Publications by authors named "Rolf T Urbanus"

74 Publications

Long-term treated HIV infection is associated with platelet mitochondrial dysfunction.

Sci Rep 2021 Mar 18;11(1):6246. Epub 2021 Mar 18.

Department of Internal Medicine, Radboud Center for Infectious Diseases, Radboud University Medical Center, 6500 HB, Nijmegen, The Netherlands.

HIV infection and antiretroviral therapy have been linked to mitochondrial dysfunction. The role of platelet mitochondrial dysfunction in thrombosis, immunoregulation and age-related diseases is increasingly appreciated. Here, we studied platelet mitochondrial DNA content (mtDNA) and mitochondrial function in people living with HIV (PLHIV) and related this to platelet function. In a cohort of 208 treated PLHIV and 56 uninfected controls, mtDNA was quantified, as well as platelet activation, platelet agonist-induced reactivity and inflammation by circulating factors and flow cytometry. In a subgroup of participants, the metabolic activity of platelets was further studied by mitochondrial function tests and the Seahorse Flux Analyzer. PLHIV had significantly lower mtDNA compared to controls (8.5 copies/platelet (IQR: 7.0-10.7) vs. 12.2 copies/platelet (IQR: 9.5-16.6); p < 0.001), also after correction for age, sex and BMI. Prior zidovudine-use (n = 46) was associated with a trend for lower mtDNA. PLHIV also had reduced ex vivo platelet reactivity and mean platelet volume compared to controls. MtDNA correlated positively with both platelet parameters and correlated negatively with inflammatory marker sCD163. Mitochondrial function tests in a subgroup of participants confirmed the presence of platelet mitochondrial respiration defects. Platelet mitochondrial function is disturbed in PLHIV, which may contribute to platelet dysfunction and subsequent complications. Interventions targeting the preservation of normal platelet mitochondrial function may ultimately prove beneficial for PLHIV.
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http://dx.doi.org/10.1038/s41598-021-85775-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973809PMC
March 2021

Anti-β2-glycoprotein I and anti-prothrombin antibodies cause lupus anticoagulant through different mechanisms of action.

J Thromb Haemost 2021 Apr 9;19(4):1018-1028. Epub 2021 Feb 9.

Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

Background: The presence of lupus anticoagulant (LA) is an independent risk factor for thrombosis. This laboratory phenomenon is detected as a phospholipid-dependent prolongation of the clotting time and is caused by autoantibodies against beta2-glycoprotein I (β2GPI) or prothrombin. How these autoantibodies cause LA is unclear.

Objective: To elucidate how anti-β2GPI and anti-prothrombin antibodies cause the LA phenomenon.

Methods: The effects of monoclonal anti-β2GPI and anti-prothrombin antibodies on coagulation were analyzed in plasma and with purified coagulation factors.

Results: Detection of LA caused by anti-β2GPI or anti-prothrombin antibodies required the presence of the procofactor factor V (FV) in plasma. LA effect disappeared when FV was replaced by activated FV (FVa), both in a model system and in patient plasma, although differences between anti-β2GPI and anti-prothrombin antibodies were observed. Further exploration of the effects of the antibodies on coagulation showed that the anti-β2GPI antibody attenuated FV activation by activated faxtor X (FXa), whereas the anti-prothrombin antibody did not. Binding studies showed that β2GPI--antibody complexes directly interacted with FV with high affinity. Anti-prothrombin complexes caused the LA phenomenon through competition for phospholipid binding sites with coagulation factors as reduced FXa binding to lipospheres was observed with flow cytometry in the presence of these antibodies.

Conclusion: Anti-β2GPI and anti-prothrombin antibodies cause LA through different mechanisms of action: While anti-β2GPI antibodies interfere with FV activation by FXa through a direct interaction with FV, anti-prothrombin antibodies compete with FXa for phospholipid binding sites. These data provide leads for understanding the paradoxical association between thrombosis and a prolonged clotting time in the antiphospholipid syndrome.
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http://dx.doi.org/10.1111/jth.15241DOI Listing
April 2021

Hemostatic changes by thrombopoietin-receptor agonists in immune thrombocytopenia patients.

