Publications by authors named "Mark Roest"

107 Publications

Flow cytometric analysis of platelet function to detect high on-treatment residual platelet reactivity in patients on dual antiplatelet therapy.

Int J Lab Hematol 2021 Oct 21. Epub 2021 Oct 21.

Coagulation Laboratory, Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium.

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http://dx.doi.org/10.1111/ijlh.13744DOI Listing
October 2021

Targeted SERPIN (TaSER): a dual-action antithrombotic agent that targets platelets for SERPIN delivery.

J Thromb Haemost 2021 Oct 15. Epub 2021 Oct 15.

CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

Background: Occlusive thrombi are not homogeneous in composition. The core of a thrombus is rich in activated platelets and fibrin while the outer shell contains resting platelets. This core is inaccessible to plasma proteins. We produced a fusion protein (targeted SERPIN - TaSER), consisting of a function-blocking V H against glycoprotein Ibα and a thrombin-inhibiting serine protease inhibitor (SERPIN; α1-antitrypsin AIAR ) to interfere with platelet-driven thrombin formation.

Aim: To evaluate the antithrombotic properties of TaSER.

Methods: Besides TaSER, we generated three analogous control variants with either a wild-type antitrypsin subunit, a non-targeting control V H, or their combination. We investigated TaSER and controls in protease activity assays, (platelet-dependent) thrombin generation assays and by western blotting. The effects of TaSER on platelet activation and von Willebrand Factor (VWF)-binding were studied by FACS, in agglutination studies and in ATP secretion experiments. We studied the influence of TaSER in whole blood 1) on platelet adhesion on VWF, 2) aggregate formation on collagen and 3) thrombus formation (after recalcification) on collagen and tissue-factor.

Results: TaSER binds platelets and inhibits thrombin activity on the platelet surface. It blocks VWF binding and disassembles platelet agglutinates. TaSER delays tissue factor-triggered thrombin generation and ATP secretion in platelet-rich plasma in a targeted manner. In flow studies, TaSER interferes with platelet adhesion and aggregate formation due to GPIbα blockade and limits thrombus formation due to targeted inhibition of platelet-dependent thrombin activity.

Conclusion: The synergy between the individual properties of TaSER makes it a highly effective antithrombotic agent with possible clinical implications.
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http://dx.doi.org/10.1111/jth.15554DOI Listing
October 2021

Kallikrein augments the anticoagulant function of the protein C system in thrombin generation.

J Thromb Haemost 2021 Sep 17. Epub 2021 Sep 17.

Synapse Research Institute, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands.

Background: Genetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)-modulated TG using plasma from the Human Functional Genomics Project.

Methods: Calibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome-wide single-nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A-IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG.

Results: Thrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome-wide level (nETP-TMsr, P = 4.27 × 10 ). In vitro supplementation of kallikrein, but not prekallikrein or apolipoprotein A-IV, into plasma dose-dependently augmented the anticoagulant effect of TM and APC in TG. Variations of rs4241819 was not associated with the plasma concentration of prekallikrein. Association between rs4241819 and nETP-TMsr was absent when TG was measured in presence of a contact pathway inhibitor corn trypsin inhibitor.

Conclusions: Our results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis.
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http://dx.doi.org/10.1111/jth.15530DOI Listing
September 2021

Von Willebrand factor, ADAMTS13 and mortality in dialysis patients.

BMC Nephrol 2021 Jun 16;22(1):222. Epub 2021 Jun 16.

Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands.

Background: Von Willebrand Factor (VWF) multimers are cleaved into smaller and less coagulant forms by the metalloprotease ADAMTS13. The aim of this study was to investigate the association between VWF and ADAMTS13 and mortality in dialysis patients.

Methods: We prospectively followed 956 dialysis patients. VWF levels and ADAMTS13 activity were measured. Cox proportional hazard analyses were used to calculate hazard ratios (HRs) with 95 % confidence intervals (CIs) to investigate the association between quartiles of VWF levels and ADAMTS13 activity and all-cause mortality. HRs were adjusted for age, sex, body mass index, cardiovascular disease, dialysis modality, primary kidney disease, use of antithrombotic medication, systolic blood pressure, albumin, C-reactive protein and residual GFR.

Results: Of the 956 dialysis patients, 288 dialysis patients died within three years (mortality rate 151 per 1000 person-years). The highest quartile of VWF as compared with lower levels of VWF was associated with a 1.4-fold (95 %CI 1.1-1.8) increased mortality risk after adjustment. The lowest quartile of ADAMTS13 activity as compared with other quartiles was associated with a 1.3-fold (95 %CI 1.0-1.7) increased mortality risk after adjustment. The combination of the highest VWF quartile and lowest ADAMTS13 quartile was associated with a 2.0-fold (95 %CI 1.3-3.0) increased mortality risk as compared with the combination of the lowest VWF quartile and highest ADAMTS13 quartile.

Conclusions: High VWF levels and low ADAMTS13 activity were associated with increased mortality risks in dialysis patients.
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http://dx.doi.org/10.1186/s12882-021-02420-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8207579PMC
June 2021

Added Value of Blood Cells in Thrombin Generation Testing.

Thromb Haemost 2021 Mar 19. Epub 2021 Mar 19.

Synapse Research Institute, Maastricht, The Netherlands.

