher than in healthy donors at all three time points. We found no significant differences in sTNFR1 or sTNFR2 when comparing patients with patent versus occluded culprit vessels, or between patients having a thrombus aspiration or not. Survival analysis was performed comparing patients with levels of biomarkers above and below the median values at that time point. We found significant differences in survival for sTNFR2 in acute samples (p = 0.0151) and for both sTNFR1 and sTNFR2 in samples 1-3 days after PCI (p = 0.0054 and p = 0.0003, respectively). Survival analyses suggest that sTNFR1 or sTNFR2 could be promising markers to predict mortality in STEMI patients after PCI.

In X-linked thrombocytopenia with thalassemia (XLTT; OMIM 314050), caused by the mutation p.R216Q in exon 4 of the GATA1 gene, male hemizygous patients display macrothrombocytopenia, bleeding diathesis and a β-thalassemia trait. Herein, we describe findings in two unrelated Swedish XLTT families with a bleeding tendency exceeding what is expected from the thrombocytopenia. Blood tests revealed low P-PAI-1 and P-factor 5, and elevated S-thrombopoietin levels. Transmission electron microscopy showed diminished numbers of platelet α- and dense granules. The proteomes of isolated blood platelets from 5 male XLTT patients, compared to 5 gender- and age matched controls, were explored. Quantitative mass spectrometry showed alterations of 83 proteins (fold change ≥±1.2, q< .05). Of 46 downregulated proteins, 39 were previously reported to be associated with platelet granules. Reduced protein levels of PTGS1 and SLC35D3 were validated in megakaryocytes of XLTT bone marrow biopsies by immunohistochemistry. Platelet function testing by flow cytometry revealed low dense- and α-granule release and fibrinogen binding in response to ligation of receptors for ADP, the thrombin receptor PAR4 and the collagen receptor GPVI. Significant reductions of a number of α-granule proteins overlapped with a previous platelet proteomics investigation in the inherited macrothrombocytopenia gray platelet syndrome (GPS). In contrast, Ca2+ transporter proteins that facilitate dense granule release were downregulated in XLTT but upregulated in GPS. Ingenuity Pathway Analysis showed altered Coagulation System and Protein Ubiquitination pathways in the XLTT platelets. Collectively, the results revealed protein and functional alterations affecting platelet α- and dense granules in XLTT, probably contributing to bleeding.

Background: A commercial MP reagent containing phospholipids is used for thrombin generation (TG) measurements to estimate the procoagulant activity of microparticles (MPs). Previous reports have shown that contact activation affects TG when TF levels are low, and that addition of phospholipids might augment this effect.

Objectives: To quantify the impact of contact activation on TG in the presence of phospholipids and low/no TF, as is the case using a commercially available MP-reagent. Methods Thrombin generation was analyzed using MP- or platelet-rich plasma (PRP)-reagent in the presence and absence of corn trypsin inhibitor and anti-TF antibodies, respectively. To quantify the impact of different experimental parameters on contact activation, microparticle-depleted plasma was analyzed in the presence of different concentrations of phospholipids, TF and/or contact activating agents (kaolin).

Results: Even with low contact activating blood collection tubes, substantial thrombin generation was observed with the MP-reagent, but this was completely inhibited by addition of corn trypsin inhibitor. Control experiments illustrate that the phospholipids in the reagent play a major role in enhancing TG initiated by FXIIa. Even with the PRP-reagent, which is recommended for determining the content of phospholipids from MPs, TG was partly dependent on contact activation.

Conclusions: Contact activation plays a major role in TG when using reagents/samples containing phospholipids but little or no tissue factor. This needs to be considered and accounted for in future clinical studies using TG to assess the procoagulant activity of MPs.

