Publications by authors named "Johan W M Heemskerk"

164 Publications

Increased platelet thrombus formation under flow conditions in whole blood from polycythaemia vera patients.

Blood Transfus 2021 Mar 30. Epub 2021 Mar 30.

Department of Immunohaematology and Transfusion Medicine, "Papa Giovanni XXIII" Hospital, Bergamo, Italy.

Background: Polycythaemia vera is a myeloproliferative neoplasm characterised by a high incidence of thrombosis. The contribution of platelets, key players in haemostasis, in this setting is still unclear. So far, the majority of studies have been focussed on specific platelet abnormalities but not on their actual capacity to form thrombi. The aim of this study was to characterise, ex vivo under flow conditions, the capacity of platelets from patients with polycythaemia vera to adhere to collagen and induce thrombus formation.

Materials And Methods: Thirty-nine patients and 30 healthy controls were studied. Thrombus formation was induced by perfusing whole blood over a collagen-coated surface, in a parallel-plate flow chamber coupled to a fluorescent microscope. This dynamic system enables platelet adhesion and thrombus formation to be followed in real time and also allows measurements of the extent of the thrombus and platelet surface antigen expression. Laboratory data were analysed in the light of the patients' main haematological parameters and therapies.

Results: Platelet adhesion was significantly greater in patients than in control subjects. Patient thrombi were usually larger and more complex than those formed by control platelets. A significant positive correlation was found between platelet adhesion and both the haematocrit and red blood cell count. These parameters remained significantly correlated with platelet adhesion also after multivariable analysis adjusted for gender, age, therapy and JAK2V617F allele burden. Furthermore, subjects with a haematocrit >45% had significantly greater platelet adhesion than subjects with a haematocrit <45%.

Discussion: Our data indicate that increased platelet adhesion participates in the thrombotic diathesis of patients with polycythaemia vera, and that the haematocrit level can affect the adhesive and thrombus forming capacities of platelets.
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http://dx.doi.org/10.2450/2021.0456-20DOI Listing
March 2021

Long-term platelet priming after glycoprotein VI stimulation in comparison to Protease-Activating Receptor (PAR) stimulation.

PLoS One 2021 3;16(3):e0247425. Epub 2021 Mar 3.

Department of Biochemistry, CARIM, Maastricht University, Maastricht, The Netherlands.

Platelets can respond to multiple antagonists and agonists, implying that their activation state is a consequence of past exposure to these substances. While platelets are often considered as one-time responsive cells, they likely can respond to sequential application of inhibitors and stimuli. We hypothesized that the ability of platelets to sequentially respond depends on the time and type of repeated agonist application. The present proof-of-concept data show that iloprost (cAMP elevation), tirofiban (integrin αIIbβ3 blocker) and Syk kinase inhibition subacutely modulated platelet aggregation, i.e. halted this process even when applied after agonist. In comparison to thrombin-activated receptor (PAR) stimulation, glycoprotein VI (GPVI) stimulation was less sensitive to time-dependent blockage of aggregation, with Syk inhibition as an exception. Furthermore, cytosolic Ca2+ measurements indicated that, when compared to PAR, prior GPVI stimulation induced a more persistent, priming activation state of platelets that influenced the response to a next agent. Overall, these data point to an unexpected priming memory of activated platelets in subacutely responding to another inhibitor or stimulus, with a higher versatility and faster offset after PAR stimulation than after GPVI stimulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928515PMC
March 2021

Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks.

Res Pract Thromb Haemost 2021 Jan 18;5(1):231-242. Epub 2020 Dec 18.

Department of Biochemistry Cardiovascular Research Institute Maastricht (CARIM) Maastricht University Maastricht The Netherlands.

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 , an African soft tick. Whereas conventional αIIbβ3 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-αIIbβ3 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) 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 ( < .01) and eptifibatide ( < .05).

Conclusions: Both αIIbβ3-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 αIIbβ3 inhibitors.
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http://dx.doi.org/10.1002/rth2.12466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845065PMC
January 2021

Platelet Membrane Receptor Proteolysis: Implications for Platelet Function.