The capacity of blood to form thrombin is a critical determinant of coagulability. Plasma thrombin generation (TG), a test that probes the capacity of plasma to form thrombin, has improved our knowledge of the coagulation system and shows promising utility in coagulation management. Although plasma TG gives comprehensive insights into the function of pro- and anticoagulation drivers, it does not measure the role of blood cells in TG. In this literature review, we discuss currently available continuous TG tests that can reflect the involvement of blood cells in coagulation, in particular the fluorogenic assays that allow continuous measurement in platelet-rich plasma and whole blood. We also provide an overview about the influence of blood cells on blood coagulation, with emphasis on the direct influence of blood cells on TG. Platelets accelerate the initiation and velocity of TG by phosphatidylserine exposure, granule content release and surface receptor interaction with coagulation proteins. Erythrocytes are also major providers of phosphatidylserine, and erythrocyte membranes trigger contact activation. Furthermore, leukocytes and cancer cells may be important players in cell-mediated coagulation because, under certain conditions, they express tissue factor, release procoagulant components and can induce platelet activation. We argue that testing TG in the presence of blood cells may be useful to distinguish blood cell-related coagulation disorders. However, it should also be noted that these blood cell-dependent TG assays are not clinically validated. Further standardization and validation studies are needed to explore their clinical usefulness.
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http://dx.doi.org/10.1055/a-1450-8300DOI 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
August 2021

Plasmatic Coagulation Capacity Correlates With Inflammation and Abacavir Use During Chronic HIV Infection.

J Acquir Immune Defic Syndr 2021 05;87(1):711-719

Department of Internal Medicine, Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, the Netherlands.

Background: D-dimer concentrations in people living with HIV (PLHIV) on combination antiretroviral therapy (cART) are increased and have been linked to mortality. D-dimer is a biomarker of in vivo coagulation. In contrast to reports on D-dimer, data on coagulation capacity in PLHIV are conflicting. In this study, we assessed the effect of cART and inflammation on coagulation capacity.

Setting: We explored coagulation capacity using calibrated thrombin generation (TG) and linked this to persistent inflammation and cART in a cross-sectional study including PLHIV with viral suppression and uninfected controls.

Methods: We used multivariate analyses to identify independent factors influencing in vivo coagulation (D-dimer) and ex vivo coagulation capacity (TG).

Results: Among 208 PLHIV, 94 (45%) were on an abacavir-containing regimen. D-dimer levels (219.1 vs 170.5 ng/mL, P = 0.001) and inflammatory makers (sCD14, sCD163, and high-sensitive C-reactive protein) were increased in PLHIV compared with those in controls (n = 56). PLHIV experienced lower TG (reflected by endogenous thrombin potential [ETP]) when compared with controls, after correction for age, sex, and antiretroviral therapy. Abacavir use was independently associated with increased ETP. Prothrombin concentrations were strongly associated with ETP and lower in PLHIV on a non-abacavir-containing regimen compared with those in controls, suggesting consumption as a possible mechanism for HIV-associated reduction in TG. D-dimer concentrations were associated with inflammation, but not TG.

Conclusions: Abacavir use was associated with increased TG and could serve as an additional factor in the reported increase in thrombotic events during abacavir use. Increased exposure to triggers that propagate coagulation, such as inflammation, likely underlie increased D-dimer concentrations found in most PLHIV.
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http://dx.doi.org/10.1097/QAI.0000000000002633DOI Listing
May 2021

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

Arterioscler Thromb Vasc Biol 2021 02 3;41(2):e97-e111. 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
February 2021

Efficacy of pro- and anticoagulant strategies in plasma of patients undergoing hepatobiliary surgery.

J Thromb Haemost 2020 11 10;18(11):2840-2851. Epub 2020 Sep 10.

Surgical Research Laboratory and Section of Hepatobiliary Surgery and Liver transplantation, Department of Surgery, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Background: In vitro efficacy of pro- and antihemostatic drugs is profoundly different in patients with compensated cirrhosis and in those who have cirrhosis and are critically ill.

Objectives: Here we assessed the efficacy of pro- and anticoagulant drugs in plasma of patients undergoing hepato-pancreato-biliary (HPB) surgery, which is associated with unique hemostatic changes.

Methods: We performed in vitro analyses on blood samples of 60 patients undergoing HPB surgery and liver transplantation: 20 orthotopic liver transplantations, 20 partial hepatectomies, and 20 pylorus-preserving pancreaticoduodenectomies. We performed thrombin generation experiments before and after in vitro addition of fresh frozen plasma (FFP), prothrombin complex concentrate (PCC), recombinant factor VIIa (rFVIIa), low molecular weight heparin (LMWH), unfractionated heparin, dabigatran, and rivaroxaban.

Results: We showed that patients undergoing HPB surgery are in a hypercoagulable state by thrombin generation testing. FFP and rFVIIa had minimal effects on thrombin generation, whereas PCC had a more pronounced procoagulant effect in patients compared with controls. Dabigatran showed a more pronounced anticoagulant effect in patients compared with controls, whereas rivaroxaban and LMWH had a decreased anticoagulant effect in patients.

Conclusion: We demonstrate profoundly altered in vitro efficacy of commonly used anticoagulants, in patients undergoing HPB surgery compared with healthy controls, which may have implications for anticoagulant dosing in the early postoperative period. In the correction of perioperative bleeding complications, PCCs appear much more potent than FFP or rFVIIa, and PCCs may require conservative dosing and caution in use in patients undergoing HPB surgery.
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http://dx.doi.org/10.1111/jth.15060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693071PMC
November 2020

Current methods of measuring platelet activity: pros and cons.

