Cisplatin Resistance and Metabolism: Simplification of Complexity.

Cisplatin is one of the most well-known anti-cancer drugs and has demonstrated efficacy against numerous tumor types for many decades. However, a key challenge with cisplatin, as with any chemotherapeutic agent, is the development of resistance with a resultant loss of efficacy. This resistance is often associated with metabolic alterations that allow insensitive cells to divide and survive under treatment. These adaptations could vary greatly among different tumor types and may seem questionable and incomprehensible at first glance. Here we discuss the disturbances in glucose, lipid, and amino acid metabolism in cisplatin-resistant cells as well as the roles of ferroptosis and autophagy in acquiring this type of drug intolerance.

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.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors.

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3-5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

Kinetics and regulation of coagulation factor X activation by intrinsic tenase on phospholipid membranes.

BACKGROUND: Factor X activation by the phospholipid-bound intrinsic tenase complex is a critical membrane-dependent reaction of blood coagulation. Its regulation mechanisms are unclear, and a number of questions regarding diffusional limitation, pathways of assembly and substrate delivery remain open. METHODS: We develop and analyze here a detailed mechanism-driven computer model of intrinsic tenase on phospholipid surfaces. Three-dimensional reaction-diffusion-advection and stochastic simulations were used where appropriate. RESULTS: Dynamics of the system was predominantly non-stationary under physiological conditions. In order to describe experimental data, we had to assume both membrane-dependent and solution-dependent delivery of the substrate. The former pathway dominated at low cofactor concentration, while the latter became important at low phospholipid concentration. Factor VIIIa-factor X complex formation was the major pathway of the complex assembly, and the model predicted high affinity for their lipid-dependent interaction. Although the model predicted formation of the diffusion-limited layer of substrate for some conditions, the effects of this limitation on the fXa production were small. Flow accelerated fXa production in a flow reactor model by bringing in fIXa and fVIIIa rather than fX. CONCLUSIONS: This analysis suggests a concept of intrinsic tenase that is non-stationary, employs several pathways of substrate delivery depending on the conditions, and is not particularly limited by diffusion of the substrate.

Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

OBJECTIVES: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. METHODS: For activation, 2.5 µM CaCl2 , 5 µM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. RESULTS: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. CONCLUSIONS: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

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.

Platelet Hemostasis Reactions at Different Temperatures Correlate with Intracellular Calcium Concentration.

Hypo- and hyperthermia affect both primary and secondary hemostasis; however, there are controversial data concerning platelet activation and the underlying mechanisms under hypo- and hyperthermia. The discrepancies in the data could be partly explained by different approaches to hemostatic reactions analysis. We applied a new LaSca-TMF laser particle analyzer for a simultaneous fluorescence and laser scattering analysis of platelet responses at different temperatures. Human platelets were activated by ADP in a wide range of temperatures, and platelet transformations (e.g., a shape change reaction, aggregation and clot formation) and the intracellular calcium concentration ([Ca2+]i) were analyzed by LaSca-TMF and confocal microscopy. The platelet shape change reaction gradually increased with a rising temperature. The platelet aggregation strongly decreased at low ADP concentrations with the augmentation of the temperature and was independent of the temperature at high ADP concentrations. In contrast, the clotting time decreased with a temperature increase. Similar to the aggregation response, a rise in [Ca2+]i triggered by low ADP concentrations was higher under hypothermic conditions and the differences were independent of the temperature at high ADP concentrations. We showed that the key reactions of cellular hemostasis are differentially regulated by temperature and demonstrated for the first time that an accelerated aggregation under hypothermic conditions directly correlated with an increased level in [Ca2+]i in platelets.

Healthy pediatric platelets are moderately hyporeactive in comparison with adults' platelets.

Studies on platelet function in children older than neonatal period are few and their results are controversial. The pediatric platelets were alternatively reported to be more active or less active than adults' ones. We compared platelet function in the several age groups of children to adults and evaluated the age when platelet function reaches the adults' status. The study included 76 healthy children and 49 healthy adult volunteers. Types of platelet activation used included: collagen-related peptide (CRP) and PAR-1 activating peptide SFLLRN; SFLLRN, PAR-4 activating peptide AYPGKF and adenosine diphosphate (ADP); ADP. The parameters determined included forward (FSC) and side scatter (SSC), CD42b, CD61, CD62P, PAC-1, annexin V binding and mepacrine release levels. Resting pediatric platelets were similar to adults' platelets except for 1.2-fold decreased FSC and dense granules volume in youngest children, and 2.5-fold increased annexin V level in children aged 1-10 years. After CRP+SFLLRN stimulation, pediatric platelets had a 1.2-fold lower alpha- and 1.1-fold lower dense granule release than adults. For SFLLRN+AYPGKF+ADP stimulation, this was observed only for youngest children. The response to ADP stimulation was identical for pediatric platelets and adults. Pediatric platelets have lower granular release than adults' platelets, which persists until the age of 18.