Front Cardiovasc Med 2020 8;7:608391. Epub 2021 Jan 8.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands.

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.
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http://dx.doi.org/10.3389/fcvm.2020.608391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820117PMC
January 2021

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro.

PLoS One 2021 7;16(1):e0244736. Epub 2021 Jan 7.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244736PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790394PMC
January 2021

Platelet calcium signaling by G-protein coupled and ITAM-linked receptors regulating anoctamin-6 and procoagulant activity.

Platelets 2020 Dec 26:1-9. Epub 2020 Dec 26.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University , Maastricht, The Netherlands.

Most agonists stimulate platelet Ca rises via G-protein coupled receptors (GPCRs) or ITAM-linked receptors (ILRs). Well studied are the GPCRs stimulated by the soluble agonists thrombin (PAR1, PAR4), ADP (P2Y, P2Y), and thromboxane A (TP), signaling via phospholipase (PLC)β isoforms. The platelet ILRs glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2), and FcγRIIa are stimulated by adhesive ligands or antibody complexes and signal via tyrosine protein kinases and PLCγ isoforms. Marked differences exist between the GPCR- and ILR-induced Ca signaling in: dependency of tyrosine phosphorylation; oscillatory versus continued Ca rises by mobilization from the endoplasmic reticulum; and smaller or larger role of extracellular Ca entry via STIM1/ORAI1. Co-stimulation of both types of receptors, especially by thrombin (PAR1/4) and collagen (GPVI), leads to a highly enforced Ca rise, involving mitochondrial Ca release, which activates the ion and phospholipid channel, anoctamin-6. This highly Ca-dependent process causes swelling, ballooning, and phosphatidylserine expression, establishing a unique platelet population swinging between vital and necrotic (procoagulant 'zombie' platelets). Additionally, the high Ca status of procoagulant platelets induces a set of additional events: Ca dependent cleavage of signaling proteins and receptors via calpain and ADAM isoforms; microvesiculation; enhanced coagulation factor binding; and fibrin-coat formation involving transglutaminases. Given the additive roles of GPCR and ILR in Ca signal generation, high-throughput screening of biomolecules or small molecules based on Ca flux measurements provides a promising way to find new inhibitors interfering with prolonged high Ca, phosphatidylserine expression, and hence platelet procoagulant activity.
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http://dx.doi.org/10.1080/09537104.2020.1859103DOI Listing
December 2020

Mild hyperlipidemia in mice aggravates platelet responsiveness in thrombus formation and exploration of platelet proteome and lipidome.

Sci Rep 2020 12 8;10(1):21407. Epub 2020 Dec 8.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.

Hyperlipidemia is a well-established risk factor for cardiovascular diseases. Millions of people worldwide display mildly elevated levels of plasma lipids and cholesterol linked to diet and life-style. While the prothrombotic risk of severe hyperlipidemia has been established, the effects of moderate hyperlipidemia are less clear. Here, we studied platelet activation and arterial thrombus formation in Apoe and Ldlr mice fed a normal chow diet, resulting in mildly increased plasma cholesterol. In blood from both knockout mice, collagen-dependent thrombus and fibrin formation under flow were enhanced. These effects did not increase in severe hyperlipidemic blood from aged mice and upon feeding a high-fat diet (Apoe mice). Bone marrow from wild-type or Ldlr mice was transplanted into irradiated Ldlr recipients. Markedly, thrombus formation was enhanced in blood from chimeric mice, suggesting that the hyperlipidemic environment altered the wild-type platelets, rather than the genetic modification. The platelet proteome revealed high similarity between the three genotypes, without clear indication for a common protein-based gain-of-function. The platelet lipidome revealed an altered lipid profile in mildly hyperlipidemic mice. In conclusion, in Apoe and Ldlr mice, modest elevation in plasma and platelet cholesterol increased platelet responsiveness in thrombus formation and ensuing fibrin formation, resulting in a prothrombotic phenotype.
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http://dx.doi.org/10.1038/s41598-020-78522-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722935PMC
December 2020

Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbβ3 in Fibrin-Mediated Microthrombus Formation.

