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In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter-and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced alpha(IIb)beta(3) activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced alpha(IIb)beta(3) activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.

Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr−/− mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions., Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr−/−) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr−/− mice and CONV-R Ldlr−/− mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr−/− mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr−/− mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr−/− mice on HFD were diminished compared to CONV-R Ldlr−/− controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr−/− mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr−/− mice relative to CONV-R Ldlr−/− mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr−/− mice on type III collagen.

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin alpha(6)beta(1) pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

GPVI is the major signalling receptor for collagen on platelets. Dimerization of GPVI is required for collagen binding and initiation of signalling through the associated FcR-gamma chain. Recently, fibrin and fibrinogen have been identified as ligands for GPVI and shown to induce signalling in support of thrombus formation and stabilization. Contrasting observations have been reported on whether fibrin binds to monomeric or dimeric GPVI, or to neither form. In this article, we discuss reasons for the contradictory results and how to reconcile these. We conclude that a lack of structural knowledge regarding the GPVI constructs that are being used, along with the use of non-standardized reagents, might be the main cause of the discrepant results. This article aims to highlight some of the key areas that need to be addressed.

We would like to promote the fact that platelets are increasingly emerging as a rich source of potential biomarkers for cancer. Blood platelets contain vast amounts of bioactive proteins, such as growth factors, chemokines, and cytokines. These proteins are either synthesized by the megakaryocytes that produce the platelets or are sequestered by the circulating platelets from the blood, in which case these proteins may originate from the tumor. Recent studies in patients have demonstrated that the presence of cancer influences multiple platelet characteristics (e.g., platelet count, volume, activation status, proteins, and RNA content). Interestingly, these changes happened already in early stages of the disease before metastasis had occurred. Additionally, exploiting these platelet alterations enabled discrimination of patients with early-stage cancer from healthy sex- and age-matched individuals. Therefore, we challenge clinicians and researchers to look beyond traditional fluid sources such as plasma or serum, and to take platelets and their content into account as they may become the holy grail in cancer blood biomarker research.

Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin &alpha, 2&beta, 1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence.

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Background In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. Methods Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2β1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. Results and Conclusion In this first comparison of four inhibitors of platelet–collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

BACKGROUND: The platelet-signaling receptor glycoprotein VI (GPVI) is a promising antithrombotic target. We have previously raised a series of high-affinity nanobodies (Nbs) against GPVI and identified Nb2, Nb21, and Nb35 as potent GPVI inhibitors. The Nb2 binding site has been mapped to the D1 domain, which is directly adjacent to the CRP binding site. Ligand-binding complementary determining region 3 has only 15% conservation between all 3 Nbs.OBJECTIVES: To map the binding sites of Nb21 and Nb35 on GPVI.METHODS: We determined the X-ray crystal structure of the D1 and D2 extracellular domains of the GPVI-Nb35 complex. We then looked at the effects of various GPVI mutations on the ability of Nbs to inhibit collagen binding and GPVI signaling using surface binding assays and transfected cell lines.RESULTS: The crystal structure of GPVI bound to Nb35 was solved. GPVI was present as a monomer, and the D1+D2 conformation was comparable to that in the dimeric structure. Arg46, Tyr47, and Ala57 are common residues on GPVI targeted by both Nb2 and Nb35. Mutating Arg46 to an Ala abrogated the ability of Nb2, Nb21, and Nb35 to inhibit collagen-induced GPVI signaling and blocked the binding of all 3 Nbs. In addition, Arg60 was found to reduce Nb21 inhibition but not the inhibition Nb2 or Nb35.CONCLUSIONS: These findings reveal key residues involved in the high-affinity binding of GPVI inhibitors and negate the idea that GPVI dimerization induces a conformational change required for ligand binding.

S100A8/A9, also known as “calprotectin” or “MRP8/14,” is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

An integrated approach based on the use of data mining methods is proposed in order to improve the efficiency of the analysis of photon counting histograms during study of the molecular composition of a substance by fluorescence fluctuation spectroscopy. The principal component method is used to test the hypothesis about cluster separability of the multidimensional experimental data. The reason for compression of the data cloud to characteristic nonlinearity, or so-called arcuate (arc-shaped) cloud, in the space of the first two principal components is investigated. Examples of simulated data sets of molecular systems with various brightness and concentration characteristics are considered. Interpretation and subsequent quantitative analysis of the data are complicated by nonlinear effects. It was established that the arcuate nature of the data cloud is a consequence of the presence of significant variation in one or more physical parameters and results and, in particular, from significant increase in the spread of the brightness or concentration parameters of the molecules. If only one type of molecule is being studied these parameters can provide additional indicators for assessing the quality of the experiments and can also be used for characterizing the system in the case of a mixture of various types of molecules. It was proposed to use normalization of local weighted smoothing of the scattering diagram to eliminate nonlinear effects in the space of the principal components.

Background: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. Methods: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry–based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. Results: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3 + T cells in the atherosclerotic lesions suggested a T-cell–related effect of homoarginine supplementation, which was mainly attributed to CD4 + T cells. Macrophages, dendritic cells, and B cells were not affected. CD4 + T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. Conclusions: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Missense mutations along the leucine-rich repeat kinase 2 (LRRK2) protein are a major contributor to Parkinson's Disease (PD), the second most commonly occurring neurodegenerative disorder worldwide. We recently reported the development of allosteric constrained peptide inhibitors that target and downregulate LRRK2 activity through disruption of LRRK2 dimerization. In this study, we designed doubly constrained peptides with the objective of inhibiting C-terminal of Roc (COR)-COR mediated dimerization at the LRRK2 dimer interface. We show that the doubly constrained peptides are cell-permeant, bind wild-type and pathogenic LRRK2, inhibit LRRK2 dimerization and kinase activity, and inhibit LRRK2-mediated neuronal apoptosis, and in contrast to ATP-competitive LRRK2 kinase inhibitors, they do not induce the mislocalization of LRRK2 to skein-like structures in cells. This work highlights the significance of COR-mediated dimerization in LRRK2 activity while also highlighting the use of doubly constrained peptides to stabilize discrete secondary structural folds within a peptide sequence.