Thrombin-induced platelet activation via PAR1 and PAR4 is an important event in haemostasis. Although the underlying mechanisms responsible for ensuring efficient PAR1 activation by thrombin have been extensively studied, the potential involvement of recognitions sites outside the active site of the protease in thrombin-induced PAR4 activation is largely unknown. In this study, we developed a new assay to assess the importance of exosite I and II for PAR4 activation with alpha- and gamma-thrombin. Surprisingly, we found that exosite II is critical for activation of PAR4. We also show that this dependency on exosite II likely represents a new mechanism, as it is unaffected by blockage of the previously known interaction between thrombin and glycoprotein Ib alpha.

Background: Although flow cytometry is often brought forward as a preferable method in the setting of thrombocytopenia, the relative effects of low sample counts on results from flow cytometry-based platelet function testing (FC-PFT) in comparison with light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) has not been reported.

Objectives: To compare the effects of different sample platelet counts (10, 50, 100, and 200x10(9)L(-1)) on platelet activation measured with FC-PFT, LTA, and MEA using the same anticoagulant and agonist concentrations as for the commercial MEA test.

Methods: Platelets were stimulated with two commonly used platelet agonists (ADP [6.5 mu molL(-1)] and PAR1-AP [TRAP, 32 mu molL(-1)]). The specified sample platelet counts were obtained by combining platelet-rich and platelet poor hirudinized plasma in different proportions with or without red blood cells.

Results: For FC, P-selectin exposure and PAC-1 binding was reduced at 10x10(9)L(-1) after stimulation with PAR1-AP (by approximately 20% and 50%, respectively), but remained relatively unchanged when ADP was used as agonist (n=9). The platelet count-dependent effects observed with PAR1-AP were eliminated when samples were pre-incubated with apyrase, implying that reduced purinergic signaling was the main underlying factor (n=5). Both aggregometry-based PFTs showed a 50% reduction at 50x10(9)L(-1) and more than 80% reduction at 10x10(9)L(-1), irrespective of agonist used (n=7).

Conclusions: Although FC-PFT is generally preferable to aggregometry-based PFTs in situations with low sample platelet counts, a careful optimization of experimental parameters is still required in order to eliminate platelet count-related effects.

Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clinical correlation between the test results and the patient phenotype. Herein, we present the results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and P-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP, and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or with the fifth percentile of the entire patient cohort. We found that the abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient cohort (p = 0.00008, hazard ratio 8.7; 95% CI 2.5-40). Our results suggest that whole-blood flow cytometry-based platelet function testing could become a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related hemostasis.

Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A(4) (LXA(4)) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA(4) on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA(4). Furthermore, we found that LXA(4) significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA(4) was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA(4) antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strainMMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased beta-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to gamma-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development.

Background: Thrombocytopenia is a common finding during viral hemorrhagic fever, which includes hemorrhagic fever with renal syndrome (HFRS). The 2 main causes for thrombocytopenia are impaired thrombopoiesis and/or increased peripheral destruction of platelets. In addition, there is an increased intravascular coagulation risk during HFRS, which could be due to platelet activation.

Methods: Thrombopoiesis was determined by quantification of platelet counts, thrombopoietin, immature platelet fraction, and mean platelet volume during HFRS. The in vivo platelet activation was determined by quantification of soluble P-selectin (sP-selectin) and glycoprotein VI (sGPVI). The function of circulating platelets was determined by ex vivo stimulation followed by flow cytometry analysis of platelet surface-bound fibrinogen and P-selectin exposure. Intravascular coagulation during disease was determined by scoring for disseminated intravascular coagulation (DIC) and recording thromboembolic complications.

Results: The levels of thrombopoietin, immature platelet fraction, and mean platelet volume all indicate increased thrombopoiesis during HFRS. Circulating platelets had reduced ex vivo function during disease compared to follow-up. Most interestingly, we observed significantly increased in vivo platelet activation in HFRS patients with intravascular coagulation (DIC and thromboembolic complications) as shown by sP-selectin and sGPVI levels. Conclusions. HFRS patients have increased thrombopoiesis and platelet activation, which contributes to intravascular coagulation.

INTRODUCTION: During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients.

AIM: To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis.

MATERIALS AND METHODS: We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations.