Arterioscler Thromb Vasc Biol 2020 Dec 3:ATVBAHA120314641. Epub 2020 Dec 3.

Department of Biochemistry, CARIM, Maastricht University, the Netherlands (G.P., J.H., I.P., F.S., P.E.J.v.d.M., R.A.S.A., S.P.W., J.W.M.H.).

Objective: Fibrin is considered to strengthen thrombus formation via integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbβ3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers.

Conclusions: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 through signaling via Syk and low-level Ca rises.
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http://dx.doi.org/10.1161/ATVBAHA.120.314641DOI Listing
December 2020

Complementary roles of platelet αβ integrin, phosphatidylserine exposure and cytoskeletal rearrangement in the release of extracellular vesicles.

Atherosclerosis 2020 10 1;310:17-25. Epub 2020 Aug 1.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands. Electronic address:

Background And Aims: Platelets can release extracellular vesicles (EVs) upon stimulation with various agonists. Interestingly, platelets from patients with Glanzmann thrombasthenia have reduced EV release. These platelets lack functional αβ integrins, indicating that αβ integrin is critical in vesicle release. Integrin activation is central in platelet function and is associated with e.g. adhesion, aggregation and cytoskeletal rearrangement. However, while platelet activation pathways are widely known, the mechanisms underlying EV release remain uncharacterized. We investigated the role of integrin αβ, phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release.

Methods: EVs were isolated from activated platelets. Platelet activation status was measured by multicolour flow cytometry. A panel of pharmacologic inhibitors was used to interfere in specific signalling pathways. EV release was quantified enzymatically based on membrane PS content and nanoparticle tracking analysis. In addition, real-time visualization of EV shedding with confocal microscopy and EVs with Cryo-TEM imaging was performed.

Results: Platelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of αβ integrin activation and subsequent closure paralleled by PS exposure. Prevention of αβ activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using αβ-deficient platelets from patients with Glanzmann thrombasthenia. Inhibition of actin cytoskeleton rearrangement decreased EV release, whereas inhibition of individual signalling targets upstream of cytoskeletal rearrangement showed no such effects.

Conclusion: Platelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.07.015DOI Listing
October 2020

Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation.

Sci Rep 2020 07 17;10(1):11910. Epub 2020 Jul 17.

Departments of Biochemistry and Internal Medicine, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre+, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.

In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet-fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping.
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http://dx.doi.org/10.1038/s41598-020-68438-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368055PMC
July 2020

Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS.

Sci Rep 2020 07 9;10(1):11389. Epub 2020 Jul 9.

Department of Biochemistry, CARIM, Maastricht University, PO Box 616, 6200 MD, Maastricht, The Netherlands.

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsα-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.
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http://dx.doi.org/10.1038/s41598-020-68379-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347634PMC
July 2020

Flow studies on human GPVI-deficient blood under coagulating and noncoagulating conditions.

Blood Adv 2020 07;4(13):2953-2961

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.

The role of glycoprotein VI (GPVI) in platelets was investigated in 3 families bearing an insertion within the GP6 gene that introduces a premature stop codon prior to the transmembrane domain, leading to expression of a truncated protein in the cytoplasm devoid of the transmembrane region. Western blotting and flow cytometry of GP6hom (homozygous) platelets confirmed loss of the full protein. The level of the Fc receptor γ-chain, which associates with GPVI in the membrane, was partially reduced, but expression of other receptors and signaling proteins was not altered. Spreading of platelets on collagen and von Willebrand factor (which supports partial spreading) was abolished in GP6hom platelets, and spreading on uncoated glass was reduced. Anticoagulated whole blood flowed over immobilized collagen or a mixture of von Willebrand factor, laminin, and rhodocytin (noncollagen surface) generated stable platelet aggregates that express phosphatidylserine (PS). Both responses were blocked on the 2 surfaces in GP6hom individuals, but adhesion was not altered. Thrombin generation was partially reduced in GP6hom blood. The frequency of the GP6het (heterozygous) variant in a representative sample of the Chilean population (1212 donors) is 2.9%, indicating that there are ∼4000 GP6hom individuals in Chile. These results demonstrate that GPVI supports aggregation and PS exposure under flow on collagen and noncollagen surfaces, but not adhesion. The retention of adhesion may contribute to the mild bleeding diathesis of GP6hom patients and account for why so few of the estimated 4000 GP6hom individuals in Chile have been identified.
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http://dx.doi.org/10.1182/bloodadvances.2020001761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362345PMC
July 2020

Clonal hematopoietic mutations linked to platelet traits and the risk of thrombosis or bleeding.