In platelets, elevated cytosolic Ca 2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca 2+ response consists of Ca 2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca 2+ entry pathways. Several channels can contribute to the Ca 2+ entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca 2+] i measurements in the presence of EGTA or CaCl 2. Per agonist condition, this resulted in sets of EGTA, CaCl 2 and Ca 2+ entry ratio curves, defined by six parameters, reflecting different Ca 2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca 2+ entry ratio of 3-7. Strikingly, in combination with Ca 2+-ATPase inhibition by thapsigargin, the maximal Ca 2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca 2+ entry. By pharmacological blockage of specific Ca 2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca 2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na +/Ca 2+ exchange (NCE) > P2× 1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca 2+ carriers regulating GPVI- and PAR-induced Ca 2+ entry in human platelets.

Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (similar to 10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIia (GPIIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.

The prevention and treatment of arterial thrombosis continue to be clinically challenging, and understanding the relevant molecular mechanisms in detail may facilitate the quest to identify novel targets and therapeutic approaches that improve protection from ischemic and bleeding events. The chemokine CXCL12 augments collagen-induced platelet aggregation by activating its receptor CXCR4. Here we show that inhibition of CXCR4 attenuates platelet aggregation induced by collagen or human plaque homogenate under static and arterial flow conditions by antagonizing the action of platelet-secreted CXCL12. We further show that platelet-specific CXCL12 deficiency in mice limits arterial thrombosis by affecting thrombus growth and stability without increasing tail bleeding time. Accordingly, neointimal lesion formation after carotid artery injury was attenuated in these mice. Mechanistically, CXCL12 activated via CXCR4 a signaling cascade involving Bruton’s tyrosine kinase (Btk) that led to integrin αIIbβ3 activation, platelet aggregation, and granule release. The heterodimeric interaction between CXCL12 and CCL5 can inhibit CXCL12-mediated effects as mimicked by CCL5-derived peptides such as [VREY]4. An improved variant of this peptide, i[VREY]4, binds to CXCL12 in a complex with CXCR4 on the surface of activated platelets, thereby inhibiting Btk activation and preventing platelet CXCL12-dependent arterial thrombosis. In contrast to standard antiplatelet therapies such as aspirin or P2Y12 inhibition, i[VREY]4 reduced CXCL12-induced platelet aggregation and yet did not prolong in vitro bleeding time. We provide evidence that platelet-derived CXCL12 is involved in arterial thrombosis and can be specifically targeted by peptides that harbor potential therapeutic value against atherothrombosis.

As novel liquid chromatography-mass spectrometry (LC-MS) technologies for proteomics offer a substantial increase in LC-MS runs per day, robust and reproducible sample preparation emerges as a new bottleneck for throughput. We introduce a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96) on a commercial positive-pressure solid-phase extraction device. PF96 allows for a five-fold increase in throughput in conjunction with extraordinary reproducibility with Pearson product-moment correlations on the protein level of r = 0.9993, as demonstrated for mouse heart tissue lysate in 40 technical replicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96 variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36-60 μg result in optimal peptide recovery, but lower amounts ≥3 μg can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.

BACKGROUND: Bruton's kinase (Btk) is critical for collagen-triggered platelet signal transduction. The Btk inhibitor ibrutinib has been shown to selectively block platelet adhesion to atherosclerotic plaque material under laminar arterial flow. However, this has not been studied under a shear gradient, which is characteristic for atherothrombosis.OBJECTIVE: To determine the effect of ibrutinib treatment on in vitro thrombus formation on collagen and atherosclerotic plaque material in the absence or presence of a shear gradient.METHODS: Blood was obtained from patients with chronic lymphocytic leukemia, mantle-cell lymphoma and Waldenström macroglobulinemia with and without ibrutinib treatment and perfused through a microfluidic channel with(out) 60% stenosis over Horm type I collagen or human atherosclerotic plaque homogenate.RESULTS: At a constant shear rate of 1500 s-1, platelet deposition was significantly decreased in blood from haematological malignancy patients treated with ibrutinib as compared to untreated patients, on atherosclerotic plaque material but not on collagen. However, thrombus size, stability, and height, were reduced on both plaque material and collagen. An increase in shear rate up to 3900 s-1, as induced by 60% stenosis, resulted in decreased platelet deposition and thrombus parameters on plaque material but not on collagen when compared to a laminar shear of 1500 s-1. Ibrutinib treatment decreased platelet deposition and thrombus parameters even further around the stenosis.CONCLUSION: Treatment of patients with haematological disorders with the Btk inhibitor ibrutinib reduces in vitro platelet deposition, thrombus size and contraction on human atherosclerotic plaque around a stenosis when compared to patients not receiving ibrutinib.

