Platelet biology and function: plaque erosion vs. rupture.

The leading cause of heart disease in developed countries is coronary atherosclerosis, which is not simply a result of ageing but a chronic inflammatory process that can lead to acute clinical events upon atherosclerotic plaque rupture or erosion and arterial thrombus formation. The composition and location of atherosclerotic plaques determine the phenotype of the lesion and whether it is more likely to rupture or to erode. Although plaque rupture and erosion both initiate platelet activation on the exposed vascular surface, the contribution of platelets to thrombus formation differs between the two phenotypes. In this review, plaque phenotype is discussed in relation to thrombus composition, and an overview of important mediators (haemodynamics, matrix components, and soluble factors) in plaque-induced platelet activation is given. As thrombus formation on disrupted plaques does not necessarily result in complete vessel occlusion, plaque healing can occur. Therefore, the latest findings on plaque healing and the potential role of platelets in this process are summarized. Finally, the clinical need for more effective antithrombotic agents is highlighted.

Endothelial Cell Dysfunction and Increased Cardiovascular Risk in Patients With Chronic Kidney Disease.

The endothelium is considered to be the gatekeeper of the vessel wall, maintaining and regulating vascular integrity. In patients with chronic kidney disease, protective endothelial cell functions are impaired due to the proinflammatory, prothrombotic and uremic environment caused by the decline in kidney function, adding to the increase in cardiovascular complications in this vulnerable patient population. In this review, we discuss endothelial cell functioning in healthy conditions and the contribution of endothelial cell dysfunction to cardiovascular disease. Further, we summarize the phenotypic changes of the endothelium in chronic kidney disease patients and the relation of endothelial cell dysfunction to cardiovascular risk in chronic kidney disease. We also review the mechanisms that underlie endothelial changes in chronic kidney disease and consider potential pharmacological interventions that can ameliorate endothelial health.

Blood Coagulation and Beyond: Position Paper from the Fourth Maastricht Consensus Conference on Thrombosis.

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.

Repeated platelet activation and the potential of previously activated platelets to contribute to thrombus formation.

BACKGROUND: Especially in disease conditions, platelets can encounter activating agents in circulation. OBJECTIVES: To investigate the extent to which previously activated platelets can be reactivated and whether in-and reactivation applies to different aspects of platelet activation and thrombus formation. METHODS: Short-and long-term effects of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) stimulation on platelet activation and aggregation potential were compared via flow cytometry and plate-based aggregation. Using fluorescence and electron microscopy, we assessed platelet morphology and content, as well as thrombus formation. RESULTS: After 30 minutes of stimulation with thrombin receptor activator peptide 6 (TRAP6) or adenosine diphosphate (ADP), platelets secondarily decreased in PAC-1 binding and were less able to aggregate. The reversibility of platelets after thrombin stimulation was concentration dependent. Reactivation was possible via another receptor. In contrast, cross-linked collagen-related peptide (CRP-XL) or high thrombin stimulation evoked persistent effects in αIIbß3 activation and platelet aggregation. However, after 60 minutes of CRP-XL or high thrombin stimulation, when αIIbß3 activation slightly decreased, restimulation with ADP or CRP-XL, respectively, increased integrin activation again. Compatible with decreased integrin activation, platelet morphology was reversed. Interestingly, reactivation of reversed platelets again resulted in shape change and if not fully degranulated, additional secretion. Moreover, platelets that were previously activated with TRAP6 or ADP regained their potential to contribute to thrombus formation under flow. On the contrary, prior platelet triggering with CRP-XL was accompanied by prolonged platelet activity, leading to a decreased secondary platelet adhesion under flow. CONCLUSION: This work emphasizes that prior platelet activation can be reversed, whereafter platelets can be reactivated through a different receptor. Reversed, previously activated platelets can contribute to thrombus formation.

Coagulation Factor XIIIa and Activated Protein C Activate Platelets via GPVI and PAR1.