Haematologica 2020 08 18;105(8):2020-2031. Epub 2020 Jun 18.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht

Platelets are key elements in thrombosis, particularly in atherosclerosis-associated arterial thrombosis (atherothrombosis), and hemostasis. Megakaryocytes in the bone marrow, differentiated from hematopoietic stem cells are generally considered as a uniform source of platelets. However, recent insights into the causes of malignancies, including essential thrombocytosis, indicate that not only inherited but also somatic mutations in hematopoietic cells are linked to quantitative or qualitative platelet abnormalities. In particular cases, these form the basis of thrombo-hemorrhagic complications regularly observed in patient groups. This has led to the concept of clonal hematopoiesis of indeterminate potential (CHIP), defined as somatic mutations caused by clonal expansion of mutant hematopoietic cells without evident disease. This concept also provides clues regarding the importance of platelet function in relation to cardiovascular disease. In this summative review, we present an overview of genes associated with clonal hematopoiesis and altered platelet production and/or functionality, like mutations in We consider how reported CHIP genes can influence the risk of cardiovascular disease, by exploring the consequences for platelet function related to (athero)thrombosis, or the risk of bleeding. More insight into the functional consequences of the CHIP mutations may favor personalized risk assessment, not only with regard to malignancies but also in relation to thrombotic vascular disease.
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http://dx.doi.org/10.3324/haematol.2019.235994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395290PMC
August 2020

Crystal Clots as Therapeutic Target in Cholesterol Crystal Embolism.

Circ Res 2020 04 24;126(8):e37-e52. Epub 2020 Feb 24.

From the Medizinische Klinik und Poliklinik IV, Klinikum der Universität, LMU München, Germany (C.S., T.K., S.S., S.R.M., L.Y., H.-J.A.).

Rationale: Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown.

Objective: We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure.

Methods And Results: C57BL/6J mice were injected with CC into the left kidney artery. Primary end point was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, that is, acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain-like deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA. Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase, or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was <3 hours after CC injection. However, combining Nec-1s (necrostatin-1s) prophylaxis given 1 hour before and DNase I 3 hours after CC injection completely prevented kidney failure and infarcts. In vitro, CC did not directly induce plasmatic coagulation but induced neutrophil extracellular trap formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. CC induced ATP release from aggregating platelets, which increased fibrin formation in a DNase-dependent manner.

Conclusions: CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315625DOI Listing
April 2020

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

Arterioscler Thromb Vasc Biol 2020 03 2;40(3):e65-e77. Epub 2020 Jan 2.

From the Institute for Molecular Cardiovascular Research (IMCAR) (M.S., C.C.F.M.J.B., J.W., W.T., B.W., J.J., H.N.), University Clinic Aachen, Germany.

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 (), but not of GLP-1-receptors (), 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.
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http://dx.doi.org/10.1161/ATVBAHA.119.313645DOI Listing
March 2020

Localized endothelial-based control of platelet aggregation and coagulation under flow: A proof-of-principle vessel-on-a-chip study.

J Thromb Haemost 2020 04 20;18(4):931-941. Epub 2020 Feb 20.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

Background: In the intact vessel wall, endothelial cells form a barrier between the blood and the remaining vascular structures, serving to maintain blood fluidity and preventing platelet activation and fibrin clot formation. The spatiotemporal space of this inhibition is largely unknown.