Patients referred for evaluation of bleeding symptoms occasionally have a prolonged platelet function analyser (PFA) closure time, without evidence for von Willebrand disease or impaired platelet aggregation. The aim of this study was to establish a shear-dependent platelet function defect in these patients. Patients were included based on high bleeding score and prior PFA prolongation. Common tests of von Willebrand factor (VWF) and platelet function and exome sequencing were performed. Microfluidic analysis of shear-dependent collagen-induced whole-blood thrombus formation was performed. In 14 PFA-only patients, compared to healthy volunteers, microfluidic tests showed significantly lower platelet adhesion and thrombus formation parameters. This was accompanied by lower integrin activation, phosphatidylserine exposure and P-selectin expression. Principal components analysis indicated VWF as primary explaining variable of PFA prolongation, whereas conventional platelet aggregation primarily explained the reduced thrombus parameters under shear. In five patients with severe microfluidic abnormalities, conventional platelet aggregation was in the lowest range of normal. No causal variants in Mendelian genes known to cause bleeding or platelet disorders were identified. Multiparameter assessment of whole-blood thrombus formation under shear indicates single or combined effects of low-normal VWF and low-normal platelet aggregation in these patients, suggesting a shear-dependent platelet function defect, not detected by static conventional haemostatic tests.

Introduction Studying arterial thrombus formation by in vitro flow assays is a widely used approach. Incorporating human atherosclerotic plaque material as a thrombogenic surface in these assays represents a method to model the pathophysiological environment of thrombus formation upon plaque disruption. Up until now, achieving a homogeneous coating of plaque material and subsequent reproducible platelet adhesion has been challenging. Here, we characterized a novel method for coating of plaque material on glass coverslips for use in thrombosis microfluidic assays. Methods A homogenate of human atherosclerotic plaques was coated on glass coverslips by conventional manual droplet coating or by spin coating. Prior to coating, a subset of coverslips was plasma treated. Water contact angle measurements were performed as an indicator for the hydrophilicity of the coverslips. Homogeneity of plaque coatings was determined using profilometric analysis and scanning electron microscopy. Thrombogenicity of the plaque material was assessed in real time by microscopic imaging while perfusing whole blood at a shear rate of 1500 s−1 over the plaque material. Results Plasma treatment of glass coverslips, prior to spin coating with plaque material, increased the hydrophilicity of the coverslip compared to no plasma treatment. The most homogeneous plaque coating and highest platelet adhesion was obtained upon plasma treatment followed by spin coating of the plaque material. Manual plaque coating on non-plasma treated coverslips yielded lowest coating homogeneity and platelet adhesion and activation. Conclusion Spin coating of atherosclerotic plaque material on plasma treated coverslips leads to a more homogenous coating and improved platelet adhesion to the plaque when compared to conventional droplet coating on non-plasma treated coverslips. These properties are beneficial in ensuring the quality and reproducibility of flow experiments.

Drugs targeting platelets play an important role in preventing or treating thrombotic events, for example stroke and heart attack, thus increasing the life quality and expectancy of patients. This thesis investigated the role of a promising target for anti-platelet therapy, Glycoprotein (GP) VI, a platelet receptor. First, the work in this thesis used in vitro models of microfluidics to compare the effect of GPVI-blockade compared to other drugs. Second, in order to study GPVI’s function in the living organism as well as possible strategies to inhibit its function, this work made use of a mouse model humanized for this platelet receptor. In addition, this thesis presented a second mouse model humanized for another platelet receptor, CLEC-2, also considered a potential therapeutic target. Finally, this thesis provided key information to further develop therapeutics targeting GPVI and provided a first in vivo model to study human CLEC-2 in the living organism.

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The “coagulome” as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited.

Receptor diffusion plays an essential role in cellular signalling via the plasma membrane microenvironment and receptor interactions, but the regulation is not well understood. To aid in understanding of the key determinants of receptor diffusion and signalling, we developed agent-based models (ABMs) to explore the extent of dimerisation of the platelet- and megakaryocyte-specific receptor for collagen glycoprotein VI (GPVI). This approach assessed the importance of glycolipid enriched raft-like domains within the plasma membrane that lower receptor diffusivity. Our model simulations demonstrated that GPVI dimers preferentially concentrate in confined domains and, if diffusivity within domains is decreased relative to outside of domains, dimerisation rates are increased. While an increased amount of confined domains resulted in further dimerisation, merging of domains, which may occur upon membrane rearrangements, was without effect. Modelling of the proportion of the cell membrane which constitutes lipid rafts indicated that dimerisation levels could not be explained by these alone. Crowding of receptors by other membrane proteins was also an important determinant of GPVI dimerisation. Together, these results demonstrate the value of ABM approaches in exploring the interactions on a cell surface, guiding the experimentation for new therapeutic avenues.

Mutations in the multidomain protein Leucine-rich-repeat kinase 2 (LRRK2) have been identified as a genetic risk factor for both sporadic and familial Parkinson’s disease (PD). LRRK2 has two enzymatic domains: a RocCOR tandem with GTPase activity and a kinase domain. In addition, LRRK2 has three N-terminal domains: ARM (Armadillo repeat), ANK (Ankyrin repeat), and LRR (Leucine-rich-repeat), and a C-terminal WD40 domain, all of which are involved in mediating protein–protein interactions (PPIs) and regulation of the LRRK2 catalytic core. The PD-related mutations have been found in nearly all LRRK2 domains, and most of them have increased kinase activity and/or decreased GTPase activity. The complex activation mechanism of LRRK2 includes at least intramolecular regulation, dimerization, and membrane recruitment. In this review, we highlight the recent developments in the structural characterization of LRRK2 and discuss these developments from the perspective of the LRRK2 activation mechanism, the pathological role of the PD mutants, and therapeutic targeting.