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the 'supernatant of (hirudin-treated) coagulated plasma' (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin αIIbß3 activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.

[Heart and diabetes : Platelet function and antiplatelet therapy in chronic kidney disease]. / Herz und Diabetes : Thrombozytenfunktion und antithrombozytäre Therapie bei chronischer Nierenerkrankung.

Patients with chronic kidney disease (CKD) have an increased risk of thrombosis and approximately 50% of patients with advanced CKD die because of a cardiovascular disease. In addition to an increased risk of thrombosis, patients with CKD and particularly with advanced CKD, have an increased risk of hemorrhage, which increases parallel to the decline of kidney function. Due to this parallel existence of the prohemorrhagic and prothrombotic phenotype, antiplatelet treatment is difficult in the daily routine and data show that CKD patients with acute coronary syndrome (ACS) are less likely to receive guideline-conform treatment. The underlying mechanisms are currently insufficiently understood and both platelet-dependent mechanisms and also platelet-independent mechanisms are under discussion. Accordingly, there is currently no specific treatment or treatment strategy for patients with CKD. In addition, CKD patients are underrepresented in registration studies on antiplatelet treatment and there are no data from randomized trials for patients with advanced CKD (CKD ≥ 4). Current guideline recommendations are therefore based on subgroup analyses and observational studies. In addition, questions on the duration of treatment, on risk scores for estimation of the risk of hemorrhage and on potential benefits of escalation and de-escalation strategies remain largely unanswered and should therefore be the focus of future studies.

MicroRNA-26b Attenuates Platelet Adhesion and Aggregation in Mice.

Platelets are key regulators of haemostasis, making platelet dysfunction a major driver of thrombosis. Numerous processes that determine platelet function are influenced by microRNAs (miRs). MiR-26b is one of the highest-expressed miRs in healthy platelets, and its expression in platelets is changed in a diseased state. However, the exact effect of this miR on platelet function has not been studied yet. In this study, we made use of a whole-body knockout of miR-26b in ApoE-deficient mice in order to determine its impact on platelet function, thrombus formation and platelet signalling both ex vivo and in vivo. We show that a whole-body deficiency of miR-26b exacerbated platelet adhesion and aggregation ex vivo. Additionally, in vivo, platelets adhered faster, and larger thrombi were formed in mice lacking miR-26b. Moreover, isolated platelets from miR-26b-deficient mice showed a hyperactivated Src and EGFR signalling. Taken together, we show here for the first time that miR-26b attenuates platelet adhesion and aggregation, possibly through Src and EGFR signalling.

Impact of Uremic Toxins on Endothelial Dysfunction in Chronic Kidney Disease: A Systematic Review.

Patients with chronic kidney disease (CKD) are at a highly increased risk of cardiovascular complications, with increased vascular inflammation, accelerated atherogenesis and enhanced thrombotic risk. Considering the central role of the endothelium in protecting from atherogenesis and thrombosis, as well as its cardioprotective role in regulating vasorelaxation, this study aimed to systematically integrate literature on CKD-associated endothelial dysfunction, including the underlying molecular mechanisms, into a comprehensive overview. Therefore, we conducted a systematic review of literature describing uremic serum or uremic toxin-induced vascular dysfunction with a special focus on the endothelium. This revealed 39 studies analyzing the effects of uremic serum or the uremic toxins indoxyl sulfate, cyanate, modified LDL, the advanced glycation end products N-carboxymethyl-lysine and N-carboxyethyl-lysine, p-cresol and p-cresyl sulfate, phosphate, uric acid and asymmetric dimethylarginine. Most studies described an increase in inflammation, oxidative stress, leukocyte migration and adhesion, cell death and a thrombotic phenotype upon uremic conditions or uremic toxin treatment of endothelial cells. Cellular signaling pathways that were frequently activated included the ROS, MAPK/NF-κB, the Aryl-Hydrocarbon-Receptor and RAGE pathways. Overall, this review provides detailed insights into pathophysiological and molecular mechanisms underlying endothelial dysfunction in CKD. Targeting these pathways may provide new therapeutic strategies reducing increased the cardiovascular risk in CKD.