RESULTS: At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients, see Figure 1.

CONCLUSIONS: Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient.

Arterial thrombosis (AT) originates through platelet-mediated thrombus formation in the blood vessel and can lead to heart attack, stroke, and peripheral vascular diseases. Restricting the thrombus growth and its simultaneous monitoring by visualisation is an unmet clinical need for a better AT prognosis. As a proof-of-concept, we have engineered a nanoparticle-based theranostic (combined therapy and monitoring) platform that has the potential to monitor and restrain the growth of a thrombus concurrently. The theranostic nanotool is fabricated using biocompatible super-paramagnetic iron oxide nanoparticles (SPIONs) as a core module tethered with the anti-platelet agent Abciximab (ReoPro) on its surface. Our in vitro feasibility results indicate that ReoPro-conjugated SPIONS (Tx@ReoPro) can effectively prevent thrombus growth by inhibiting fibrinogen receptors (GPIIbIIIa) on the platelet surface, and simultaneously, it can also be visible through non-invasive magnetic resonance imaging (MRI) for potential reporting of the real-time thrombus status.

Since their introduction, tyrosine kinase inhibitors (TKIs, eg, imatinib, nilotinib, dasatinib, bosutinib, ponatinib) have revolutionized the treatment of chronic myeloid leukemia (CML). However, long-term treatment with TKIs is associated with serious adverse events including both bleeding and thromboembolism. Experimental studies have shown that TKIs can cause platelet dysfunction. Herein, we present the first side-by-side investigation comparing the effects of currently used TKIs on platelet function and thrombin generation when used in clinically relevant concentrations. A flow cytometry multiparameter protocol was used to study a range of significant platelet activation events (fibrinogen receptor activation, alpha granule, and lysosomal exocytosis, procoagulant membrane exposure, and mitochondrial permeability changes). In addition, thrombin generation was measured in the presence of TKIs to assess the effects on global hemostasis. Results show that dasatinib generally inhibited platelet function, while bosutinib, nilotinib, and ponatinib showed less consistent effects. In addition to these general trends for each TKI, we observed a large degree of interindividual variability in the effects of the different TKIs. Interindividual variation was also observed when blood from CML patients was studied ex vivo with whole blood platelet aggregometry, free oscillation rheometry (FOR), and flow cytometry. Based on the donor responses in the side-by-side TKI study, a TKI sensitivity map was developed. We propose that such a sensitivity map could potentially become a valuable tool to help in decision-making regarding the choice of suitable TKIs for a CML patient with a history of bleeding or atherothrombotic disease.

The recent claim that stimulated platelets activate the intrinsic pathway of coagulation by the release of polyphosphates has been considered a breakthrough in hemostasis research. In little more than 3 years, the original publication by Müller et al has been cited >100 times. However, none of the citing articles has sought to independently validate this potentially paradigm-shifting concept. To this end, we performed extensive experimentation in vitro and in vivo in an attempt to verify the claim that factor XII (FXII) is primarily activated by stimulated platelets. In contrast to the original assertion, platelet-derived polyphosphates were found to be weak activators of FXII, with a FXIIa-generating activity of <10% compared with equivalent concentrations of kaolin. Using different coagulation assays, it was shown that platelet contribution to whole blood coagulation was unrelated to the generation of activated FXII in vitro. Additionally, key results used to verify the hypothesis in the original study in vivo were found to be irreproducible. We conclude that platelet-derived polyphosphates are not physiologically relevant activators of FXII.