Kinetochores connect chromosomes to spindle microtubules to ensure their correct segregation during cell division. Kinetochores of human and yeast are largely homologous, their ability to track depolymerizing microtubules however is carried out by the non-homologous complexes Ska1-C and Dam1-C, respectively. We previously reported the unique anti-correlating phylogenetic profiles of Dam1-C and Ska-C found amongst a wide variety of eukaryotes. Based on these profiles and the limited presence of Dam1-C, we speculated that horizontal gene transfer (HGT) could have played a role in the evolutionary history of Dam1-C.Here, we present expanded analyses of Dam1-c evolution, using additional genome as well as transcriptome sequences and recently acquired 3D structure data. This analysis revealed a wider and more complete presence of Dam1-C in Cryptista, Rhizaria, Ichthyosporea, CRuMs, and Colponemidia. The fungal Dam1-C cryo-EM structure supports earlier hypothesized intra-complex homologies, which enables the reconstruction of rooted and unrooted phylogenies. The rooted tree of concatenated Dam1-C subunits is statistically consistent with the species tree of eukaryotes, suggesting that Dam1-C is ancient, and that the present-day phylogenetic distribution is best explained by multiple, independent losses and no HGT was involved. Furthermore, we investigated the ancient origin of Dam1-C via profile-profile searches. Homology among eight out of the ten Dam1-C subunits suggested that the complex largely evolved from a single multimerizing subunit that diversified into an octameric core via stepwise duplication and sub-functionalization of the subunits before the origin of the Last Common Ancestor.SignificanceThe Dam1 complex has a crucial role in cell division yet its distribution across species is very patchy. To resolve the evolutionary origin of this peculiar distribution, we used the recently acquired 3D structure to obtain a rooted phylogeny. This study makes an important step in discovering the evolutionary history of the Dam1 complex, by determining that Dam1-C was part of the Last eukaryotic Common Ancestor and arose via stepwise duplications during the transition from prokaryotes to eukaryotes.

Dysfunction of the immune system and mitochondrial metabolism has been associated with Parkinson’s disease (PD) pathology. Mutations and increased kinase activity of leucine-rich repeat kinase 2 (LRRK2) are linked to both idiopathic and familial PD. However, the function of LRRK2 in the immune cells under inflammatory conditions is contradictory. Our results showed that lipopolysaccharide (LPS) stimulation increased the kinase activity of LRRK2 in parental RAW 264.7 (WT) cells. In addition to this, LRRK2 deletion in LRRK2 KO RAW 264.7 (KO) cells altered cell morphology following LPS stimulation compared to the WT cells, as shown by an increase in the cell impedance as observed by the xCELLigence measurements. LPS stimulation caused an increase in the cellular reactive oxygen species (ROS) levels in both WT and KO cells. However, WT cells displayed a higher ROS level compared to the KO cells. Moreover, LRRK2 deletion led to a reduction in interleukin-6 (IL-6) inflammatory cytokine and cyclooxygenase-2 (COX-2) expression and an increase in lactate production after LPS stimulation compared to the WT cells. These data illustrate that LRRK2 has an effect on inflammatory processes in RAW macrophages upon LPS stimulation.

Background. The Glycoprotein VI (GPVI) receptor and the protease-activated receptor 1 (PAR1) are key platelet receptors and are activated by collagen and fibrin, or via thrombin, respectively. GPVI and PAR1 play a role in platelet activation and thrombus formation, two crucial processes for thrombosis and haemostasis. Furthermore, they are currently interesting targets for novel antiplatelet therapy. There is, therefore, a need to increase the understanding of the working mechanisms and the potential ligands for GPVI and PAR1. Aims. The goal of this thesis was to increase the understanding of acute and persistent effects of platelet activation mediated through GPVI and PAR1. Results. The role of GPVI and Syk in thrombus formation on collagens and collagen�like peptides was studied. Using the Syk inhibitor PRT060318, we showed that Syk supports platelet activation induced by collagens and collagen-like peptides, regardless of the presence of the GPVI binding motif GPO. More pronounced platelet activating effects of collagens were observed after immobilization, compared to when collagens were soluble. Since GPVI is also known to be the receptor for the coagulation-generated product fibrin, the role of GPVI in other coagulation proteins was studied. Through the use of GPVI and Syk inhibitors, it was evident that GPVI and Syk were involved in platelet activation induced by FXIIIa. Moreover, we found that APC induced platelet spreading through PAR1, which had previously only been shown for endothelial cells. In line with platelet activation induced by collagens, effects induced by FXIIIa and APC were more pronounced when the proteins were immobilized on a surface compared to in solution. Short- and long-term effects through the GPVI and the GPCR receptors PAR1 and P2Y1/12 were compared. Platelet activation through GPVI was more persistent, while responses evoked by PAR1 or P2Y1/12 stimulation were rather transient. Interestingly, pre-activated platelets, which started to return to a resting state (integrin inactivation, morphological change), but still expressed P-selectin on their surface, could be activated again upon restimulation. Conclusions. Taken together, the data presented in this thesis increase our understanding of how platelet functions are regulated through GPVI and PAR1. FXIIIa and APC were identified as novel ligands for GPVI and PAR1, respectively. Further, stimulation through GPVI was persistent, while stimulation through GPCRs (PAR1 or P2Y1/12) was transient. In addition, when platelets reverse, they can be reactivated. Since platelet function is highly regulated through GPVI and PAR1, the increased knowledge of the working mechanisms of these receptors, as well as on short- and long-term effects after activation, will contribute to an enhanced understanding of pre-activated circulating platelets in vivo in patients who are continuously exposed to activating components and will contribute to the improvement and development of more effective antiplatelet therapy.

