Mathematical models of coagulation-are we there yet?

BACKGROUND: Mathematical models of coagulation have been developed to mirror thrombin generation in plasma, with the aim of investigating how variation in coagulation factor levels regulates hemostasis. However, current models vary in the reactions they capture and the reaction rates used, and their validation is restricted by a lack of large coherent datasets, resulting in questioning of their utility. OBJECTIVES: To address this debate, we systematically assessed current models against a large dataset, using plasma coagulation factor levels from 348 individuals with normal hemostasis to identify the causes of these variations. METHODS: We compared model predictions with measured thrombin generation, quantifying and comparing the ability of each model to predict thrombin generation, the contributions of the individual reactions, and their dependence on reaction rates. RESULTS: We found that no current model predicted the hemostatic response across the whole cohort and all produced thrombin generation curves that did not resemble those obtained experimentally. Our analysis has identified the key reactions that lead to differential model predictions, where experimental uncertainty leads to variability in predictions, and we determined reactions that have a high influence on measured thrombin generation, such as the contribution of factor XI. CONCLUSION: This systematic assessment of models of coagulation, using large dataset inputs, points to ways in which these models can be improved. A model that accurately reflects the effects of the multiple subtle variations in an individual's hemostatic profile could be used for assessing antithrombotics or as a tool for precision medicine.

Plasma Myeloperoxidase as a Potential Biomarker of Patient Response to Anti-Dementia Treatment in Alzheimer's Disease.

BACKGROUND: Myeloperoxidase (MPO), a neutrophil-derived pro-inflammatory protein, co-localizes with amyloid-ß (Aß) plaques in Alzheimer's disease (AD). Anti-dementia treatment may facilitate efflux of Aß and associated plaque proteins from the brain to the peripheral circulation, therefore providing potential biomarkers for the monitoring of donor response to drug treatment. OBJECTIVE: We investigated the diagnostic utility of MPO as a biomarker of AD, and how anti-dementia treatment alters plasma MPO concentration. METHODS: Thirty-two AD patients were recruited, and plasma collected pre-drug administration (baseline), and 1- and 6-months post-treatment. All patients received cholinesterase inhibitors (ChEIs). At baseline and 6 months, patients underwent neuropsychological assessment. Forty-nine elderly healthy individuals with normal cognitive status served as controls. Plasma MPO concentration was measured by ELISA. RESULTS: AD drug naïve patients had similar plasma MPO concentration to their control counterparts (p > 0.05). Baseline MPO levels positively correlated with Neuropsychiatric Inventory score (r = 0.5080; p = 0.011) and carer distress (r = 0.5022; p = 0.012). Following 1-month ChEI treatment, 84.4% of AD patients exhibited increased plasma MPO levels (p < 0.001), which decreased at 6 months (p < 0.001). MPO concentration at 1 month was greatest in AD patients whose memory deteriorated during the study period (p = 0.028), and for AD patients with deterioration in Cornell assessment score (p = 0.044). CONCLUSION: Whereas baseline MPO levels did not differentiate between healthy and AD populations, baseline MPO positively correlated with initial Neuropsychiatric Inventory evaluation. Post-treatment, transient MPO upregulation in ChEI-treated patients may reflect worse therapeutic outcome. Further studies are required to assess the potential of plasma MPO as an AD therapeutic biomarker.

A System-Wide Investigation and Stratification of the Hemostatic Proteome in Premature Myocardial Infarction.

Introduction: Advancing understanding of key factors that determine the magnitude of the hemostatic response may facilitate the identification of individuals at risk of generating an occlusive thrombus as a result of an atherothrombotic event such as an acute Myocardial Infarction (MI). While fibrinogen levels are a recognized risk factor for MI, the association of thrombotic risk with other coagulation proteins is inconsistent. This is likely due to the complex balance of pro- and anticoagulant factors in any individual. Methods: We compared measured levels of pro- and anticoagulant proteins in plasma from 162 patients who suffered an MI at an early age (MI <50 y) and 186 age- and gender-matched healthy controls with no history of CAD. We then used the measurements from these individuals as inputs for an established mathematical model to investigate how small variations in hemostatic factors affect the overall amplitude of the hemostatic response and to identify differential key drivers of the hemostatic response in male and female patients and controls. Results: Plasma from the MI patients contained significantly higher levels of Tissue Factor (P = 0.007), the components of the tenase (FIX and FVIII; P < 0.0001 for both) and the prothrombinase complexes (FX; P = 0.003), and lower levels of Tissue Factor Pathway Inhibitor (TFPI; P = 0.033) than controls. The mathematical model, which generates time-dependent predictions describing the depletion, activation, and interaction of the main procoagulant factors and inhibitors, identified different patterns of hemostatic response between MI patients and controls, and additionally, between males and females. Whereas, in males, TF, FVIII, FIX, and the inhibitor TFPI contribute to the differences seen between case and controls, and in females, FII, FVIII, and FIX had the greatest influence on the generation of thrombin. We additionally show that further donor stratification may be possible according to the predicted donor response to anticoagulant therapy. Conclusions: We suggest that modeling could be of value in enhancing our prediction of risk of premature MI, recurrent risk, and therapeutic efficacy.

The voltage-gated K+ channel Kv1.3 modulates platelet motility and α2ß1 integrin-dependent adhesion to collagen.