Platelet Abnormalities in CKD and Their Implications for Antiplatelet Therapy.

Patients with CKD display a significantly higher risk of cardiovascular and thromboembolic complications, with around half of patients with advanced CKD ultimately dying of cardiovascular disease. Paradoxically, these patients also have a higher risk of hemorrhages, greatly complicating patient therapy. Platelets are central to hemostasis, and altered platelet function resulting in either platelet hyper- or hyporeactivity may contribute to thrombotic or hemorrhagic complications. Different molecular changes have been identified that may underlie altered platelet activity and hemostasis in CKD. In this study, we summarize the knowledge on CKD-induced aberrations in hemostasis, with a special focus on platelet abnormalities. We also discuss how prominent alterations in vascular integrity, coagulation, and red blood cell count in CKD may contribute to altered hemostasis in these patients who are high risk. Furthermore, with patients with CKD commonly receiving antiplatelet therapy to prevent secondary atherothrombotic complications, we discuss antiplatelet treatment strategies and their risk versus benefit in terms of thrombosis prevention, bleeding, and clinical outcome depending on CKD stage. This reveals a careful consideration of benefits versus risks of antiplatelet therapy in patients with CKD, balancing thrombotic versus bleeding risk. Nonetheless, despite antiplatelet therapy, patients with CKD remain at high cardiovascular risk. Thus, deep insights into altered platelet activity in CKD and underlying mechanisms are important for the optimization and development of current and novel antiplatelet treatment strategies, specifically tailored to these patients who are high risk. Ultimately, this review underlines the importance of a closer investigation of altered platelet function, hemostasis, and antiplatelet therapy in patients with CKD.

Effects of Platelet Agonists and Priming on the Formation of Platelet Populations.

Platelets from healthy donors display heterogeneity in responsiveness to agonists. The response thresholds of platelets are controlled by multiple bioactive molecules, acting as negatively or positively priming substances. Higher circulating levels of priming substances adenosine and succinate, as well as the occurrence of hypercoagulability, have been described for patients with ischaemic heart disease. Here, we present an improved methodology of flow cytometric analyses of platelet activation and the characterisation of platelet populations following activation and priming by automated clustering analysis.Platelets were treated with adenosine, succinate, or coagulated plasma before stimulation with CRP-XL, 2-MeSADP, or TRAP6 and labelled for activated integrin αIIbß3 (PAC1), CD62P, TLT1, CD63, and GPIX. The Super-Enhanced Dmax subtraction algorithm and 2% marker (quadrant) setting were applied to identify populations, which were further defined by state-of-the-art clustering techniques (tSNE, FlowSOM).Following activation, five platelet populations were identified: resting, aggregating (PAC1 + ), secreting (α- and dense-granules; CD62P + , TLT1 + , CD63 + ), aggregating plus α-granule secreting (PAC1 + , CD62P + , TLT1 + ), and fully active platelet populations. The type of agonist determined the distribution of platelet populations. Adenosine in a dose-dependent way suppressed the fraction of fully activated platelets (TRAP6 > 2-MeSADP > CRP-XL), whereas succinate and coagulated plasma increased this fraction (CRP-XL > TRAP6 > 2-MeSADP). Interestingly, a subset of platelets showed a constant response (aggregating, secreting, or aggregating plus α-granule secreting), which was hardly affected by the stimulus strength or priming substances.

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin.

BACKGROUND: Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. METHODS: Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. RESULTS: Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. CONCLUSION: Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.

Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions.

Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43-0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease.

Platelet Function in CKD: A Systematic Review and Meta-Analysis.