Blood Coagul Fibrinolysis 2020 Oct;31(7):426-433

Synapse Research Institute, Maastricht, the Netherlands.

: Platelets play a pivotal role in controlling hemorrhaging from vessels of the human body. The impairment of platelets may lead to the development of bleeding manifestations. Unraveling the precise defects of platelets by means of suitable laboratory methods paves the way for the effective control and management of platelet disorders. Choosing the most appropriate approach for the detection of platelet disorders may be difficult for a researcher or clinical internist when faced with ordering a platelet-function test. The aim of the current study was to provide a user-friendly overview of the advantages and disadvantages of the available detection systems. To reach this goal, 11 commonly used methods of studying platelet activity were evaluated and compared in detail. A literature search, with no time or language limitations, was conducted in Google Scholar and Medline. All publications published before June 2019 were analyzed. The following laboratory methods were compared: number and size of platelets, bleeding time, clot retraction time, platelet function assay 100 & 200, Rapid platelet function assay, flow cytometry, light transmission aggregometry, multiple electrode aggregometry, 96-well plate aggregometry, cone and plate(let) analyzer (Impact-R), and Plateletworks (single platelet counting system). This article provides the reader with a rapid comparison of the different systems used to study platelets activities.
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http://dx.doi.org/10.1097/MBC.0000000000000941DOI Listing
October 2020

Flow cytometric analysis of platelet function to improve the recognition of thrombocytopathy.

Thromb Res 2020 10 24;194:183-189. Epub 2020 Jun 24.

Coagulation Laboratory, Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium; Department of Diagnostic Sciences, Ghent University, Ghent, Belgium.

Introduction: Light transmission aggregometry (LTA) is the gold standard for diagnosing bleeding disorders. Although LTA is laborious, requires large volumes of blood and is relatively insensitive to small changes in platelet function, there is still no competing alternative approach to replace LTA for the diagnosis of platelet bleeding disorders.

Materials And Methods: This study investigates the correlation between flow cytometry-based whole blood platelet activation test (WB-PACT) and LTA and whether WB-PACT is of additional value for the identification of bleeding disorders. In total, 161 patients with suspected bleeding diathesis were tested.

Results: A correlation of 0.41 between LTA and WB-PACT was found, and there was agreement between tests in 62% of cases (κ = 0.23). The WB-PACT is of additional value to LTA to detect platelet function disorders (PFD) as 10 patients with elevated bleeding score (BS) were detected with WB-PACT, 4 with LTA and 7 patients were positive with both tests. Interestingly, in contrast to LTA, WB-PACT has an additional option to detect VWF disfunctions.

Conclusion: WB-PACT may have added value for the routine diagnostic work-up in patients who need to have platelet function tested.
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http://dx.doi.org/10.1016/j.thromres.2020.06.037DOI Listing
October 2020

Acute exacerbations of COPD are associated with a prothrombotic state through platelet-monocyte complexes, endothelial activation and increased thrombin generation.

Respir Med 2020 09 25;171:106094. Epub 2020 Jul 25.

Department of Respiratory Medicine, Gelre Ziekenhuizen, Apeldoorn, the Netherlands; Department of Respiratory Medicine, Maastricht University Medical Centre, Maastricht, the Netherlands; NUTRIM School of Nutrition and Translational Research in Metabolism, University of Maastricht, Maastricht, the Netherlands.

Introduction: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk for cardiovascular events, particularly following an acute exacerbation (AE-COPD). Exacerbations are associated with increased systemic inflammation, which may drive coagulation. This prospective cohort study aimed to determine how an AE-COPD affects platelet activation, the endothelium, plasmatic coagulation and fibrinolysis, and its association with systemic inflammation.

Materials And Methods: Fifty-two patients with an AE-COPD were included. Blood samples at admission, at day 3 of treatment and at convalescence were available for 32 patients. Platelet-monocyte complex (PMC) formation, monocyte Mac-1 expression and platelet (re)activity (P-selectin expression, αIIbβ3 activation) were measured by flow cytometry. Von Willebrand Factor (VWF), thrombin generation (TG) and clot lysis time (CLT) were determined as measures of endothelial activation, plasmatic coagulation and fibrinolysis, respectively.

Results: Exacerbations were associated with increased PMCs (MFI 31.3 vs 23.8, p = 0.004) and Mac-1 (MFI 38.2 vs 34.8, p = 0.006) compared to convalescence, but not with changes in platelet (re)activity. VWF (antigen, activity, active fraction) and TG (peak, ETP and velocity index) were all significantly higher during AE-COPD compared to convalescence. PMCs, Mac-1, VWF and TG were positively associated with systemic inflammation (CRP). CLT was prolonged in AE-COPD patients with systemic inflammation. Moreover, platelet hyperreactivity on admission was associated with an increased risk for exacerbation relapse.

Conclusions: Acute exacerbations are associated with an inflammation-associated prothrombotic state, characterized by increased PMCs, endothelial activation and plasmatic coagulation. Our findings provide direction for future studies on biomarkers predicting the risk of exacerbation relapse and cardiovascular events.
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http://dx.doi.org/10.1016/j.rmed.2020.106094DOI Listing
September 2020

Interplay between platelets and coagulation.