Kv1.3 is a voltage-gated K+-selective channel with roles in immunity, insulin-sensitivity, neuronal excitability and olfaction. Despite being one of the largest ionic conductances of the platelet surface membrane, its contribution to platelet function is poorly understood. Here we show that Kv1.3-deficient platelets display enhanced ADP-evoked platelet aggregation and secretion, and an increased surface expression of platelet integrin αIIb. In contrast, platelet adhesion and thrombus formation in vitro under arterial shear conditions on surfaces coated with collagen were reduced for samples from Kv1.3-/- compared to wild type mice. Use of collagen-mimetic peptides revealed a specific defect in the engagement with α2ß1. Kv1.3-/- platelets developed significantly fewer, and shorter, filopodia than wild type platelets during adhesion to collagen fibrils. Kv1.3-/- mice displayed no significant difference in thrombus formation within cremaster muscle arterioles using a laser-induced injury model, thus other pro-thrombotic pathways compensate in vivo for the adhesion defect observed in vitro. This may include the increased platelet counts of Kv1.3-/- mice, due in part to a prolonged lifespan. The ability of Kv1.3 to modulate integrin-dependent platelet adhesion has important implications for understanding its contribution to normal physiological platelet function in addition to its reported roles in auto-immune diseases and thromboinflammatory models of stroke.

Why do platelets express K+ channels?

Potassium ions have widespread roles in cellular homeostasis and activation as a consequence of their large outward concentration gradient across the surface membrane and ability to rapidly move through K+-selective ion channels. In platelets, the predominant K+ channels include the voltage-gated K+ channel Kv1.3, and the intermediate conductance Ca2+-activated K+ channel KCa3.1, also known as the Gardos channel. Inwardly rectifying potassium GIRK channels and KCa1.1 large conductance Ca2+-activated K+ channels have also been reported in the platelet, although they remain to be demonstrated using electrophysiological techniques. Whole-cell patch clamp and fluorescent indicator measurements in the platelet or their precursor cell reveal that Kv1.3 sets the resting membrane potential and KCa3.1 can further hyperpolarize the cell during activation, thereby controlling Ca2+ influx. Kv1.3-/- mice exhibit an increased platelet count, which may result from an increased splenic megakaryocyte development and longer platelet lifespan. This review discusses the evidence in the literature that Kv1.3, KCa3.1. GIRK and KCa1.1 channels contribute to a number of platelet functional responses, particularly collagen-evoked adhesion, procoagulant activity and GPCR function. Putative roles for other K+ channels and known accessory proteins which to date have only been detected in transcriptomic or proteomic studies, are also discussed.

The TICONC (Ticagrelor-Oncology) Study: Implications of P2Y12 Inhibition for Metastasis and Cancer-Associated Thrombosis.

BACKGROUND: In cancer, platelets may facilitate metastatic spread by a number of mechanisms as well as contribute to thrombotic complications. Ticagrelor, a platelet antagonist- that blocks adenosine diphosphate activation of platelet P2Y12 receptors, is widely used in the treatment of cardiovascular disease, but its efficacy in cancer remains unknown. OBJECTIVES: This study sought to evaluate the effect of aspirin and ticagrelor monotherapy, as well as dual antiplatelet therapy, on platelet activation in cancer. METHODS: This study consisted of 2 phases: first, an in vitro study of human platelet-tumor cell interaction; and second, a randomized crossover clinical trial of 22 healthy donors and 16 patients with metastatic breast or colorectal cancer. Platelet activation and inhibition were measured by aggregometry and flow cytometry. RESULTS: In vitro, tumor cells induced cellular clusters that were predominantly platelet-platelet aggregates. Ticagrelor significantly inhibited formation of large tumor cell-induced platelet-platelet aggregates: 65.4 ± 4.8% to 50.9 ± 5.9% (p = 0.002) and 62.3 ± 3.1% to 48.3 ± 7.3% (p = 0.014) for MCF-7 and HT-29-induced aggregation, respectively. Supporting this finding, cancer patients on ticagrelor had significantly reduced levels of spontaneous platelet aggregation and activation compared with baseline; 14.8 ± 2.7% at baseline to 7.8 ± 2.3% with ticagrelor (p = 0.012). CONCLUSIONS: Our findings suggested that P2Y12 inhibition with ticagrelor might reduce spontaneous platelet aggregation and activation in patients with metastatic cancer and merits further investigation in patients at high risk of cancer-associated thrombosis. (Ticagrelor-Oncology [TICONC] Study; EudraCT: 2014-004049-29).

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line.

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s-1) induced a sustained increase in [Ca2+] i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+ Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+] i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

Regulation of Pannexin-1 channel activity.

Pannexin-1 (Panx1) forms anion-selective channels with a permeability up to 1 kDa and represents a pathway for the release of cytosolic ATP. Several structurally similar connexin (Cx) proteins have been identified in platelets and shown to play roles in haemostasis and thrombosis. More recently, functional Panx1 channels have been demonstrated on the surface of human platelets [Taylor et al. (2014) J. Thromb. Haemost. 12, 987-998]. Since their identification in the year 2000, several mechanisms have been reported to activate Panx1 channels, including mechanical stimulation, oxygen-glucose deprivation, a rise of [Ca2+]i, caspase cleavage and phosphorylation. Within this review, the regulation of Panx1 channels is discussed, with a focus on how they may contribute to platelet function.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis.

BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.