Objective: To assess the local inhibitory roles of a discontinuous endothelium, we developed a vessel-on-a-chip model, consisting of a microfluidic chamber coated with the thrombogenic collagen and tissue factor (TF), and covered with patches of human endothelial cells. By flow perfusion of human blood and plasma, the heterogeneous formation of platelet aggregates and fibrin clots was monitored by multicolor fluorescence microscopy.

Results: On collagen/TF coatings, a coverage of 40% to 60% of human umbilical vein endothelial cells resulted in a strong overall delay in platelet deposition and fibrin fiber formation under flow. Fibrin formation colocalized with the deposited platelets, and was restricted to regions in between endothelial cells, thus pointing to immediate local suppression of the clotting process. Fibrin kinetics were enhanced by treatment of the cells with heparinase III, partially disrupting the glycocalyx, and to a lesser degree by antagonism of the endothelial thrombomodulin. Co-coating of purified thrombomodulin and collagen had a similar coagulation-suppressing effect as endothelial thrombomodulin.

Conclusions: In this vessel-on-a-chip system with patches of endothelial cells on thrombogenic surfaces, the coagulant activity under flow is regulated by: (a) the residual exposure of trigger (collagen/TF), (b) the endothelial glycocalyx, and (c) to a lesser degree the endothelial thrombomodulin.
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http://dx.doi.org/10.1111/jth.14719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187151PMC
April 2020

Whole Blood Based Multiparameter Assessment of Thrombus Formation in Standard Microfluidic Devices to Proxy In Vivo Haemostasis and Thrombosis.

Micromachines (Basel) 2019 Nov 16;10(11). Epub 2019 Nov 16.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands.

Microfluidic assays are versatile tests which, using only small amounts of blood, enable high throughput analyses of platelet function in several minutes. In combination with fluorescence microscopy, these flow tests allow real-time visualisation of platelet activation with the possibility of examining combinatorial effects of wall shear rate, coagulation and modulation by endothelial cells. In particular, the ability to use blood and blood cells from healthy subjects or patients makes this technology promising, both for research and (pre)clinical diagnostic purposes. In the present review, we describe how microfluidic devices are used to assess the roles of platelets in thrombosis and haemostasis. We place emphasis on technical aspects and on experimental designs that make the concept of "blood-vessel-component-on-a-chip" an attractive, rapidly developing technology for the study of the complex biological processes of blood coagulability in the presence of flow.
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http://dx.doi.org/10.3390/mi10110787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915499PMC
November 2019

The Microbiota Promotes Arterial Thrombosis in Low-Density Lipoprotein Receptor-Deficient Mice.

mBio 2019 10 22;10(5). Epub 2019 Oct 22.

Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany

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 ( ) 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 mice and CONV-R 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 mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF mice on HFD were diminished compared to CONV-R controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF 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 mice relative to CONV-R mice. , this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF mice on type III collagen. 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 mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished thrombus formation under arterial flow conditions.
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http://dx.doi.org/10.1128/mBio.02298-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805995PMC
October 2019

Impact of Deficiency of Intrinsic Coagulation Factors XI and XII on Ex Vivo Thrombus Formation and Clot Lysis.

TH Open 2019 Jul 10;3(3):e273-e285. Epub 2019 Sep 10.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Center, Maastricht, The Netherlands.

The contributions of coagulation factor XI (FXI) and FXII to human clot formation is not fully known. Patients with deficiency in FXI have a variable mild bleeding risk, whereas FXII deficiency is not associated with bleeding. These phenotypes make FXII and FXI attractive target proteins in anticoagulant therapy. Here, we studied the mechanisms of fibrin clot formation, stability, and fibrinolytic degradation in patients with severe FXI or FXII deficiency. Thrombin generation was triggered in platelet-poor (PPP) and platelet-rich plasma (PRP) with the biological FXII trigger sulfatides. Intrinsic and extrinsic thrombus formation and degradation in whole blood were determined with rotational thromboelastometry (ROTEM). Clot formation under flow was assessed by perfusion of whole blood over collagen microspots with(out) tissue factor (TF). Thrombin generation and clot formation were delayed in FXII- and FXI-deficient patients triggered with sulfatides. In FXI-deficient plasma, this delay was more pronounced in PRP compared to PPP. In whole blood of FXII-deficient patients, clots were smaller but resistance to fibrinolysis was normal. In whole blood of FXI-deficient patients, clot formation was normal but the time to complete fibrinolysis was prolonged. In flow chamber experiments triggered with collagen/TF, platelet coverage was reduced in severe compared with moderate FXI deficiency, and fibrin formation was impaired. We conclude that quantitative defects in FXII and FXI have a substantial impact on contact activation-triggered coagulation. Furthermore, FXI deficiency has a dose-dependent suppressing effect on flow-mediated and platelet/TF-dependent clot formation. These last data highlight the contribution of particularly FXI to hemostasis.
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http://dx.doi.org/10.1055/s-0039-1693485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736668PMC
July 2019