Flow cytometry is increasingly used in the study of platelets in inherited and acquired disorders of platelet number and function. However, wide variation exists in specific reagents, methods, and equipment used, making interpretation and comparison of results difficult. The goal of the present study was to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet disorders. A modified RAND/UCLA survey method was used to obtain a consensus among 11 experts from 10 countries across four continents, on the appropriateness of statements relating to clinical utility, pre-analytical variables, instrument and reagent standardization, methods, reporting, and quality control for platelet flow cytometry. Feedback from the initial survey revealed that uncertainty was sometimes due to lack of expertise with a particular test condition rather than unavailable or ambiguous data. To address this, the RAND method was modified to allow experts to self-identify statements for which they could not provide expert input. There was uniform agreement among experts in the areas of instrument and reagent standardization, methods, reporting, and quality control and this agreement is used to suggest best practices in these areas. However, 25.9% and 50% of statements related to pre-analytical variables and clinical utility, respectively, were rated as uncertain. Thus, while citrate is the preferred anticoagulant for many flow cytometric platelet tests, expert opinions differed on the acceptability of other anticoagulants, particularly heparin. Lack of expert consensus on the clinical utility of many flow cytometric platelet tests indicates the need for rigorous multicenter clinical outcome studies.

BACKGROUND: Procoagulant platelets are a subpopulation of highly activated platelets that promote coagulation through surface-exposed, negatively charged phospholipids, especially phosphatidylserine (PS). Procoagulant platelets are important for clot stabilization during haemostasis and an increased number of these platelets is associated with thrombotic risk. There is a need for harmonisation in this area since many of the markers and methods used to assess procoagulant platelets are not specific when used in isolation but are also associated with platelet apoptosis.

OBJECTIVE: We initiated this project to identify a minimum set of markers and/or methods that can detect and distinguish procoagulant platelets from apoptotic platelets.

METHODS AND RESULTS: The study design involved a primary panel with twenty-seven international experts participating in an online survey and moderated virtual focus group meetings. Primary and secondary panel members were then invited to provide input on themes and statements generated from the focus groups. This led to a recommendation to use flow cytometry and a combination of the following three surface markers to differentiate procoagulant from apoptotic platelets: P-selectin (CD62P), PS (recognized by annexin V), and a platelet-specific receptor GPIX (CD42a) or αIIb integrin (CD41, GPIIb).

CONCLUSION: Procoagulant platelets are expected to be positive for all three markers, while apoptotic platelets will be positive for annexin V and the platelet specific surface receptor(s) but negative for P-selectin.

Background: Platelet count alone does not reliably predict bleeding risk, suggesting platelet function is important to monitor in patients with thrombocytopenia. There is still an unmet need for improved platelet function diagnostics in patients with low platelet count in many clinical situations. Flow cytometry is a promising tool allowing reliable platelet function study in this setting.

Objectives: The goal of this joint project between the International Society on Thrombosis and Haemostasis (ISTH) Scientific Standardization Committee (SSC) Subcommittees on Platelet Physiology and Platelet Immunology is to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet function, particularly activation, in patients with low platelet counts.

Methods: A literature review was performed to identify relevant questions and areas of interest. An electronic expression of interest form was thereafter announced on the ISTH webpage, followed by a survey encompassing 37 issues regarding preanalytical, analytical, postanalytical, and performance aspects. Areas of disagreement or uncertainty were identified and formed the basis for 2 focus group discussions.

Results: Consensus recommendations relative to patient sample collection, preanalytical variables, sample type, platelet-count cutoff, any potential specific modification of the standard flow cytometry protocol, and results expression and reporting are proposed based on the current practices of experts in the field as well as on literature review.

Conclusion: The proposed consensus recommendations would allow standardization of protocols in upcoming clinical studies. The clinical utility of platelet function testing using flow cytometry to predict bleeding risk still needs rigorous multicenter outcome studies in patients with thrombocytopenia.

Platelet-derived polyphosphates (polyP), stored in dense granule and released upon platelet activation, have been claimed to enhance thrombin activation of coagulation factor XI (FXI) and to activate FXII directly. The latter claim is controversial and principal results leading to these conclusions are probably influenced by methodological problems. It is important to consider that low-grade contact activation is initiated by all surfaces and is greatly amplified by the presence of phospholipids simulating the procoagulant membranes of activated platelets. Thus, proper use of inhibitors of the contact pathway and a careful choice of materials for plates and tubes is important to avoid artefacts. The use of phosphatases used to degrade polyP has an important drawback as it also degrades the secondary activators ADP and ATP, which are released from activated platelets. In addition, the use of positively charged inhibitors, such as polymyxin B, to inhibit polyP in platelet-rich plasma and blood is problematic, as polymyxin B also slows coagulation in the absence of polyP. In conclusion we hope awareness of the above caveats may improve research on the physiological roles of polyP in coagulation.