Blood Rev 2021 03 12;46:100733. Epub 2020 Jul 12.

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

Haemostasis stops bleeding at the site of vascular injury and maintains the integrity of blood vessels through clot formation. This regulated physiological process consists of complex interactions between endothelial cells, platelets, von Willebrand factor and coagulation factors. Haemostasis is initiated by a damaged vessel wall, followed with a rapid adhesion, activation and aggregation of platelets to the exposed subendothelial extracellular matrix. At the same time, coagulation factors aggregate on the procoagulant surface of activated platelets to consolidate the platelet plug by forming a mesh of cross-linked fibrin. Platelets and coagulation mutually influence each other and there are strong indications that, thanks to the interplay between platelets and coagulation, haemostasis is far more effective than the two processes separately. Clinically this is relevant because impaired interaction between platelets and coagulation may result in bleeding complications, while excessive platelet-coagulation interaction induces a high thrombotic risk. In this review, platelets, coagulation factors and the complex interaction between them will be discussed in detail.
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http://dx.doi.org/10.1016/j.blre.2020.100733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354275PMC
March 2021

Beta-2-glycoprotein I as a biomarker for sepsis in critically ill patients in the intensive care unit: a prospective cohort study.

Crit Care 2020 06 15;24(1):341. Epub 2020 Jun 15.

Department of Intensive Care Medicine, University Medical Centre, University of Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

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http://dx.doi.org/10.1186/s13054-020-03066-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296730PMC
June 2020

A novel assay for studying the involvement of blood cells in whole blood thrombin generation.

J Thromb Haemost 2020 06 30;18(6):1291-1301. Epub 2020 Mar 30.

Synapse Research Institute, Maastricht, The Netherlands.

Background: Fluorogenic thrombin generation (TG) assays are commonly used to determine global coagulation phenotype in plasma. Whole blood (WB)-TG assays reach one step closer to physiology by involving the intrinsic blood cells, but erythrocytes cause variable quenching of the fluorescence signals, hampering its routine application.

Objective: To develop a new assay for continuous WB-TG measurement.

Methods: In the new WB-TG assay, the erythrocyte-caused distortion of signal was solved by continuously mixing the sample during the measurement. The assay was validated by evaluating the reproducibility and comparing with the paper-based WB-TG assay. Reconstituted human blood and WB from 119 healthy donors was tested to explore the influences of hematocrit and platelet count on TG.

Results: This novel WB-TG assay showed good reproducibility while being less affected by contact activation compared with the previous paper-based assay. Reconstitution experiments showed that the lag time of TG was shortened by the addition of platelets but not erythrocytes. Increasing hematocrit strongly augmented the peak thrombin, even in the presence of high platelet counts. The lag time and peak of WB-TG of 119 healthy donors were positively related to erythrocyte count after adjusting for age, sex, and oral contraceptive use with multiple linear regression analyses. The reference range and interindividual variation of WB-TG were determined in the healthy cohort.

Conclusions: A novel WB-TG assay was developed, which is a straightforward tool to measure the involvement of platelets and erythrocytes in TG and may assist the research of blood cell-associated coagulation disorders.
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http://dx.doi.org/10.1111/jth.14786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317846PMC
June 2020

Whole blood thrombin generation profiles of patients with cirrhosis explored with a near patient assay.

J Thromb Haemost 2020 04 18;18(4):834-843. Epub 2020 Feb 18.

Institute of Liver Studies, King's College Hospital, London, UK.

Background And Aims: Patients with cirrhosis have a rebalanced hemostasis, often with normal or elevated thrombin-generating (TG) capacity in plasma. Whole blood (WB) TG allows faster determination and, importantly, includes the influence of all circulating blood cells. We aimed to study the TG profile of patients with cirrhosis in WB and in platelet poor plasma.

Methods: Thrombin-generating capacity in WB and plasma were assessed with a near-patient WB-TG assay and the calibrated automated thrombinography assay, respectively. TG assays were tested in presence and absence of thrombomodulin. Conventional coagulation tests were also performed.

Results: Thirty-four patients with cirrhosis and twenty-two controls were analyzed. Compared with controls, patients had substantially deranged results in conventional coagulation tests. Comparable WB-TG capacity (endogenous thrombin potential until peak, ETPp) but significantly lower peak thrombin were found in patients, and these results persisted when thrombomodulin was present. TG of the patients was more resistant to thrombomodulin than controls in both WB and plasma, although the inhibitory effect of thrombomodulin was drastically weaker in WB than in plasma. The peak of WB-TG in patients correlated moderately with their hematocrit and platelet count. Significant correlations were found between TG results in WB and plasma.

Conclusions: The WB-TG assay shows a normal to hypocoagulable state in patients with cirrhosis with a decreased anticoagulant activity of TM compared to plasma-TG. The clinical value of this assay needs further validation.
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http://dx.doi.org/10.1111/jth.14751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186949PMC
April 2020

Osteoprotegerin is Higher in Sepsis Than in Noninfectious SIRS and Predicts 30-Day Mortality of SIRS Patients in the Intensive Care.

J Appl Lab Med 2019 01 16;3(4):559-568. Epub 2018 Jul 16.

Intensive Care Center, University Medical Center Utrecht, University Utrecht, Utrecht, the Netherlands.