BACKGROUND: Patients with CKD are at high risk for thrombotic and hemorrhagic complications. Abnormalities in platelet function are central to these complications, but reports on platelet function in relation to CKD are conflicting, and vary from decreased platelet reactivity to normal or increased platelet responsiveness. The direct effects of uremic toxins on platelet function have been described, with variable findings. METHODS: To help clarify how CKD affects platelet function, we conducted a systematic review and meta-analysis of platelet activity in CKD, with a focus on nondialysis-induced effects. We also performed an extensive literature search for the effects of individual uremic toxins on platelet function. RESULTS: We included 73 studies in the systematic review to assess CKD's overall effect on platelet function in patients; 11 of them described CKD's effect on ex vivo platelet aggregation and were included in the meta-analysis. Although findings on platelet abnormalities in CKD are inconsistent, bleeding time was mostly prolonged and platelet adhesion mainly reduced. Also, the meta-analysis revealed maximal platelet aggregation was significantly reduced in patients with CKD upon collagen stimulation. We also found that relatively few uremic toxins have been examined for direct effects on platelets ex vivo; ex vivo analyses had varying methods and results, revealing both platelet-stimulatory and inhibitory effects. However, eight of the 12 uremic toxins tested in animal models mostly induced prothrombotic effects. CONCLUSIONS: Overall, most studies report impaired function of platelets from patients with CKD. Still, a substantial number of studies find platelet function to be unchanged or even enhanced. Further investigation of platelet reactivity in CKD, especially during different CKD stages, is warranted.

Comparison of inhibitory effects of irreversible and reversible Btk inhibitors on platelet function.

All irreversible Bruton tyrosine kinase (Btk) inhibitors including ibrutinib and acalabrutinib induce platelet dysfunction and increased bleeding risk. New reversible Btk inhibitors were developed, like MK-1026. The mechanism underlying increased bleeding tendency with Btk inhibitors remains unclear. We investigated the effects of ibrutinib, acalabrutinib and MK-1026 on platelet function in healthy volunteers, patients and Btk-deficient mice, together with off-target effects on tyrosine kinase phosphorylation. All inhibitors suppressed GPVI- and CLEC-2-mediated platelet aggregation, activation and secretion in a dose-dependent manner. Only ibrutinib inhibited thrombus formation on vWF-co-coated surfaces, while on collagen this was not affected. In blood from Btk-deficient mice, collagen-induced thrombus formation under flow was reduced, but preincubation with either inhibitor was without additional effects. MK-1026 showed less off-target effects upon GPVI-induced TK phosphorylation as compared to ibrutinib and acalabrutinib. In ibrutinib-treated patients, GPVI-stimulated platelet activation, and adhesion on vWF-co-coated surfaces were inhibited, while CLEC-2 stimulation induced variable responses. The dual inhibition of GPVI and CLEC-2 signalling by Btk inhibitors might account for the increased bleeding tendency, with ibrutinib causing more high-grade bleedings due to additional inhibition of platelet-vWF interaction. As MK-1026 showed less off-target effects and only affected activation of isolated platelets, it might be promising for future treatment.

Vitamin K antagonist use induces calcification and atherosclerotic plaque progression resulting in increased hypercoagulability.

