Publications by authors named "Constance C F M J Baaten"

16 Publications

  • Page 1 of 1

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

Thromb Haemost 2021 Jun 15. Epub 2021 Jun 15.

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

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 [Ca], 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 Ca 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.
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http://dx.doi.org/10.1055/s-0041-1730312DOI Listing
June 2021

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

Sci Rep 2021 Jun 11;11(1):12358. Epub 2021 Jun 11.

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

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.
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http://dx.doi.org/10.1038/s41598-021-91661-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196183PMC
June 2021

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

J Am Soc Nephrol 2021 May 3. Epub 2021 May 3.

Institute for Molecular Cardiovascular Research, University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule Aachen University, Aachen, Germany

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 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 ; 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.
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http://dx.doi.org/10.1681/ASN.2020101440DOI Listing
May 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

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

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

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

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

Platelet heterogeneity in activation-induced glycoprotein shedding: functional effects.

Blood Adv 2018 09;2(18):2320-2331

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

The platelet receptors glycoprotein Ibα (GPIbα) and GPVI are known to be cleaved by members of a disintegrin and metalloprotease (ADAM) family (ADAM10 and ADAM17), but the mechanisms and consequences of this shedding are not well understood. Our results revealed that (1) glycoprotein shedding is confined to distinct platelet populations showing near-complete shedding, (2) the heterogeneity between (non)shed platelets is independent of agonist type but coincides with exposure of phosphatidylserine (PS), and (3) distinct pathways of shedding are induced by elevated Ca, low Ca protein kinase C (PKC), or apoptotic activation. Furthermore, we found that receptor shedding reduces binding of von Willebrand factor, enhances binding of coagulation factors, and augments fibrin formation. In response to Ca-increasing agents, shedding of GPIbα was abolished by ADAM10/17 inhibition but not by blockage of calpain. Stimulation of PKC induced shedding of only GPIbα, which was annulled by kinase inhibition. The proapoptotic agent ABT-737 induced shedding, which was caspase dependent. In Scott syndrome platelets that are deficient in Ca-dependent PS exposure, shedding occurred normally, indicating that PS exposure is not a prerequisite for ADAM activity. In whole-blood thrombus formation, ADAM-dependent glycoprotein shedding enhanced thrombin generation and fibrin formation. Together, these findings indicate that 2 major activation pathways can evoke ADAM-mediated glycoprotein shedding in distinct platelet populations and that shedding modulates platelet function from less adhesive to more procoagulant.
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http://dx.doi.org/10.1182/bloodadvances.2017011544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156892PMC
September 2018

Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy.

Haematologica 2018 09 7;103(9):1557-1567. Epub 2018 Jun 7.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, the Netherlands

Severe thrombocytopenia (≤50×10 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca flux, integrin a β activation and P-selectin expression when stimulated with a panelof agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca store content was unaffected, the patients' platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (<0.001) and low oxygen consumption (<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, =0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients.
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http://dx.doi.org/10.3324/haematol.2017.185165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119160PMC
September 2018

Platelet populations and priming in hematological diseases.

Blood Rev 2017 11 22;31(6):389-399. Epub 2017 Jul 22.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Electronic address:

In healthy subjects and patients with hematological diseases, platelet populations can be distinguished with different response spectra in hemostatic and vascular processes. These populations partly overlap, and are less distinct than those of leukocytes. The platelet heterogeneity is linked to structural properties, and is enforced by inequalities in the environment. Contributing factors are variability between megakaryocytes, platelet ageing, and positive or negative priming of platelets during their time in circulation. Within a hemostatic plug or thrombus, platelet heterogeneity is enhanced by unequal exposure to agonists, with populations of contracted platelets in the thrombus core, discoid platelets at the thrombus surface, patches of ballooned and procoagulant platelets forming thrombin, and coated platelets binding fibrin. Several pathophysiological hematological conditions can positively or negatively prime the responsiveness of platelet populations. As a consequence, in vivo and in vitro markers of platelet activation can differ in thrombotic and hematological disorders.
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http://dx.doi.org/10.1016/j.blre.2017.07.004DOI Listing
November 2017

Platelet Control of Fibrin Distribution and Microelasticity in Thrombus Formation Under Flow.

Arterioscler Thromb Vasc Biol 2016 Apr 4;36(4):692-9. Epub 2016 Feb 4.

From the Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands (F.S., C.C.F.M.J.B., R.V., T.G.M., J.M.E.M.C., J.W.M.H., P.E.J.v.d.M.); Research and Development, Optics11, Amsterdam, The Netherlands (N.R., K.O.v.d.L., E.J.B.); Arthur Bloom Haemophilia Centre, Cardiff Institute of Infection & Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom (P.W.C.); and Central Diagnostic Laboratory (Y.M.C.H.), Departments of Anaesthesiology (M.D.L.) and Internal Medicine (Y.M.C.H.), Maastricht University Medical Center, Maastricht, The Netherlands.

Objective: Platelet- and fibrin-dependent thrombus formation is regulated by blood flow and exposure of collagen and tissue factor. However, interactions between these blood-borne and vascular components are not well understood.

Approach And Results: Here, we developed a method to assess whole-blood thrombus formation on microspots with defined amounts of collagen and tissue factor, allowing determination of the mechanical properties and intrathrombus composition. Confining the collagen content resulted in diminished platelet deposition and fibrin formation at high shear flow conditions, but this effect was compensated by a larger thrombus size and increased accumulation of fibrin in the luminal regions of the thrombi at the expense of the base regions. These thrombi were more dependent on tissue factor-triggered thrombin generation. Microforce nanoindentation analysis revealed a significantly increased microelasticity of thrombi with luminal-oriented fibrin. At a low shear rate, fibrin fibers tended to luminally cover the thrombi, again resulting in a higher microelasticity. Studies with blood from patients with distinct hemostatic insufficiencies indicated an impairment in the formation of a platelet-fibrin thrombus in the cases of dilutional coagulopathy, thrombocytopenia, Scott syndrome, and hemophilia B.