Platelets are blood cells that prevent the loss of extensive blood volumes, but also contribute to arterial thrombosis. After a vessel is injured, platelets become activated by the exposed collagen and aggregate together, forming a thrombus. Whilst this is a crucial process in hemostasis, platelets also become activated due to rupture of an atherosclerotic plaque in atherothrombosis. Herein, leading to for example, myocardial infarction, stroke or transient ischemic attacks, mediated by collagen that is present in the atherosclerotic plaque. These events are still leading causes of death world-wide, and patients are usually prescribed anti-platelet drugs to prevent a second thrombosis. Currently used drugs are effective in preventing thrombosis, but they are prone to causing unwanted bleeding events in some of the patients. Therefore, other treatment options are currently investigated. A promising approach is to prevent platelet-collagen interactions via inhibition of the collagen receptor, glycoprotein VI (GPVI). In this thesis, we compared the various approaches used to interfere in the GPVI-dependent interaction of platelets with collagens. This was done by a whole blood flow chamber set up with collagen coatings, assessing the effects on thrombus formation.

Charge interactions play a critical role in the activation of the innate immune system by damage- and pathogen-associated molecular pattern receptors. The ability of these receptors to recognize a wide spectrum of ligands through a common mechanism is critical in host defense. In this article, we argue that platelet glycoprotein receptors that signal through conserved tyrosine-based motifs function as pattern recognition receptors (PRRs) for charged endogenous and exogenous ligands, including sulfated polysaccharides, charged proteins and nanoparticles. This is exemplified by GPVI, CLEC-2 and PEAR1 which are activated by a wide spectrum of endogenous and exogenous ligands, including diesel exhaust particles, sulfated polysaccharides and charged surfaces. We propose that this mechanism has evolved to drive rapid activation of platelets at sites of injury, but that under some conditions it can drive occlusive thrombosis, for example, when blood comes into contact with infectious agents or toxins. In this Opinion Article, we discuss mechanisms behind charge-mediated platelet activation and opportunities for designing nanoparticles and related agents such as dendrimers as novel antithrombotics.

Introduction Hormonal contraception is a well-known risk factor for venous thromboembolism (VTE). APC resistance and impaired functions of protein S and TFPI are thought to play an important role in the pathogenesis of hormone-related VTE. It is unknown, whether women, who develop VTE during hormonal contraception possess a vulnerability in these pathways, making them susceptible to thrombosis. Materials and methods Plasma samples were obtained from 57 premenopausal women in average 15.3 years after hormone-associated VTE and from 31 healthy controls. Thrombin generation at high tissue factor (TF) in the absence and in the presence of activated protein C (APC) and at low TF without and with inhibiting anti-protein S- and anti-TFPI-antibodies was measured via calibrated automated thrombography. Results Women with previous hormone-related thrombosis had higher thrombin generation at low TF, higher APC resistance, protein S- and TFPI ratios, differences: 219.9 nM IIa.min (95%CI:90.4 to 349.3); 1.88 (95%CI:0.71 to 3.05); 0.13 (95%CI:0.01 to 0.26) and 0.19 (95%CI:0.08 to 0.30), respectively. Thrombin generation at high TF without APC did not differ between the groups. Smoking decreased thrombin generation at low TF by −222.6 nM IIa.min (95%CI: −381.1 to −64.1), the APC sensitivity ratio by −2.20 (95%CI: −3.63 to −0.77) and the TFPI ratio by −0.16 (95%CI: −0.29 to −0.03), but did not influence thrombin generation at high TF. Discussion We demonstrated impairment of the protein S/TFPI system and increased APC resistance in women with previous hormone-induced VTE. Smoking decreased thrombin generation at assay conditions, dependent on the function of the TFPI system.

In Dictyostelium, chemoattractants induce a fast cGMP response that mediates myosin filament formation in the rear of the cell. The major cGMP signaling pathway consists of a soluble guanylyl cyclase sGC, a cGMP-stimulated cGMP-specific phosphodiesterase, and the cGMP-target protein GbpC. Here we combine published experiments with many unpublished experiments performed in the past 45 years on the regulation and function of the cGMP signaling pathway. The chemoattractants stimulate heterotrimeric G alpha beta gamma and monomeric Ras proteins. A fraction of the soluble guanylyl cyclase sGC binds with high affinity to a limited number of membrane binding sites, which is essential for sGC to become activated by Ras and G alpha proteins. sGC can also bind to F-actin; binding to branched F-actin in pseudopods enhances basal sGC activity, whereas binding to parallel F-actin in the cortex reduces sGC activity. The cGMP pathway mediates cell polarity by inhibiting the rear: in unstimulated cells by sGC activity in the branched F-actin of pseudopods, in a shallow gradient by stimulated cGMP formation in pseudopods at the leading edge, and during cAMP oscillation to erase the previous polarity and establish a new polarity axis that aligns with the direction of the passing cAMP wave.

Funder: Canadian Institutes of Health Research (Grant: FRN-142349) Natural Sciences and Engineering Research Council of Canada (Grant: RGPIN 05871-2014), Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

© 2022 The AuthorsFerroptosis is an iron-dependent regulated cell death pathway characterized by excessive lipid peroxidation. It is implicated in many neurodegenerative diseases, including Parkinson's Disease (PD). Mutations and increased leucine-rich repeat kinase 2 (LRRK2) kinase activity are associated with both familial and idiopathic PD pathology. Increased iron deposition was observed in the substantia nigra of LRRK2 mutation-carrying PD patients compared to healthy individuals, suggesting a potential link between LRRK2 and ferroptosis. However, the role of LRRK2 in the immune cells is still not well-understood. This study aims to investigate the effect of LRRK2 on ferroptosis-induced cell death in immune cells. We used LRRK2 parental (WT) and LRRK2 KO (KO) RAW 264.7 murine macrophages. Cells were challenged with the ferroptosis inducer, erastin, and the kinase activity was investigated using the LRRK2 kinase inhibitor, MLi2. Cell metabolism and viability analysis showed that WT cells were more resistant to ferroptosis than the KO cells. Lipid peroxidation and cellular reactive oxygen species (ROS) generation were significantly elevated in the KO cells. Furthermore, mitochondrial membrane potential and mitochondrial respiration were decreased in the KO cells after erastin treatment compared to the WT cells. Inhibition of the LRRK2 kinase function resulted in increased cell sensitivity to erastin. Cell and mitochondrial substrates utilization were altered in the KO and kinase inhibited WT cells compared to WT cells. These results indicate a protective role of LRRK2 against erastin-induced ferroptosis in RAW macrophages and point towards the importance of LRRK2 kinase function in the protective mechanism.