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.

In vitro megakaryocyte culture from human bone marrow aspirates as a research and diagnostic tool.

Megakaryocytes (MKs) are relatively rare in bone marrow, comprising <0.05% of the nucleated cells, which makes direct isolation from human bone marrow impractical. As such, in vitro expansion of primary MKs from patient samples offers exciting fundamental and clinical opportunities. As most of the developed ex vivo methods require a substantial volume of biomaterial, they are not widely performed on young patients. Here we propose a simple, robust, and adapted method of primary human MK culture from 1 mL of bone marrow aspirate. Our technique uses a small volume of bone marrow per culture, uses straightforward isolation methods, and generates approximately 6 × 105 mature MKs per culture. The relative high cell purity and yield achieved by this technique, combined with efficient use of low volumes of bone marrow, make this approach suitable for diagnostic and basic research of human megakaryopoiesis.

Determination of fibrin clot growth and spatial thrombin propagation in the presence of different types of phospholipid surfaces.

In this work, we present a new method-Thrombodynamics-4D-for the assessment of both plasma and platelet contributions to clotting. Thrombodynamics-4D potentially allows for the determination of plasma or platelet disorders and the effects of various drugs on plasma clotting or on platelet procoagulant function. In this assay, clot formation in platelet-rich plasma or platelet-free plasma supplemented with phospholipids is activated with tissue factor immobilized on a surface. Spatial fibrin clot growth and thrombin concentration dynamics are registered by measuring light scattering of the fibrin clot and fluorescence of the product formed by cleavage of the synthetic fluorogenic substrate by thrombin, respectively. Here, we describe the preanalytical requirements, measurement methodology and calculation principles of assay parameters. Preanalytical and analytical variability and reference ranges of the assay are given. Additionally, we show some clinical examples, which determine the effect of anticoagulants, measure clotting dysfunction in patients with platelet or coagulation disorders and evaluate the effect of surgery.

Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

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.

Platelet-derived extracellular vesicles infiltrate and modify the bone marrow during inflammation.

During inflammation, steady-state hematopoiesis switches to emergency hematopoiesis to repopulate myeloid cells, with a bias toward the megakaryocytic lineage. Soluble inflammatory cues are thought to be largely responsible for these alterations. However, how these plasma factors rapidly alter the bone marrow (BM) is not understood. Inflammation also drives platelet activation, causing the release of platelet-derived extracellular vesicles (PEVs), which package diverse cargo and reprogram target cells. We hypothesized that PEVs infiltrate the BM, providing a direct mode of communication between the plasma and BM environments. We transfused fluorescent, wild-type (MPL+) platelets into recipient cMpl-/-mice before triggering systemic inflammation. Twenty hours postinfusion, we observed significant infiltration of donor platelet-derived particles in the BM, which we tracked immunophenotypically (MPL+ immunohistochemistry staining) and quantified by flow cytometry. To determine if this phenomenon relates to humans, we extensively characterized both megakaryocyte-derived and PEVs generated in vitro and in vivo, and found enrichment of extracellular vesicles in bone marrow compared with autologous peripheral blood. Last, BM from cMpl-/- mice was cultured in the presence or absence of wild-type (MPL+) PEVs. After 72 hours, flow cytometry revealed increased megakaryocytes only in cultures with added PEVs. The majority of CD41+ cells were bound to PEVs, suggesting a PEV-mediated rescue of megakaryopoiesis. In conclusion, we report for the first time that plasma-residing PEVs infiltrate the BM. Further, PEVs interact with BM cells in vivo and in vitro, causing functional reprogramming that may represent a novel model of inflammation-induced hematopoiesis.

Platelet function and bleeding in chronic lymphocytic leukemia and mantle cell lymphoma patients on ibrutinib.