LIM-only protein FHL2 attenuates vascular tissue factor activity, inhibits thrombus formation in mice and genetic variation associates with human venous thrombosis.

Haematologica 2020 06 29;105(6):1677-1685. Epub 2019 Aug 29.

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our observations, decreases TF activity in endothelial and smooth muscle cells whereas knockdown or deficiency results in enhanced TF activity. Finally, the single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify as a novel gene associated with venous thrombosis in humans.
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http://dx.doi.org/10.3324/haematol.2018.203026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271603PMC
June 2020

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

Front Cardiovasc Med 2019 30;6:99. Epub 2019 Jul 30.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands.

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 αβ 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 Ca homeostasis. The majority of mice demonstrating an antithrombotic phenotype displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation . Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis , this was accompanied by impaired hemostasis. This was also reflected by comparing thrombus formation (by microfluidics) with alterations in 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 arterial thrombosis and hemostasis.
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http://dx.doi.org/10.3389/fcvm.2019.00099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682619PMC
July 2019

Clinical Protocol to Prevent Thrombogenic Effect of Liver-Derived Mesenchymal Cells for Cell-Based Therapies.

Cells 2019 08 7;8(8). Epub 2019 Aug 7.

Laboratoire d'Hépatologie Pédiatrique et Thérapie Cellulaire, Unité PEDI, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain (UCLouvain), 1200 Brussels, Belgium.

The efficacy of mesenchymal stem cell infusion is currently tested in numerous clinical trials. However, therapy-induced thrombotic consequences have been reported in several patients. The aim of this study was to optimize protocols for heterologous human adult liver-derived progenitor cell (HHALPC) infusion, in order to eliminate acute thrombogenesis in liver-based metabolic or acute decompensated cirrhotic (ADC) patients. In rats, thrombotic effects were absent when HHALPCs were infused at low cell dose (5 × 10 cells/kg), or at high cell dose (5 × 10 cells/kg) when combined with anticoagulants. When HHALPCs were exposed to human blood in a whole blood perfusion assay, blocking of the tissue factor (TF) coagulation pathway suppressed fibrin generation and platelet activation. In a Chandler tubing loop model, HHALPCs induced less explosive activation of coagulation with blood from ADC patients, when compared to blood from healthy controls, without alterations in coagulation factor levels other than fibrinogen. These studies confirm a link between TF and thrombogenesis, when TF-expressing cells are exposed to human blood. This phenomenon however, could be controlled using either a low, or a high cell dose combined with anticoagulants. In clinical practice, this points to the suitability of a low HHALPC dose infusion to cirrhotic patients, provided that platelet and fibrinogen levels are monitored.
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http://dx.doi.org/10.3390/cells8080846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721739PMC
August 2019

Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces.

Int J Mol Sci 2019 Jun 7;20(11). Epub 2019 Jun 7.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands.

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 αβ. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca. 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) and Horm-type collagen evoked Syk-dependent Ca rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO) 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) sequence.
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http://dx.doi.org/10.3390/ijms20112788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600290PMC
June 2019

Defective Zn homeostasis in mouse and human platelets with α- and δ-storage pool diseases.

Sci Rep 2019 06 6;9(1):8333. Epub 2019 Jun 6.

Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany.