Aims: Vascular calcification is a hallmark of atherosclerotic burden and can predict the cardiovascular outcome. Vitamin K antagonists (VKA) are widely used anticoagulant drugs to treat patients at risk of arterial and venous thrombosis but are also associated with increase vascular calcification progression. We aim to unravel the paradox that VKA suppresses plasma coagulation but promotes vascular calcification and subsequent atherosclerosis-dependent coagulability of the vessel wall. Methods and results: Apoe -/- mice were placed on western-type diet enriched with the VKA warfarin for 18 weeks to measure atherosclerotic plaque burden, calcification, and coagulation. Patients (n = 54) displaying paroxysmal atrial fibrillation with a low cardiovascular risk, who were treated with VKA were included to measure pre-thrombotic state. Finally, primary vascular smooth muscle cells (VSMC) derived from human tissue explants were used for in vitro experiments. In Apoe -/- mice, VKA increases both atherosclerotic plaque size and calcification. Higher plaque calcification was associated with increased plasma levels of thrombin-antithrombin and factor IXa-antithrombin complexes in mice and patients treated with VKA. Mechanistically, phenotypic switching of VSMC into synthetic VSMC promotes thrombin generation, which is enhanced in a tissue-factor (TF)-dependent manner by VSMC calcification. Moreover, calcified VSMC exposed to whole blood under flow significantly enhanced platelet deposition and TF-dependent fibrin formation. Conclusions: Oral anticoagulation with VKA aggravates vascular calcification and atherosclerosis. VSMC phenotype differentiation impacts coagulation potential in a TF-dependent manner. VKA-induced vascular calcification increases hypercoagulability and could thereby potentially positively affect atherothrombosis.

Native, Intact Glucagon-Like Peptide 1 Is a Natural Suppressor of Thrombus Growth Under Physiological Flow Conditions.

OBJECTIVE: In patients with diabetes mellitus, increased platelet reactivity predicts cardiac events. Limited evidence suggests that DPP-4 (dipeptidyl peptidase 4) influences platelets via GLP-1 (glucagon-like peptide 1)-dependent effects. Because DPP-4 inhibitors are frequently used in diabetes mellitus to improve the GLP-1-regulated glucose metabolism, we characterized the role of DPP-4 inhibition and of native intact versus DPP-4-cleaved GLP-1 on flow-dependent thrombus formation in mouse and human blood. Approach and Results: An ex vivo whole blood microfluidics model was applied to approach in vivo thrombosis and study collagen-dependent platelet adhesion, activation, and thrombus formation under shear-flow conditions by multiparameter analyses. In mice, in vivo inhibition or genetic deficiency of DPP-4 (Dpp4-/-), but not of GLP-1-receptors (Glp1r-/-), suppressed flow-dependent platelet aggregation. In human blood, GLP-1(7-36), but not DPP-4-cleaved GLP-1(9-36), reduced thrombus volume by 32% and impaired whole blood thrombus formation at both low/venous and high/arterial wall-shear rates. These effects were enforced upon ADP costimulation and occurred independently of plasma factors and leukocytes. Human platelets did not contain detectable levels of GLP-1-receptor transcripts. Also, GLP-1(7-36) did not inhibit collagen-induced aggregation under conditions of stirring or stasis of platelets, pointing to a marked flow-dependent role. CONCLUSIONS: Native, intact GLP-1 is a natural suppressor of thrombus growth under physiological flow conditions, with DPP-4 inhibition and increased intact GLP-1 suppressing platelet aggregation under flow without a main relevance of GLP-1-receptor on platelets.

Platelet Membrane Receptor Proteolysis: Implications for Platelet Function.

The activities of adhesion and signaling receptors in platelets are controlled by several mechanisms. An important way of regulation is provided by proteolytic cleavage of several of these receptors, leading to either a gain or a loss of platelet function. The proteases involved are of different origins and types: (i) present as precursor in plasma, (ii) secreted into the plasma by activated platelets or other blood cells, or (iii) intracellularly activated and cleaving cytosolic receptor domains. We provide a comprehensive overview of the proteases acting on the platelet membrane. We describe how these are activated, which are their target proteins, and how their proteolytic activity modulates platelet functions. The review focuses on coagulation-related proteases, plasmin, matrix metalloproteinases, ADAM(TS) isoforms, cathepsins, caspases, and calpains. We also describe how the proteolytic activities are determined by different platelet populations in a thrombus and conversely how proteolysis contributes to the formation of such populations.

Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis.

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 α6ß1 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.

High-throughput elucidation of thrombus formation reveals sources of platelet function variability.

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 αIIbß3 activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced αIIbß3 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.