Background: Systemic inflammatory response syndrome (SIRS) is a complex disease involving multiple pathways and organs. Biomarkers reflecting these pathways and organ function could correlate with the severity of the disease. Osteoprotegerin (OPG), mainly known for its role in bone metabolism, is also involved in the immune and vascular system and is therefore an interesting biomarker to study in SIRS patients. In this prospective observational study, we investigated the correlation of plasma OPG concentrations, sepsis, and 30-day mortality of SIRS patients in the intensive care unit (ICU).

Methods: This observational, single-center, cohort study included 313 consecutive patients admitted to the ICU, with an anticipated stay of more than 48 h and SIRS on admission. Data from included patients were collected daily until discharge or death for a maximum of 10 days. Thirty-day mortality was retrospectively assessed. OPG concentrations were measured in the first 48 h after admission. The relation of OPG with no sepsis, sepsis, and septic shock was assessed with the Kruskal-Wallis test and the Mann-Whitney -test. Cox proportional hazards regression was used to study OPG concentrations and 30-day mortality.

Results: OPG concentrations were higher in patients with sepsis and septic shock than in patients without sepsis. Furthermore, patients with OPG concentrations in the highest tertile at admission in the ICU have an increased risk of mortality within 30 days when compared to patients with OPG concentrations in the lowest and middle tertiles, independent of acute physiologic and chronic health evaluation (APACHE) and sequential organ failure assessment (SOFA) scores.

Conclusions: We show that OPG is a biomarker that correlates with sepsis and predicts mortality of SIRS patients in the ICU.
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http://dx.doi.org/10.1373/jalm.2018.026559DOI Listing
January 2019

A Synthetic Triple Helical Collagen Peptide as a New Agonist for Flow Cytometric Measurement of GPVI-Specific Platelet Activation.

Thromb Haemost 2019 Dec 21;119(12):2005-2013. Epub 2019 Oct 21.

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

Synthetic cross-linked collagen-related peptide (CRP-XL) is a glycoprotein VI (GPVI) receptor activator for platelet activation. This triple helical peptide, widely used in platelet function tests, is synthesized and cross-linked through cysteine residues at its N-terminus and C-terminus. Currently, there is only one laboratory, which is capable to produce this valuable peptide for clinical applications. In an attempt to provide a standardized alternative for CRP-XL, we developed a synthetic triple helical collagen peptide (STH-CP) with the same primary sequence as CRP-XL (GPC-(GPO)-GPCG-amide), which was both on the C-terminus and on the N-terminus fixed on a scaffold with a binding side for each of the three peptides. The performance of STH-CP on platelet function was studied using flow cytometry and compared with CRP-XL. We found that platelet activation pattern in response to STH-CP and CRP-XL is similar, although the STH-CP requires sixfold higher concentrations to activate platelets to the same state. The intra-assay percent coefficient of variation of STH-CP and CRP-XL were both < 5% and the interindividual variation measured in 118 individuals for both peptides was around 23 and 21% for αIIbβ3 activation and P-selectin expression, respectively. The STH-CP in ready-to-use reaction mix has lower variation than CRP-XL over 1-year storage. In reference values and seasonal variation study, the platelet activation response showed a strong correlation between STH-CP and CRP-XL.Our findings show that this new STH-CP is a stable and potent platelet GPVI agonist which can induce the same reproducible platelet activation as CRP-XL and that STH-CP can be considered as a good alternative for CRP-XL.
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http://dx.doi.org/10.1055/s-0039-1697660DOI Listing
December 2019

Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor.

PLoS One 2019 13;14(2):e0211961. Epub 2019 Feb 13.

Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands.

Background: Interaction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ''active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.

Methods: We developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.

Results: Intra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding.

Conclusions: We developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211961PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373957PMC
November 2019

The anticoagulant effect of dabigatran is reflected in the lag time and time-to-peak, but not in the endogenous thrombin potential or peak, of thrombin generation.

Thromb Res 2018 11 6;171:160-166. Epub 2018 Oct 6.

Université catholique de Louvain, CHU UCL Namur, Namur Thrombosis and Hemostasis Center, Hematology Laboratory, Namur, Belgium.

Introduction: Calibrated automated thrombinography (CAT) is a sensitive method to assess coagulation. Dabigatran inhibits both free thrombin and the αmacroglobulin (αM)-thrombin complex, which results in an erroneously increased peak and endogenous thrombin potential (ETP) without affecting lag time and time-to-peak. The aim of this study was to elucidate the artefacts in CAT when dabigatran is present.

Materials And Methods: Thrombin generation (TG) was measured in vitro by using CAT in the presence or absence of 6 μM idarucizumab in plasma spiked with dabigatran. Additionally, ex vivo measurements were performed in plasmas of 63 patients using dabigatran in the presence and absence of idarucizumab.

Results: The in vitro experiments confirmed that the ETP, peak and velocity index were artificially increased. This was mainly due to the inhibition of the calibrator by dabigatran and partly due to CAT algorithms. The calibration artefact could be resolved by adding idarucizumab to the calibrator well. However, the second, mathematical artefact remains when dabigatran is present in the TG well. These findings were corroborated by ex vivo experiments i.e. the lag time and time-to-peak were significantly reduced in patients upon addition of idarucizumab, but the ETP and peak were not significantly affected. The velocity index did change significantly, since this is a combination of time-dependent factors and the peak.