Widespread monitoring of platelet function and the effect of antiplatelet drugs will improve outcomes in cardiovascular patients, but platelet function testing is not routine in clinical practice. We report a rapid, accurate methodology to quantify platelet-protein interactions: a microarray of contact-printed 6-mu m fibrinogen dots on a transparent substrate binds platelets from whole blood, one platelet per dot. The fractional occupancy of an array of fibrinogen dots after a predefined incubation time quantitatively assays platelet adhesion to the protein matrix. We demonstrate this technique by measurement of platelet adhesion to fibrinogen as a means to quantify the effect of the P2Y(12) and alpha IIb beta 3 receptor inhibitors cangrelor and abciximab, respectively, both in vitro-by incubating the drug with a freshly drawn blood sample-and in blood from patients treated with antiplatelet agents. The effects of single- and dual-antiplatelet therapy are also assessed. Results from this platelet-binding assay are well correlated with standard techniques including flow cytometry and light transmission aggregometry. This assay technology, readily integrated with microfluidic platforms, is generally applicable to the assay of cell-protein interactions and promises more effective, rapid assay of drug effects in cardiovascular disease patients.

As platelet activation is an irreversible and potentially harmful event, platelet stimulatory signaling must be tightly regulated to ensure the filtering-out of inconsequential fluctuations of agonist concentrations in the vascular milieu. Herein, we show that platelet activation via G protein-coupled receptors is gradient-dependent that is determined not only by agonist concentrations per se but also by how rapidly concentrations change over time. We demonstrate that gradient-dependent inhibition is a common feature of all major platelet stimulatory G protein-coupled receptors, while platelet activation via the non- G protein-coupled receptor glycoprotein VI is strictly concentration-dependent. By systematically characterizing the effects of variations in temporal agonist concentration gradients on different aspects of platelet activation, we demonstrate that gradient-dependent inhibition of protease-activated receptors exhibit different kinetics, with platelet activation occurring at lower agonist gradients for protease-activated receptor 4 than for protease-activated receptor 1, but share a characteristic bimodal effect distribution, as gradient-dependent inhibition increases over a narrow range of gradients, below which aggregation and granule secretion is effectively shut off. In contrast, the effects of gradient-dependent inhibition on platelet activation via adenosine diphosphate and thromboxane receptors increase incrementally over a large range of gradients. Further, depending on the affected activation pathway, gradient-dependent inhibition results in different degrees of refractoriness to subsequent autologous agonist stimulation. Mechanistically, our study identifies an important role for the cyclic adenosine monophosphate-dependent pathway in gradient-dependent inhibition. Together, our findings suggest that gradient-dependent inhibition may represent a new general mechanism for hemostatic regulation in platelets.

Introduction: Arterial thrombi contain more platelets than venous thrombi and are more resistant to fibrinolysis. This resistance could partly be due to plasminogen activator inhibitor 1 (PAI-1) secreted by platelets. The aim of this study was to elucidate differences between thrombin receptors protease-activated receptor (PAR) 1 and 4 and platelet storage, secretion and synthesis of platelet PAI-1, as compared to other platelet alpha-granule proteins such as VEGF and endostatin.