Parkinson’s disease (PD) is the most common movement disorder affecting the elderly. The disease results from the death of neurons in the brain that secret dopamine leading to impaired movement. Although PD symptoms have been described for decades, no cure exists to prevent or stop the disease. Over the last two decades, several genes have been linked to PD. Among them, mutations in leucine-rich-repeat kinase 2 (LRRK2) stand as the most common genetic cause. LRRK2 is a large multidomain protein with a double enzymatic function (kinase and GTPase). LRRK2 kinase activity is increased in PD patients. Although PD is considered as a brain disease, recent evidence showed that the immune system plays an important role in the development of PD. LRRK2 is highly expressed in immune cells and both the LRRK2 level and kinase activity are upregulated in PD patients’ immune cells. In addition, recent studies showed that mutations in LRRK2 resulted in defects in the function of mitochondria, the powerhouse of the cell. Yet, the exact mechanism by which LRRK2 regulates mitochondrial function in immune cells is unclear.In this thesis, we investigated LRRK2 regulation of mitochondrial function and energy production in immune cells. We proved that LRRK2 has a conserved role in mitochondrial energy generation. Mitochondrial defects could impair energy production resulting in cell death. Furthermore, we presented a better understanding of the influence of LRRK2 on stimulated immune cells. Additionally, we identified a novel protective effect of LRRK2 and its kinase function against the unique iron-dependent form of programmed cell death, ferroptosis, involved in PD development. Together these findings mean that LRRK2 contributes to the inflammatory process that may lead to the death of dopamine-secreting neurons.

Fibrinogen γ' accounts for 3% to 40% of plasma fibrinogen. Earlier studies indicated that fibrinogen γ' forms altered fibrin clots under static conditions, whereas clinically, altered plasma γ' levels are associated with arterial and venous thrombosis. However, the effects of static vs flow conditions on the role of γ′ throughout the pathophysiological range is unknown. This study explores the effects of γ' levels on clot formation and structure in static and flow conditions. Coagulation of plasma samples with low (n = 41; 3%), normal (n = 45; 10%), or high (n = 33; 30%) γ′ levels were compared with that of purified fibrinogen mixtures with increasing ratios of γ′ (3%, 10%, 30%). Clots were analyzed by confocal microscopy, permeation, turbidity, and lysis techniques. In a novel 2-step flow-perfusion model, fibrinogen-deficient plasma repleted with increasing ratios of γ′ (3%, 10%, 30%) or plasmas with low (n = 5, 3%) or high (n = 5, 30%) γ′ were flowed over preformed platelet aggregates at arterial (500 s−1) and venous (150 s−1) shear rates. Increasing γ′ percentages within the pathophysiological range (3%-30%) did not result in any change in clot-formation rates; however, it led to significantly higher clot density, thinner fibers, and slower lysis in static conditions. Under flow at arterial shear, high γ′ (30%) led to faster (+44.1%-75.3%) and increased (+104%-123%) fibrin deposition, with clots exhibiting a larger volume (+253%-655%) and height (+130%-146%). These trends were magnified at venous shear. Overall, our findings demonstrate the significant impact of pathophysiological fibrinogen γ′ levels on clot structure and provide new flow-dependent mechanisms to explain how γ′ increases thrombosis risk.

Introduction: Current developments to assess qualitative and quantitative platelet traits in flowed whole-blood are based on microfluidic devices that mostly operate at room temperature. However, operation at physiological temperature (37 degrees;C) may increase the assay's sensitivity, and facilitates the comparison to other platelet function tests of the diagnostic laboratory.Materials and methods: We adapted the conventional microspot-based microfluidic device with a simple thermocoupled pre-heating module. Automated analysis of microscopic images assisted in obtaining five timedependent parameters of thrombus formation over collagen microspots (shear rate 1000 s- 1). These modifications allowed rapid testing of control and patient blood samples at physiological temperature.Results and conclusion: The higher temperature enhanced platelet adhesion and aggregation as well as late thrombus characteristics such as size and contraction, when compared to room temperature. Moreover, assessment at 37 degrees C indicated a time-dependent impairment of the thrombus parameters in blood from patients taking common antiplatelet medication, i.e. aspirin and/or clopidogrel. This pointed to increased contribution of the autocrine platelet agonists thromboxane A2 and ADP in the buildup of contracted thrombi under flow. Overall, this study underlined the advantage of multiparameter assessment of microfluidic thrombus formation in detecting an acquired platelet dysfunction, when operating at physiological temperature. This work may bring microfluidics tests closer to the diagnostic laboratory.Introduction: Current developments to assess qualitative and quantitative platelet traits in flowed whole-blood are based on microfluidic devices that mostly operate at room temperature. However, operation at physiological temperature (37 degrees C) may increase the assay's sensitivity, and facilitates the comparison to other platelet function tests of the diagnostic laboratory.Materials and methods: We adapted the conventional microspot-based microfluidic device with a simple thermocoupled pre-heating module. Automated analysis of microscopic images assisted in obtaining five timedependent parameters of thrombus formation over collagen microspots (shear rate 1000 s- 1). These modifications allowed rapid testing of control and patient blood samples at physiological temperature.Results and conclusion: The higher temperature enhanced platelet adhesion and aggregation as well as late thrombus characteristics such as size and contraction, when compared to room temperature. Moreover, assessment at 37 degrees C indicated a time-dependent impairment of the thrombus parameters in blood from patients taking common antiplatelet medication, i.e. aspirin and/or clopidogrel. This pointed to increased contribution of the autocrine platelet agonists thromboxane A2 and ADP in the buildup of contracted thrombi under flow. Overall, this study underlined the advantage of multiparameter assessment of microfluidic thrombus formation in detecting an acquired platelet dysfunction, when operating at physiological temperature. This work may bring microfluidics tests closer to the diagnostic laboratory.

Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexanparasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimalstructure with limited control over chromosome segregation. In this study, we use interactomics combined with deephomology searches to identify 13 previously unknown components of kinetochores in Apicomplexa. Apicomplexankinetochores are highly divergent in sequence and composition from animal and fungal models. The nanoscale organizationincludes at least four discrete compartments, each displaying different biochemical interactions, subkinetochorelocalizations and evolutionary rates across the phylum. We reveal alignment of kinetochores at the metaphase plate in bothPlasmodium berghei and Toxoplasma gondii, suggestive of a conserved “hold signal” that prevents precocious entry intoanaphase. Finally, we show unexpected plasticity in kinetochore composition and segregation between apicomplexanlifecycle stages, suggestive of diverse requirements to maintain fidelity of chromosome segregation across parasite modes ofdivision.

Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.

C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. CLEC-2 has been shown to support thrombus formation at sites of inflammation, but to have a minor/negligible role in hemostasis. This identifies CLEC-2 as a promising therapeutic target in thromboinflammatory disorders, without hemostatic detriment. We utilized a GPIbα-Cre recombinase mouse for more restricted deletion of platelet-CLEC-2 than the previously used PF4-Cre mouse. clec1bfl/flGPIbα-Cre+ mice are born at a Mendelian ratio, with a mild reduction in platelet count, and present with reduced thrombus size post-FeCl3-induced thrombosis, compared to littermates. Antibody-mediated depletion of platelet count in C57BL/6 mice, to match clec1bfl/flGPIbα-Cre+ mice, revealed that the reduced thrombus size post-FeCl3-injury was due to the loss of CLEC-2, and not mild thrombocytopenia. Similarly, clec1bfl/flGPIbα-Cre+ mouse blood replenished with CLEC-2-deficient platelets ex vivo to match littermates had reduced aggregate formation when perfused over collagen at arterial flow rates. In contrast, platelet-rich thrombi formed following perfusion of human blood under flow conditions over collagen types I or III, atherosclerotic plaque, or inflammatory endothelial cells were unaltered in the presence of CLEC-2-blocking antibody, AYP1, or recombinant CLEC-2-Fc. The reduction in platelet aggregation observed in clec1bfl/flGPIbα-Cre+ mice during arterial thrombosis is mediated by the loss of CLEC-2 on mouse platelets. In contrast, CLEC-2 does not support thrombus generation on collagen, atherosclerotic plaque, or inflamed endothelial cells in human at arterial shear.

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C′ loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C′ loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.

Circulating biomarkers reflecting cardiac inflammation are needed to improve the diagnostics and guide the treatment of heart failure patients. The cardiac production and shedding of the transmembrane proteoglycan syndecan-4 is upregulated by innate immunity signaling pathways. Here, we investigated the potential of syndecan-4 as a blood biomarker of cardiac inflammation. Serum syndecan-4 was measured in patients with (i) non-ischemic, non-valvular dilated cardiomyopathy (DCM), with (n = 71) or without (n = 318) chronic inflammation; (ii) acute myocarditis (n = 15), acute pericarditis (n = 3) or acute perimyocarditis (23) and (iii) acute myocardial infarction (MI) at day 0, 3 and 30 (n = 119). Syndecan-4 was investigated in cultured cardiac myocytes and fibroblasts (n = 6–12) treated with the pro-inflammatory cytokines interleukin (IL)-1β and its inhibitor IL-1 receptor antagonist (IL-1Ra), or tumor necrosis factor (TNF)α and its specific inhibitor infliximab, an antibody used in treatment of autoimmune diseases. The levels of serum syndecan-4 were comparable in all subgroups of patients with chronic or acute cardiomyopathy, independent of inflammation. Post-MI, syndecan-4 levels were increased at day 3 and 30 vs. day 0. IL-1Ra attenuated IL-1β-induced syndecan-4 production and shedding in vitro, while infliximab had no effect. In conclusion, syndecan-4 shedding from cardiac myocytes and fibroblasts was attenuated by immunomodulatory therapy. Although its circulating levels were increased post-MI, syndecan-4 did not reflect cardiac inflammatory status in patients with heart disease.