Conclusions: The peak, ETP and velocity index do not represent the anticoagulant effect of dabigatran on TG measured with CAT. The lag time and time-to-peak, however, do reflect the effect of dabigatran.
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http://dx.doi.org/10.1016/j.thromres.2018.10.005DOI Listing
November 2018

Effects of Repeated Bouts of Exercise on the Hemostatic System.

Semin Thromb Hemost 2018 Nov 5;44(8):710-722. Epub 2018 Oct 5.

Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht University, Maastricht, The Netherlands.

Physical activity is beneficial for health, for example, by lowering the risk of cardiovascular events. However, vigorous exercise is associated with the occurrence of thromboembolic events and sudden cardiac death, in particular in untrained individuals. Whereas acute exercise is known to cause a hypercoagulable state, repeated exposure to (strenuous) exercise by means of training may actually condition the hemostatic response to exercise. To date, the effects of exercise training on blood coagulability and the underlying mechanisms have yet to be fully discerned. In this review, the authors provide an overview of existing literature on how training programs and training status influence hemostasis in healthy individuals. Furthermore, they present data of a pilot study in which we studied the effects of repetitive submaximal intensity cycling on procoagulant and anticoagulant processes. It is known that factor VIII (FVIII) and von Willebrand factor (VWF) increase after exercise, but we found that this increase in FVIII and VWF (antigen, propeptide, and VWF in active conformation) was smaller on each of three subsequent days, suggesting either adaptation of endothelial activation or exhaustion of endothelial VWF supplies. With respect to thrombin generation, elevated FVIII significantly increased the thrombin generation peak but not the endogenous thrombin potential. In contrast, platelet activation in terms of P-selectin expression after stimulation with protease-activated receptor-1 and glycoprotein VI agonists decreased after exercise and did not recover, indicating exhaustion of the platelet response to repetitive exercise.
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http://dx.doi.org/10.1055/s-0038-1673619DOI Listing
November 2018

Thrombin generation and platelet activation in cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy - A prospective cohort study.

PLoS One 2018 21;13(6):e0193657. Epub 2018 Jun 21.

Department of Anesthesiology & Pain Treatment, Maastricht University Medical Centre, Maastricht, The Netherlands.

Background And Objectives: Cytoreductive surgery (CRS) with hyperthermic intraperitoneal peroperative chemotherapy (HIPEC), indicated for patients with peritoneal metastases from digestive or gynecological malignancies alike, demonstrates a considerable impact on hemostatic metabolism, both on platelet and on coagulation level. The potential hemostatic interference in CRS and HIPEC is phase dependent. The hypothesis of this prospective cohort study is that the procedure exposed an increased thrombotic risk, resulting in a faster and increased thrombin generation and hyper platelet function.

Methods: This study explores the combined use of ROTEM (rotational thromboelastometry), PACT (platelet activation test) and CAT (thrombin generation test) assays during CRS and HIPEC with a follow-up of 7 days postoperative in 27 patients with confirmed histological diagnosis of peritoneal disease.

Results: Platelet reactivity (relative to before incision values) to CRP (collagen-related peptide) (p value 0.02) and TRAP (thrombin receptor activator peptide) (p value 0.048) seems to be slightly reduced during CRS and HIPEC with regard to αIIbβ3 activation, while P-selectin expression is not affected. During surgery, CAT demonstrates that, the LT (lagtime) (p value 0.0003) and TTP (time-to-thrombin peak) values (p value 0.002) decrease while and the TP (thrombin peak) (p value 0.004) and ETP (endogenous thrombin potential) (p value 0.02) increase. Subsequently, after surgery, the LT and TTP increase and ETP and TP decrease in time. ROTEM EXTEM (extrinsic) MCF (maximum clot firmness) (p value 0.005), INTEM (intrinsic) MCF (p value 0.003) and FIBTEM (fibrinogen) MCF (p value <0.001) decreased during CRS. At day 7 INTEM and FIBTEM MCF values (p values of 0.004 and <0.001) were significantly higher than before surgery. No considerable changes in platelet count and hemoglobin concentration and absence of leukopenia are noticed.

Conclusion: This approach detects changes in coagulation much earlier than noticed by standard coagulation tests.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193657PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013150PMC
November 2018

No indications for platelet activation in acute clinical myocardial or renal ischemia/reperfusion injury.

Am J Transl Res 2018 15;10(3):816-826. Epub 2018 Mar 15.

Department of Surgery, Leiden University Medical CenterLeiden, The Netherlands.

The pathophysiology of ischemia/reperfusion (I/R) injury is complex and poorly understood. Animal studies imply platelet activation as an initiator of the inflammatory response upon reperfusion. However, it remains unclear whether and how these results translate to clinical I/R. This study evaluates putative platelet activation in the context of two forms of clinical I/R (heart valve surgery with aortic-cross clamping, n = 39 and kidney transplantation, n = 34). The technique of sequential selective arteriovenous (AV) measurements over the reperfused organs was applied to exclude the influence of systemic changes occurring during surgery while simultaneously maximizing sensitivity. Platelet activation and degranulation was evaluated by assessing the expression levels of established markers, i.e. RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), β-thromboglobulin (β-TG), platelet-derived growth factor (PDGF)-BB and CXCL8 (known as interleukin-8), and by employing an in-vitro assay that specifically tests for platelet excitability. Moreover, a histological analysis was performed by means of CD41 staining. Results show stable RANTES, β-TG, PDGF-BB and CXCL8 AV-concentrations within the first half hour over the reperfused organs, suggesting that myocardial and renal I/R are not associated with platelet activation. Results from the platelet excitability assay were in line with these findings and indicated reduced and stable platelet excitability following renal and myocardial reperfusion, respectively. Histological analysis yield evidence of platelet marginalization in the reperfused organs. In conclusion, results from this study do not support a role for platelet activation in early phases of clinical I/R injury.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5883122PMC
March 2018

Platelet function is disturbed by the angiogenesis inhibitors sunitinib and sorafenib, but unaffected by bevacizumab.