Materials and methods: Human isolated platelets were incubated with thrombin (0.5 U/ml), PAR1-activating peptide (AP) (0.4-30 mu M) or PAR4-AP (1.5-300 mu M) for up to 24 hours. ELISA, western blot and fluorescence microscopy were used to measure secretion, contents and localization of PAI-1, VEGF and endostatin. Results: Our results show that PAI-1 and VEGF might be co-localized and that endostatin does not co-localize with either PAI-1 or VEGF. PAI-1 and VEGF show a similar secretion pattern, being more sensitive to low grade PAR1 activation, but secretion was also observed with higher concentrations of PAR4-APs. PAI-1 is secreted in an active form. PAI-1 mRNA was found in platelets, and elevated levels of PAI-1 were detected after 24 hours incubation of platelets.

Conclusions: PAI-1 and VEGF, but not endostatin, might be stored in the same alpha-granule in human platelets. PAI-1 and VEGF also show a similar secretion pattern, being more sensitive to PAR1 than to PAR4 activation, but the secretion is not exclusively selective. Our results also show that platelet PAI-1 is increased if incubated for 24 hours, both with addition of PAR1-activating peptide and without activation, which could indicate de novo synthesis.

Introduction: Residual pump blood from the cardiopulmonary bypass (CPB) circuit is often collected into an infusion bag (IB) and re-transfused. An alternative is to chase the residual blood into the circulation through the arterial cannula with Ringer’s acetate. Our aim was to assess possible differences in hemostatic blood quality between these two techniques.

Methods: Forty adult patients undergoing elective coronary artery bypass graft surgery with CPB were randomized to receive the residual pump blood by either an IB or through the Ringer’s chase (RC) technique. Platelet activation and function (impedance aggregometry), coagulation and hemolysis variables were assessed in the re-transfused blood and in the patients before, during and after surgery. Results are presented as median (25-75 quartiles).

Results: Total hemoglobin and platelet levels in the re-transfused blood were comparable with the two methods, as were soluble platelet activation markers P-selectin and soluble glycoprotein VI (GPVI). Platelet aggregation (U) in the IB blood was significantly lower compared to the RC blood, with the agonists adenosine diphosphate (ADP) 24 (10-32) vs 46 (33-65), p<0.01, thrombin receptor activating peptide (TRAP) 50 (29-73) vs 69 (51-92), p=0.04 and collagen 24 (17-28) vs 34 (26-59), p<0.01. The IB blood had higher amounts of free hemoglobin (mg/L) (1086 (891-1717) vs 591(517-646), p<0.01) and D-dimer 0.60 (0.33-0.98) vs 0.3 (0.3-0.48), p<0.01. Other coagulation variables showed no difference between the groups. Conclusions: The handling of blood after CPB increases hemolysis, impairs platelet function and activates coagulation and fibrinolysis. The RC technique preserved the blood better than the commonly used IB technique.

Introduction: Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated.

Methods: The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry.

Results: PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca2+ mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca2+ chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it.

Conclusion: The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca2+ signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.

The search for biomarkers associated with obesity-related diseases is ongoing, but it is not clear whether plasma and serum can be used interchangeably in this process. Here we used high-throughput screening to analyze 358 proteins and 76 lipids, selected because of their relevance to obesity-associated diseases, in plasma and serum from age- and sex-matched lean and obese humans. Most of the proteins/lipids had similar concentrations in plasma and serum, but a subset showed significant differences. Notably, a key marker of cardiovascular disease PAI-1 showed a difference in concentration between the obese and lean groups only in plasma. Furthermore, some biomarkers showed poor correlations between plasma and serum, including PCSK9, an important regulator of cholesterol homeostasis. Collectively, our results show that the choice of biofluid may impact study outcome when screening for obesity-related biomarkers and we identify several markers where this will be the case.