Hemodynamics play a central role in hemostasis and thrombosis by affecting all aspects linked to platelet functions and coagulation. In vitro flow devices are extensively used in basic research, pharmacological studies, antiplatelet agent screening, and development of diagnostic tools. Because hemodynamic conditions vary tremendously throughout the vascular tree and among different (patho)physiological processes, it is important to use flow conditions based on relevant biorheological reference ranges. Surprisingly, it is particularly difficult to find a concise overview of relevant hemodynamic parameters in various human and mouse vessels. To our knowledge, this is the first time an inventory of flow conditions in healthy, non-diseased, human and mouse vessels has been created. The objective of providing such a repertoire is to aid researchers in the field of hemostasis and thrombosis in choosing rheological conditions relevant in in vitro flow experiments and to promote harmonization of flow-based assays to facilitate comparative evaluations between studies. With reference to the human, we discuss relevant similarities and discrepancies in wall shear rates in the mouse, which are typically one order of magnitude greater in agreement with allometric scaling laws between species. Importantly, we bring the attention of the researchers to the fact that the relevant range of average wall shear rates in human arteries where clinically relevant arterial thrombosis occurs may fall as low as 100 to 200 s(-1), thus significantly overlapping with what are considered "venous" shear rates. The same range for the murine arteries used for arterial thrombosis models may significantly exceed 1000 s(-1) reaching values considered to be "pathological."

In vitro flow-based assays are widely used to investigate the role of platelets and coagulation in hemostasis and thrombosis. Their main advantage over other assays relies on the fact that they integrate blood flow that regulates many aspects of platelet function, including adhesion, activation, and aggregation. Blood flow is also central in the regulation of coagulation through its ability to modulate the local concentrations of coagulation factors within and around thrombi. The most broadly used assay to study thrombus formation consists in perfusing whole blood over immobilized fibrillar collagen through a single channel, which helps to reproduce thrombus formation as it occurs in vivo after vascular injury, with platelets adhering, becoming activated, and forming a mural thrombus. This process can also be studied under conditions of thrombin generation, notably by recalcifying blood collected in sodium citrate. In this manuscript, we briefly discuss the advantages and limits of this broadly used "in vitro thrombus formation model." The main emphasis is on the description of the most recent developments regarding design of new flow models and new techniques, and how these may advance the landscape of in vitro studies into the formation of physiological or pathophysiological thrombi. Challenges linked to mimicking the formation of a hemostatic plug in a healthy vessel or a thrombus in diseased arteries and the complexity of reproducing the various aspects of venous thrombosis are discussed. Future directions are proposed to improve the physiological or pathophysiological relevance of current flow-based assays.

Background: Ticks puncture the skin of their hosts and secrete saliva, containing antiplatelet proteins, into the blood. Here, we studied disagregin, a potent platelet-inhibiting protein derived from the salivary glands of Ornithodoros moubata, an African soft tick. Whereas conventional alpha IIb beta 3 antagonists contain an Arg-Gly-Asp (RGD) sequence for platelet integrin binding, disagregin contains an Arg-Glu-Asp (RED) sequence, hypothesizing a different mode of inhibitory action.Objectives: We aimed to compare the inhibitory effects of disagregin and its RGD variant (RGD-disagregin) on platelet activation and to unravel the molecular basis of disagregin-alpha IIb beta 3 integrin interactions.Methods: Disagregin and RGD-disagregin were synthesized by tert-butyloxycarbonyl -based solid-phase peptide synthesis. Effects of both disagregins on platelet aggregation were assessed by light transmission aggregometry in human platelet-rich plasma. Whole-blood thrombus formation was investigated by perfusing blood over collagen I with and without tissue factor at a high wall-shear rate (1000 s(-1)) in the presence of disagregin, RGD-disagregin, or eptifibatide.Results: Disagregin showed inhibition of collagen- and ADP-induced platelet aggregation with half maximal inhibitory concentration values of 64 and 99 nM, respectively. This resembled the complete antiaggregatory effect of eptifibatide. Multiparameter assessment of thrombus formation showed highly suppressed platelet adhesion and aggregate formation with both disagregins, in contrast to eptifibatide treatment, which incompletely blocked aggregation under flow. Fibrin formation under flow was delayed by both disagregin and RGD-disagregin (P Conclusions: Both alpha IIb beta 3-blocking disagregins have a strong potential to suppress collagen-tissue factor-mediated platelet adhesion, thrombus formation, and fibrin formation. Both disagregins can be seen as potential new alpha IIb beta 3 inhibitors.

BACKGROUND: The collagen receptor glycoprotein VI (GPVI) is an attractive antiplatelet target due to its critical role in thrombosis but minor involvement in hemostasis.OBJECTIVE: To investigate GPVI receptor involvement in platelet activation by collagen-I and atherosclerotic plaque using novel blocking and non-blocking anti-GPVI nanobodies (Nbs).METHODS: Nb effects on GPVI-mediated signaling and function were assessed by western blot and whole blood thrombus formation under flow. GPVI clustering was visualized in thrombi using fluorescently labeled Nb28.RESULTS: Under arterial shear, inhibitory Nb2 blocks thrombus formation and platelet activation on collagen and plaque, but only reduces adhesion on plaque. In contrast, adhesion on collagen, but not plaque, is decreased by blocking integrin α2β1. Adhesion on plaque is maintained despite inhibition of integrins αvβ3, α5β1, α6β1, and αIIbβ3. Only combined αIIbβ3 and α2β1 blockade inhibits adhesion and thrombus formation to the same extent as Nb2 alone. Nb2 prevents GPVI signaling, with loss of Syk, Lat, and PLCɣ2 phosphorylation, especially to plaque stimulation. Non-blocking fluorescently labeled Nb28 reveals distinct GPVI distribution patterns on collagen and plaque, with GPVI clustering clearly apparent on collagen fibers and less frequent on plaque. Clustering on collagen fibers is lost in the presence of Nb2.CONCLUSIONS: This work emphasizes the critical difference in GPVI-mediated platelet activation by plaque and collagen; it highlights the importance of GPVI clustering for downstream signaling and thrombus formation. Labeled Nb28 is a novel tool for providing mechanistic insight into this process and the data suggest Nb2 warrants further investigation as a potential anti-thrombotic agent.