Zinc (Zn) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn storage and release. To visualize Zn storage in human and mouse platelets, the Zn specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn release upon activation. Platelets from Nbeal2 mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2 and Unc13d mice, and the impairment could be partially restored by extracellular Zn. Altogether, we conclude that the release of ionic Zn store from secretory granules upon platelet activation contributes to the procoagulant role of Zn in platelet-dependent fibrin formation.
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http://dx.doi.org/10.1038/s41598-019-44751-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554314PMC
June 2019

Comparison of the GPVI inhibitors losartan and honokiol.

Platelets 2020 8;31(2):187-197. Epub 2019 Mar 8.

Institute of Cardiovascular Sciences, IBR Building, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.
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http://dx.doi.org/10.1080/09537104.2019.1585526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034533PMC
September 2020

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

Haematologica 2019 06 13;104(6):1256-1267. Epub 2018 Dec 13.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands

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 αβ activation and secretion. Common sequence variation of and , associated with GPVI-induced αβ 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.
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http://dx.doi.org/10.3324/haematol.2018.198853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545858PMC
June 2019

Store-operated calcium entry in thrombosis and thrombo-inflammation.

Cell Calcium 2019 01 23;77:39-48. Epub 2018 Nov 23.

Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Centre, University of Würzburg, Würzburg, Germany. Electronic address:

Cytosolic free calcium (Ca) is a second messenger regulating a wide variety of functions in blood cells, including adhesion, activation, proliferation and migration. Store-operated Ca entry (SOCE), triggered by depletion of Ca from the endoplasmic reticulum, provides a main mechanism of regulated Ca influx in blood cells. SOCE is mediated and regulated by isoforms of the ion channel proteins ORAI and TRP, and the transmembrane Ca sensors stromal interaction molecules (STIMs), respectively. This report provides an overview of the (patho)physiological importance of SOCE in blood cells implicated in thrombosis and thrombo-inflammation, i.e. platelets and immune cells. We also discuss the physiological consequences of dysregulated SOCE in platelets and immune cells and the potential of SOCE inhibition as a therapeutic option to prevent or treat arterial thrombosis as well as thrombo-inflammatory disease states such as ischemic stroke.
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http://dx.doi.org/10.1016/j.ceca.2018.11.005DOI Listing
January 2019

Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients.

Front Cardiovasc Med 2018 16;5:142. Epub 2018 Oct 16.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands.

Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients.
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http://dx.doi.org/10.3389/fcvm.2018.00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232667PMC
October 2018

Platelet biology and functions: new concepts and clinical perspectives.

Nat Rev Cardiol 2019 03;16(3):166-179

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

Platelets - blood cells continuously produced from megakaryocytes mainly in the bone marrow - are implicated not only in haemostasis and arterial thrombosis, but also in other physiological and pathophysiological processes. This Review describes current evidence for the heterogeneity in platelet structure, age, and activation properties, with consequences for a diversity of platelet functions. Signalling processes of platelet populations involved in thrombus formation with ongoing coagulation are well understood. Genetic approaches have provided information on multiple genes related to normal haemostasis, such as those encoding receptors and signalling or secretory proteins, that determine platelet count and/or responsiveness. As highly responsive and secretory cells, platelets can alter the environment through the release of growth factors, chemokines, coagulant factors, RNA species, and extracellular vesicles. Conversely, platelets will also adapt to their environment. In disease states, platelets can be positively primed to reach a pre-activated condition. At the inflamed vessel wall, platelets interact with leukocytes and the coagulation system, interactions mediating thromboinflammation. With current antiplatelet therapies invariably causing bleeding as an undesired adverse effect, novel therapies can be more beneficial if directed against specific platelet responses, populations, interactions, or priming conditions. On the basis of these novel concepts and processes, we discuss several initiatives to target platelets therapeutically.
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http://dx.doi.org/10.1038/s41569-018-0110-0DOI Listing
March 2019

A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Blood 2018 12 1;132(24):e35-e46. Epub 2018 Oct 1.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (, , , ) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes.
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http://dx.doi.org/10.1182/blood-2018-02-831982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293874PMC
December 2018