The use of arachidonic acid (AA) to stimulate platelets is considered as a specific approach to study aspirin treatment efficacy. However, very high concentrations of AA are used, and it has been previously reported that AA can induce cell lysis in other settings. Several clinical studies have reported decreased responses to AA in whole blood tests in the presence of clopidogrel. Our aim was to investigate whether unspecific effects contribute to AA-induced aggregation and platelet activation in light transmission aggregometry (LTA) in platelet-rich plasma (PRP), and in assays using whole blood, multiple electrode aggregometry (MEA, Multiplate®), and flow cytometry. We report that cell lysis, especially of red blood cells, does occur at concentrations of AA used in the clinical tests and that ADP is very important for the AA-induced platelet activation responses. In flow cytometry, very limited platelet activation was detected before reaching AA concentrations in the millimolar range, where cell lysis also occurred, making it problematic to develop a reliable flow cytometry assay using AA as reagent. We conclude that cell lysis and ADP release contribute to AA-induced platelet responses, most markedly in whole blood assays. This finding could potentially explain some differences between studies comparing methods using whole blood and PRP and also how clopidogrel treatment could influence AA-induced aggregation results in previously published studies. Our findings highlight some issues with AA as reagent for platelet activation, which also have an impact on how platelet activation assays using AA should be interpreted.

Platelets bind annexin V when stimulated with combinations of platelet agonists such as collagen and thrombin. Previous studies have demonstrated significant heterogeneity of platelets binding annexin V. The relative role of the thrombin protease-activated receptors (PARs), PAR1 and PAR4, together with different methods of platelet preparation on annexin V binding to platelets is unclear. We therefore investigated the role of PAR1- and PAR4-activating peptides in combination with collagen-related peptide on annexin V binding. In diluted whole blood, PAR1- and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding. However, in washed platelets, PAR-activating peptides were less potent than thrombin at inducing annexin V binding. This difference was more pronounced when experiments were performed at 37 degrees C compared to room temperature. In studies of diluted whole blood, platelet rich plasma and washed platelets, platelets incubated at room temperature bound more annexin V than platelets incubated at 37 degrees C. We also saw a significant effect of time on annexin V binding, in that more annexin V bound to platelets with longer incubation times. In conclusion, PAR1 and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding in combination with collagen-related peptide in diluted whole blood and platelet rich plasma, but not in washed platelets. In addition, incubation temperature and time has a strong influence on annexin V binding to platelets. Thus variations in these conditions may explain the differences observed between previous studies.

Flow cytometry enables studies of several different aspects of platelet function in response to a variety of platelet agonists. This can be done using only a small volume of whole blood, and also in blood with low platelet counts. These properties, together with the increasing number of flow cytometers available in hospitals worldwide, make flow cytometry an interesting option for laboratories interested in studies of platelet function in different clinical settings. This review focuses on practical issues regarding the use of flow cytometry for platelet function testing. It provides an overview of available activation markers, platelet agonists, and experimental setup issues. The review summarizes previous experience and factors important to consider to perform high-quality platelet function testing by flow cytometry. It also discusses its current use and possibilities and challenges for future use of flow cytometry in clinical settings.

BACKGROUND: Administration of agents that enhance platelet reactivity may reduce the perioperative bleeding risk in patients treated with the adenosine diphosphate (ADP)-receptor antagonist ticagrelor. Adrenaline potentiates ADP-induced aggregation and activation in blood samples from ticagrelor-treated patients, but it has not previously been evaluated in vivo.

METHODS: Ten healthy male subjects were included in an interventional study. A loading dose of ticagrelor (180 mg) was administered, followed 2 hours later by a gradually increased intravenous adrenaline infusion (0.01, 0.05, 0.10 and 0.15 µg/kg/min; 15 minutes at each step). Blood pressure, heart rate, platelet aggregation (impedance aggregometry), platelet activation (flow cytometry), clot formation (rotational thromboelastometry) and adrenaline plasma concentration were determined before and after ticagrelor administration and at the end of each adrenaline step.

RESULTS:  = 0.007).

CONCLUSION: Infusion of adrenaline at clinically relevant doses improves in vivo platelet reactivity and clot formation in ticagrelor-treated subjects. Adrenaline could thus potentially be used to prevent perioperative bleeding complications in ticagrelor-treated patients. Studies in patients are necessary to determine the clinical importance of our observations.

TRIAL REGISTRY NUMBER: ClinicalTrials.gov NCT03441412.