BACKGROUND: Therapy with irreversible Bruton's tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) is associated with bleeding. OBJECTIVES: To propose the predictive markers of such bleeding, as well as mechanisms responsible for decreased bleeding at later therapy stages. PATIENTS/METHODS: We investigate platelet functional activity in 50 CLL and 16 MCL patients on ibrutinib using flow cytometry and light transmission aggregometry. RESULTS: Prior to treatment, both patient groups had decreased platelet counts; impaired aggregation with adenosine diphosphate (ADP); and decreased binding of CD62P, PAC1, and annexin V upon stimulation. Bleeding in patients treated with ibrutinib was observed in 28 (56%) CLL patients, who had decreased aggregation with ADP and platelet count before therapy. Their platelet count on therapy did not change, platelet aggregation with ADP steadily improved, and aggregation with collagen first decreased and then increased in anticorrellation with bleeding. Bleeding in MCL was observed in 10 (62%) patients, who had decreased dense granule release before therapy. ADP and ristocetin induced platelet aggregation in ibrutinib-treated MCL patients increased on therapy, while collagen-induced aggregation evolved similarly to CLL patients. CONCLUSIONS: Our results suggest that ibrutinib-dependent bleeding in CLL patients involves three mechanisms: decreased platelet count (the most important discriminator between bleeding and non-bleeding patients), impaired platelet response to ADP caused by CLL, and inhibition by ibrutinib. Initially, ibrutinib shifts the balance to bleeding, but then it is restored because of the improved response to ADP.

Platelet function and blood coagulation system status in childhood essential thrombocythemia.

Childhood essential thrombocythemia (ET) is a rare chronic myeloproliferative disorder. The quality of life of ET patients may decrease as a result of ischemic and hemorrhagic complications of unclear origin. Our goal was to characterize the hemostatic system in children with ET. We genotyped and investigated blood samples from 20 children with ET in a prospective case series study using platelet aggregation, functional flow cytometry (FC) assay and standard clotting assays. Three children had a JAK2V617F mutation, 4 had mutations in CALR and 13 were triple-negative. Myelofibrosis in stage 1-2 was detected in 3 children. Three patients had bleeding episodes and seven had ischemic events. Aggregation in response to collagen, adenosine diphosphate, and ristomycin was decreased in all patients. In FC, significant changes in the whole patient group compared to the healthy children control group were decrease in the resting forward scatter and PAC1 binding (activated GPIIb/IIIa) level. For the activated platelets, dense granules release (by mepacrine), PAC1, and GPIIb/IIIa levels were significantly decreased. GPIb/V/IX, P-selectin, and phosphatidylserine levels manifested only moderate differences. Forward and side scatter changes in response to stimulation (representing shape change) and dense granules release were significantly lower in the 3 patients with bleeding than in the 17 patients without hemorrhage. Activated partial thromboplastin time was slightly prolonged, prothrombin index was slightly shortened and thrombin time was normal, while fibrinogen was mildly decreased in the ET patients. It could be concluded that the observed platelet function defects could be related to bleeding in ET, and be potentially used as a marker.

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.

Flow cytometry for pediatric platelets.

The ability of platelets to carry out their hemostatic function can be impaired in a wide range of inherited and acquired conditions: trauma, surgery, inflammation, pre-term birth, sepsis, hematological malignancies, solid tumors, chemotherapy, autoimmune disorders, and many others. Evaluation of this impairment is vitally important for research and clinical purposes. This problem is particularly pronounced in pediatric patients, where these conditions occur frequently, while blood volume and the choice of blood collection methods could be limited. Here we describe a simple flow cytometry-based screening method of comprehensive whole blood platelet function testing that was validated for a range of pediatric and adult samples (n = 31) in the hematology hospital setting including but not limited to: classic inherited platelet function disorders (Glanzmann's thrombasthenia; Bernard-Soulier, Wiscott-Aldrich, and Hermasky-Pudlak syndromes, MYH9-dependent thrombocytopenia), healthy and pre-term newborns, acute and chronic immune thrombocytopenia, chronic lympholeukemia, effects of therapy on platelet function, etc. The method output includes levels of forward and side scatter, levels of major adhesion and aggregation glycoproteins Ib and IIb-IIIa, active integrins' level based on PAC-1 binding, major alpha-granule component P-selectin, dense granule function based on mepacrine uptake and release, and procoagulant activity quantified as a percentage of annexin V-positive platelets. This analysis is performed for both resting and dual-agonist-stimulated platelets. Preanalytical and analytical variables are provided and discussed. Parameter distribution within the healthy donor population for adults (n = 72) and children (n = 17) is analyzed.