Dissecting thrombus-directed chemotaxis and random movement in neutrophil near-thrombus motion in flow chambers.

BACKGROUND: Thromboinflammation is caused by mutual activation of platelets and neutrophils. The site of thromboinflammation is determined by chemoattracting agents release by endothelium, immune cells, and platelets. Impaired neutrophil chemotaxis contributes to the pathogenesis of Shwachman-Diamond syndrome (SDS). In this hereditary disorder, neutrophils are known to have aberrant chemoattractant-induced F-actin properties. Here, we aim to determine whether neutrophil chemotaxis could be analyzed using our previously developed ex vivo assay of the neutrophils crawling among the growing thrombi. METHODS: Adult and pediatric healthy donors, alongside with pediatric patients with SDS, were recruited for the study. Thrombus formation and granulocyte movement in hirudinated whole blood were visualized by fluorescent microscopy in fibrillar collagen-coated parallel-plate flow chambers. Alternatively, fibrinogen, fibronectin, vWF, or single tumor cells immobilized on coverslips were used. A computational model of chemokine distribution in flow chamber with a virtual neutrophil moving in it was used to analyze the observed data. RESULTS: The movement of healthy donor neutrophils predominantly occurred in the direction and vicinity of thrombi grown on collagen or around tumor cells. For SDS patients or on coatings other than collagen, the movement was characterized by randomness and significantly reduced velocities. Increase in wall shear rates to 300-500 1/s led to an increase in the proportion of rolling neutrophils. A stochastic algorithm simulating leucocyte chemotaxis movement in the calculated chemoattractant field could reproduce the experimental trajectories of moving neutrophils for 72% of cells. CONCLUSIONS: In samples from healthy donors, but not SDS patients, neutrophils move in the direction of large, chemoattractant-releasing platelet thrombi growing on collagen.

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

BACKGROUND: Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. METHODS: Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. RESULTS: We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. CONCLUSIONS: Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. IMPACT: Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

Ex vivo observation of granulocyte activity during thrombus formation.

BACKGROUND: The process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors. RESULTS: Thrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s-1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90-120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients' platelets were replaced with healthy donor's platelets the granulocytes' crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes' behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers. CONCLUSIONS: We have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.

Longitudinal multiparametric characterization of platelet dysfunction in COVID-19: Effects of disease severity, anticoagulation therapy and inflammatory status.

INTRODUCTION: Defects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19. MATERIALS AND METHODS: Analysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed. RESULTS AND CONCLUSIONS: All patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber. We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus.

Platelet function and bleeding at different phases of childhood immune thrombocytopenia.

Immune thrombocytopenia (ITP) is believed to be associated with platelet function defects. However, their mechanisms are poorly understood, in particular with regard to differences between ITP phases, patient age, and therapy. We investigated platelet function and bleeding in children with either persistent or chronic ITP, with or without romiplostim therapy. The study included 151 children with ITP, of whom 56 had disease duration less than 12 months (grouped together as acute/persistent) and 95 were chronic. Samples of 57 healthy children were used as controls, while 5 patients with leukemia, 5 with aplastic anemia, 4 with MYH9-associated thrombocytopenia, and 7 with Wiskott-Aldrich syndrome were used as non-ITP thrombocytopenia controls. Whole blood flow cytometry revealed that platelets in both acute/persistent and chronic ITP were increased in size compared with healthy donors. They were also pre-activated as assessed by PAC1, CD62p, cytosolic calcium, and procoagulant platelet levels. This pattern was not observed in other childhood thrombocytopenias. Pre-activation by CD62p was higher in the bleeding group in the chronic ITP cohort only. Romiplostim treatment decreased size and pre-activation of the patient platelets, but not calcium. Our data suggest that increased size, pre-activation, and cytosolic calcium are common for all ITP platelets, but their association with bleeding could depend on the disease phase.

Effects of bacterial lipopolysaccharides on platelet function: inhibition of weak platelet activation.

Platelets are anucleate blood cells with reported roles in hemostasis and immune responses, which possess a functional receptor for bacterial lipopolysaccharides (LPSs), the well-known inducers of inflammation. However, LPSs effects on platelets are contradictory. Here we aim to investigate mechanisms of platelet functioning in the presence of LPS and to find the cause of the discrepancy in the previously published data. Cell activity was analyzed by flow cytometry, western blotting, and aggregometry. Thrombus growth was assessed by fluorescent microscopy. LPS' activity was checked by their capability to induce PMN activation. However, LPSs did not substantially affect either thrombus growth in flow chambers, irreversible platelet aggregation, or platelet responses to strong activation. Platelet aggregation in response to 1 µM of ADP was significantly inhibited by LPSs. Flow cytometry analysis revealed that platelet activation responses to weak stimulation were also diminished by LPSs, while VASP phosphorylation was weakly increased. Additionally, LPSs were capable of inhibition of ADP-induced P2-receptor desensitization. Incubation of platelets with a pan-PDE inhibitor IBMX significantly enhanced the LPSs-induced platelet inhibition, implying cAMP/cGMP dependent mechanism. The discrepancy in the previously published data could be explained by LPS-induced weak inhibition of platelet activation and the prevention of platelet desensitization.