Angiogenesis 2018 05 12;21(2):325-334. Epub 2018 Mar 12.

Department of Medical Oncology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan, 1117, 1081 HV, Amsterdam, The Netherlands.

Introduction: At the clinical introduction of antiangiogenic agents as anticancer agents, no major toxicities were expected as merely just endothelial cells (ECs) in tumors would be affected. However, several (serious) toxicities became apparent, of which underlying mechanisms are largely unknown. We investigated to what extent sunitinib (multitargeted antiangiogenic tyrosine kinase inhibitor (TKI)), sorafenib (TKI) and bevacizumab [specific antibody against vascular endothelial growth factor (VEGF)] may impair platelet function, which might explain treatment-related bleedings.

Materials And Methods: In vitro, the influence of sunitinib, sorafenib, and bevacizumab on platelet aggregation, P-selectin expression and fibrinogen binding, platelet-EC interaction, and tyrosine phosphorylation of c-Src was studied by optical aggregation, flow cytometry, real-time perfusion, and western blotting. Ex vivo, platelet aggregation was analyzed in 25 patients upon sunitinib or bevacizumab treatment. Concentrations of sunitinib, VEGF, and platelet and EC activation markers were measured by LC-MS/MS and ELISA.

Results: In vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20 μM sunitinib: 71.3%, p < 0.001; 25 μM sorafenib: 55.8%, p = 0.042). Sorafenib and sunitinib significantly inhibited P-selectin expression on platelets. Exposure to both TKIs resulted in a reduced tyrosine phosphorylation of c-Src. Ex vivo, within 24 h sunitinib impaired platelet aggregation (83.0%, p = 0.001, N = 8). Plasma concentrations of sunitinib, VEGF, and platelet/EC activation markers were not correlated with disturbed aggregation. In contrast, bevacizumab only significantly impaired platelet aggregation in vitro at high concentrations, but not ex vivo.

Conclusion: Sunitinib significantly inhibits platelet aggregation in patients already after 24 h of first administration, whereas bevacizumab had no effect on aggregation. These findings may explain the clinically observed bleedings during treatment with antiangiogenic TKIs.
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http://dx.doi.org/10.1007/s10456-018-9598-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5878190PMC
May 2018

Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test.

PLoS One 2018 1;13(2):e0192079. Epub 2018 Feb 1.

Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands.

Introduction: Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals.

Methods: Platelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbβ3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined.

Results: Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbβ3 activation, respectively.

Conclusions: The current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192079PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794146PMC
March 2018

Myeloperoxidase can differentiate between sepsis and non-infectious SIRS and predicts mortality in intensive care patients with SIRS.

Intensive Care Med Exp 2017 Sep 15;5(1):43. Epub 2017 Sep 15.

Department of Intensive Care Medicine, University Medical Centre, University of Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

Background: Systemic inflammatory response syndrome (SIRS) is a clinical syndrome following inflammation. Clinically, it is difficult to distinguish SIRS following an infection, i.e., sepsis, from non-infectious SIRS. Myeloperoxidase is a hemeprotein stored in the neutrophil azurophilic granules and is one of the main pillars of neutrophil attack. Therefore, we hypothesized that myeloperoxidase can differentiate between sepsis and non-infectious SIRS in patients with systemic inflammatory response syndrome in the intensive care unit (ICU).

Methods: An observational single-center cohort study was conducted measuring myeloperoxidase in patients with SIRS in the first 48 h after admission. The outcomes were established using predefined definitions. Thirty-day mortality was retrospectively assessed.

Results: We found significantly higher levels of myeloperoxidase in patients with sepsis and septic shock compared to patients without sepsis (60 ng/ml versus 43 ng/ml, P = 0.002). Myeloperoxidase levels were related to 30-day mortality (P = 0.032), and high MPO levels on top of a high APACHE IV score further increased mortality risk.

Conclusions: We show that myeloperoxidase is a potentially novel biomarker for sepsis in the ICU. Myeloperoxidase could eventually help in diagnosing sepsis and predicting mortality. However, more research is necessary to confirm our results.
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http://dx.doi.org/10.1186/s40635-017-0157-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602808PMC
September 2017

Interdependence between osteoprotegerin and active von Willebrand factor in long-term cardiovascular mortality prediction in patients undergoing percutaneous coronary intervention.

Thromb Haemost 2017 08 20;117(9):1730-1738. Epub 2017 Jul 20.