Conclusions: Taken together, our data indicate that (1) thrombin increases the platelet thrombus volume; (2) tissue factor drives the formation of fibrin outside of the platelet thrombus; (3) limitation of platelet adhesion redirects fibrin from bottom to top of the thrombus; (4) a lower shear rate promotes thrombus coverage with fibrin; (5) the fibrin distribution pattern determines thrombus microelasticity; and (6) the thrombus-forming process is reduced in patients with diverse hemostatic defects.
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http://dx.doi.org/10.1161/ATVBAHA.115.306537DOI Listing
April 2016

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII.

Haematologica 2016 Apr 31;101(4):427-36. Epub 2015 Dec 31.

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

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)β3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)β3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)β3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)β3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)β3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)β3.
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http://dx.doi.org/10.3324/haematol.2015.131441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004391PMC
April 2016

Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage.

FASEB J 2016 Feb 19;30(2):727-37. Epub 2015 Oct 19.

*Department of Cell Biochemistry of Thrombosis and Haemostasis Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, The Netherlands; Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany; Walter Brendel Centre of Experimental Medicine and German Centre of Cardiovascular Research, Munich Heart Alliance, Ludwig-Maximilians-Universität München, München, Germany; Department of Developmental Biology, Centre for Medical Biotechnology, University of Duisburg-Essen, Duisburg-Essen, Germany; Aragon Institute of Health Sciences I+CS/IIS and ARAID, Zaragoza, Spain; School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom; Institute of Physiology, University of Regensburg, Regensburg, Germany; **Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, Cardiff, United Kingdom

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.
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http://dx.doi.org/10.1096/fj.15-280446DOI Listing
February 2016

Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication.

Haematologica 2015 Sep 25;100(9):1131-8. Epub 2015 Jun 25.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands

In patients with acute coronary syndrome, dual antiplatelet therapy with aspirin and a P2Y12 inhibitor like prasugrel is prescribed for one year. Here, we investigated how the hemostatic function of platelets recovers after discontinuation of prasugrel treatment. Therefore, 16 patients who suffered from ST-elevation myocardial infarction were investigated. Patients were treated with aspirin (100 mg/day, long-term) and stopped taking prasugrel (10 mg/day) after one year. Blood was collected at the last day of prasugrel intake and at 1, 2, 5, 12 and 30 days later. Platelet function in response to ADP was normalized between five and 30 days after treatment cessation and in vitro addition of the reversible P2Y12 receptor antagonist ticagrelor fully suppressed the regained activation response. Discontinuation of prasugrel resulted in the formation of an emerging subpopulation of ADP-responsive platelets, exhibiting high expression of active integrin αIIbβ3. Two different mRNA probes, thiazole orange and the novel 5'Cy5-oligo-dT probe revealed that this subpopulation consisted of juvenile platelets, which progressively contributed to platelet aggregation and thrombus formation under flow. During offset, juvenile platelets were overall more reactive than older platelets. Interestingly, the responsiveness of both juvenile and older platelets increased in time, pointing towards a residual inhibitory effect of prasugrel on the megakaryocyte level. In conclusion, the gradual increase in thrombogenicity after cessation of prasugrel treatment is due to the increased activity of juvenile platelets.
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http://dx.doi.org/10.3324/haematol.2014.122457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4800712PMC
September 2015

Normal platelet activation profile in patients with peripheral arterial disease on aspirin.

Thromb Res 2015 Mar 6;135(3):513-20. Epub 2015 Jan 6.

Departments of Biochemistry and Internal Medicine, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, The Netherlands. Electronic address:

Background: Peripheral arterial disease (PAD) is a progressive vascular disease associated with a high risk of cardiovascular morbidity and death. Antithrombotic prevention is usually applied by prescribing the antiplatelet agent aspirin. However, in patients with PAD aspirin fails to provide protection against myocardial infarction and death, only reducing the risk of ischemic stroke. Platelets may play a role in disease development, but this has not been tested by proper mechanistic studies. In the present study, we performed a systematic evaluation of platelet reactivity in whole blood from patients with PAD using two high-throughput assays, i.e. multi-agonist testing of platelet activation by flow cytometry and multi-parameter testing of thrombus formation on spotted microarrays.

Methods: Blood was obtained from 40 patients (38 on aspirin) with PAD in majority class IIa/IIb and from 40 age-matched control subjects. Whole-blood flow cytometry and multiparameter thrombus formation under high-shear flow conditions were determined using recently developed and validated assays.

Results: Flow cytometry of whole blood samples from aspirin-treated patients demonstrated unchanged high platelet responsiveness towards ADP, slightly elevated responsiveness after glycoprotein VI stimulation, and decreased responsiveness after PAR1 thrombin receptor stimulation, compared to the control subjects. Most parameters of thrombus formation under flow were similarly high for the patient and control groups. However, in vitro aspirin treatment caused a marked reduction in thrombus formation, especially on collagen surfaces. When compared per subject, markers of ADP- and collagen-induced integrin activation (flow cytometry) strongly correlated with parameters of collagen-dependent thrombus formation under flow, indicative of a common, subject-dependent regulation of both processes.

Conclusion: Despite of the use of aspirin, most platelet activation properties were in the normal range in whole-blood from class II PAD patients. These data underline the need for more effective antithrombotic pharmacoprotection in PAD.
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http://dx.doi.org/10.1016/j.thromres.2014.12.029DOI Listing
March 2015
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