Development of a Simple Kinetic Mathematical Model of Aggregation of Particles or Clustering of Receptors.

The process of clustering of plasma membrane receptors in response to their agonist is the first step in signal transduction. The rate of the clustering process and the size of the clusters determine further cell responses. Here we aim to demonstrate that a simple 2-differential equation mathematical model is capable of quantitative description of the kinetics of 2D or 3D cluster formation in various processes. Three mathematical models based on mass action kinetics were considered and compared with each other by their ability to describe experimental data on GPVI or CR3 receptor clustering (2D) and albumin or platelet aggregation (3D) in response to activation. The models were able to successfully describe experimental data without losing accuracy after switching between complex and simple models. However, additional restrictions on parameter values are required to match a single set of parameters for the given experimental data. The extended clustering model captured several properties of the kinetics of cluster formation, such as the existence of only three typical steady states for this system: unclustered receptors, receptor dimers, and clusters. Therefore, a simple kinetic mass-action-law-based model could be utilized to adequately describe clustering in response to activation both in 2D and in 3D.

Control of Platelet CLEC-2-Mediated Activation by Receptor Clustering and Tyrosine Kinase Signaling.

Platelets are blood cells responsible for vascular integrity preservation. The activation of platelet receptor C-type lectin-like receptor II-type (CLEC-2) could partially mediate the latter function. Although this receptor is considered to be of importance for hemostasis, the rate-limiting steps of CLEC-2-induced platelet activation are not clear. Here, we aimed to investigate CLEC-2-induced platelet signal transduction using computational modeling in combination with experimental approaches. We developed a stochastic multicompartmental computational model of CLEC-2 signaling. The model described platelet activation beginning with CLEC-2 receptor clustering, followed by Syk and Src family kinase phosphorylation, determined by the cluster size. Active Syk mediated linker adaptor for T cell protein phosphorylation and membrane signalosome formation, which resulted in the activation of Bruton's tyrosine kinase, phospholipase and phosphoinositide-3-kinase, calcium, and phosphoinositide signaling. The model parameters were assessed from published experimental data. Flow cytometry, total internal reflection fluorescence and confocal microscopy, and western blotting quantification of the protein phosphorylation were used for the assessment of the experimental dynamics of CLEC-2-induced platelet activation. Analysis of the model revealed that the CLEC-2 receptor clustering leading to the membrane-based signalosome formation is a critical element required for the accurate description of the experimental data. Both receptor clustering and signalosome formation are among the rate-limiting steps of CLEC-2-mediated platelet activation. In agreement with these predictions, the CLEC-2-induced platelet activation, but not activation mediated by G-protein-coupled receptors, was strongly dependent on temperature conditions and cholesterol depletion. Besides, the model predicted that CLEC-2-induced platelet activation results in cytosolic calcium spiking, which was confirmed by single-platelet total internal reflection fluorescence microscopy imaging. Our results suggest a refined picture of the platelet signal transduction network associated with CLEC-2. We show that tyrosine kinase activation is not the only rate-limiting step in CLEC-2-induced activation of platelets. Translocation of receptor-agonist complexes to the signaling region and linker adaptor for T cell signalosome formation in this region are limiting CLEC-2-induced activation as well.

Heterogeneity of Integrin αIIbß3 Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis.

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin αIIbß3 functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group.

Quantitative dynamics of reversible platelet aggregation: mathematical modelling and experiments.

Although reversible platelet aggregation observed in response to ADP stimulation in the presence of calcium is a well-known phenomenon, its mechanisms are not entirely clear. To study them, we developed a simple kinetic mass-action-law-based mathematical model to use it in combination with experiments. Light transmission platelet aggregometry (LTA) induced by ADP was performed for platelet-rich plasma or washed platelets using both conventional light transmission and aggregate size monitoring method based on optical density fluctuations. Parameter values of the model were determined by means of parameter estimation techniques implemented in COPASI software. The mathematical model was able to describe reversible platelet aggregation LTA curves without assuming changes in platelet aggregation parameters over time, but with the assumption that platelet can enter the aggregate only once. In the model, the mean size of platelet aggregates correlated with the solution transparency. This corresponded with flow cytometry analysis and with optical density fluctuations data on aggregate size. The predicted values of model parameters correlated with ADP concentration used in experiments. These data suggest that, at the start of the aggregation, when platelet integrins switch "on", large unstable platelet aggregates are rapidly formed, which leads to an increase in light transmission. However, upon fragmentation of these aggregates, the probability of the post-aggregate platelets' attachment to each other decreases preventing new aggregation and resulting in the reversible aggregation phenomenon.