Jolanta Siller-Matula, MD, PhD, Department of Cardiology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria, E-mail: , or, Bernd Jilma, MD, Department of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria, E-mail:

The interdependence of the predictive accuracy of serum osteoprotegerin (OPG) and von Willebrand factor (vWF) levels for long-term cardiovascular outcomes has not been investigated so far. This was a prospective observational cohort study in 361 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI). Baseline levels of OPG, vWF, active vWF (act vWF) and ristocetin cofactor activity (vWF:RICO) were measured. Cardiovascular mortality was recorded over a median of five years. OPG concentrations >3.7 µg/ml emerged as the strongest predictor of cardiovascular (CV) death: 30 % of patients died during the five-year follow-up in this group, as compared to 10 % in patients with OPG ≤3.7 µg/ml (p<0.001). Act vWF had a significant prognostic impact on CV mortality when OPG levels were low (≤3.7 µg/ml): patients with act vWF concentration >1 µg/ml died in 14 %, whereas those with act vWF values ≤1 µg/ml had a mortality rate of 1 % (p=0.015). We stratified patients into three groups: high OPG, low OPG/high act vWF and low OPG/low act vWF. Patients with high OPG values had a 13-fold higher risk for CV death than those with low OPG/low act vWF concentrations (adj. HR: 12.6; 95 %CI: 1.7-94.7; p=0.014), and a two-fold higher risk as compared to those patients with low OPG/high act vWF concentrations (adj. HR: 2.0; 95 %CI: 1.1-3.7; p=0.03) in the adjusted Cox regression analysis. In conclusion, elevated OPG at the time of PCI was a strong independent predictor of five-years cardiovascular mortality, whereas act vWF had a significant prognostic impact on CV mortality when OPG levels were low.
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http://dx.doi.org/10.1160/TH17-02-0087DOI Listing
August 2017

Soluble P-selectin as a Biomarker for Infection and Survival in Patients With a Systemic Inflammatory Response Syndrome on the Intensive Care Unit.

Biomark Insights 2017 7;12:1177271916684823. Epub 2017 Feb 7.

Department of Intensive Care Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.

Aims: This study investigated the ability of soluble platelet selectin (sP-selectin) to identify infection and predict 30-day mortality in patients with a systemic inflammatory response syndrome (SIRS) on the intensive care unit.

Methods: Soluble platelet selectin levels were measured daily in the first 48 hours in patients presenting with SIRS. The outcome, proven infection, was established using predefined definitions. The 30-day mortality was retrospectively assessed.

Results: In a total of 313 patients with SIRS, sP-selectin levels were measured. Of these, 114 patients had proven infection on admission or developing during their intensive care unit (ICU) stay. Patients with proven infection had moderately higher levels of sP-selectin (147 ng/mL; interquartile range [IQR], 93.4-203 ng/mL) compared with noninfected patients (143.8 ng/mL; IQR, 89.6-194.7 ng/mL). This difference was not statistically significant ( = .072). However, in patients who were not admitted for infection (n = 235), sP-selectin levels were significantly related to the subsequent development of infection ( = .013). Soluble platelet selectin levels were particularly high in patients with abdominal sepsis and skin infections. Higher sP-selectin levels were associated with higher mortality (although not statistically significant, = .08).

Conclusions: This study shows that in patients with SIRS not admitted for infection, sP-selectin levels are significantly related to the subsequent development of infection. Furthermore, patients with higher sP-selectin levels in the first 2 days of admission had higher 30-day mortality, although this association is not statistically significant. Therefore, we conclude that sP-selectin is a potential future biomarker for both mortality and infection in patients with SIRS, but more research is needed to confirm its prognostic role.
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http://dx.doi.org/10.1177/1177271916684823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345948PMC
February 2017

Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT): Monitoring platelet activation in platelet concentrates.

PLoS One 2017 16;12(2):e0172265. Epub 2017 Feb 16.

Synapse Research Institute, Maastricht, the Netherlands.

Background: Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage.

Study Design And Methods: The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference.

Results: Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis.

Conclusion: PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172265PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313179PMC
August 2017

Baseline thrombospondin-1 concentrations are not associated with mortality in septic patients: a single-center cohort study on the intensive care unit.

Intensive Care Med Exp 2017 Dec 25;5(1). Epub 2017 Jan 25.

Intensive Care Department, University Medical Center Utrecht, University Utrecht, Utrecht, the Netherlands.

Background: The initial phase of sepsis is characterized by hyperinflammation. Levels of thrombospondin-1 (TSP-1) rise rapidly during acute inflammation. The purpose of this clinical study was to study the association between plasma TSP-1 levels and mortality in patients with sepsis on the intensive care unit.

Methods: Critically ill adult patients with sepsis, severe sepsis, or septic shock were included. They were further divided into tertiles based on their baseline plasma TSP-1 concentrations. Primary outcome measure was 28-day mortality. Furthermore, associations with severity of sepsis and platelet counts were studied.

Results: Two hundred thirty-five patients were included. Median plasma TSP-1 concentrations of the tertiles were 194, 463 and 874 ng/mL, respectively. There were no baseline differences. Mortality rates (26.6, 16.7, and 16.7%, p = 0.20) and cumulative survival curves (p = 0.22) were not statistically different between the tertiles. There was no association of baseline TSP-1 with severity of sepsis (p = 0.08). TSP-1 and platelet counts were positively correlated (159, 198, and 295 × 10/L, p = 0.04).

Conclusions: Baseline plasma levels of TSP-1 were not associated with mortality and severity of sepsis in mixed population of septic ICU patients. Further research is needed to clarify the expression of TSP-1 and to unravel the potential prognostic value of this biomarker in human sepsis.
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http://dx.doi.org/10.1186/s40635-017-0120-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267614PMC
December 2017
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