Blood Rev 2020 Nov 10:100774. Epub 2020 Nov 10.

Department of Hematology, Van Creveldkliniek, University Medical Centre Utrecht, Postbox 85500, 3508 GA Utrecht, The Netherlands. Electronic address:

Thrombopoietin receptor agonist (TPO-RA) treatment increases the thrombosis rate in immune thrombocytopenia (ITP). We hypothesize that TPO-RAs influence platelet function, global and secondary hemostasis and/or fibrinolysis. A systematic review was performed. If possible, data were compared between responders (relevant increase in platelet count), and non-responders. Twelve observational studies with 305 patients were included (responders (127/150 (85%))). There were indications that TPO-RA treatment enhanced platelet function, with respect to platelet-monocyte aggregates, soluble P-selectin, GPVI expression, and adhesion under flow. Studies addressing global and secondary hemostasis and fibrinolysis were scarce. Overall, no changes were found during TPO-RA treatment, apart from an accelerated clot formation and conflicting data on levels of plasminogen activator inhibitor (PAI)-1. The parameters that increased have previously been associated with thrombosis in other patient groups, and might contribute to the increased rate of thrombosis observed in TPO-RA-treated ITP patients.
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http://dx.doi.org/10.1016/j.blre.2020.100774DOI Listing
November 2020

Fasciola hepatica serine protease inhibitor family (serpins): Purposely crafted for regulating host proteases.

PLoS Negl Trop Dis 2020 08 6;14(8):e0008510. Epub 2020 Aug 6.

Centre for One Health and Ryan Institute, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.

Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in Fasciola hepatica excretory-secretory products indicates that the parasite exploits these to regulate proteases encountered during its development within vertebrate hosts. Interrogation of the F. hepatica genome identified a multi-gene serpin family of seven members that has expanded by gene duplication and divergence to create an array of inhibitors with distinct specificities. We investigated the molecular properties and functions of two representatives, FhSrp1 and FhSrp2, highly expressed in the invasive newly excysted juvenile (NEJ). Consistent with marked differences in the reactive centre loop (RCL) that executes inhibitor-protease complexing, the two recombinant F. hepatica serpins displayed distinct inhibitory profiles against an array of mammalian serine proteases. In particular, rFhSrp1 efficiently inhibited kallikrein (Ki = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (Ki = 0.07 nM). FhSrp1 and FhSrp2 are both expressed on the NEJ surface, predominantly around the oral and ventral suckers, suggesting that these inhibitors protect the parasites from the harmful proteolytic effects of host proteases, such as chymotrypsin, during invasion. Furthermore, the unusual inhibition of kallikrein suggests that rFhSrp1 modulates host responses such as inflammation and vascular permeability by interfering with the kallikrein-kinin system. A vaccine combination of rFhSrp1 and rFhSrp2 formulated in the adjuvant Montanide ISA 206VG elicited modest but non-significant protection against a challenge infection in a rat model, but did induce some protection against liver pathogenesis when compared to a control group and a group vaccinated with two well-studied vaccine candidates, F. hepatica cathepsin L2 and L3. This work highlights the importance of F. hepatica serpins to regulate host responses that enables parasite survival during infection and, coupled with the vaccine data, encourages future vaccine trials in ruminants.
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http://dx.doi.org/10.1371/journal.pntd.0008510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437470PMC
August 2020

Sex differences in flow cytometry-based platelet reactivity in stable outpatients suspected of myocardial ischemia.

Res Pract Thromb Haemost 2020 Jul 15;4(5):879-885. Epub 2020 May 15.

Department of Cardiology University Medical Center Utrecht Utrecht The Netherlands.

Background: Antiplatelet therapy is the mainstay of secondary prevention of cardiovascular events. Studies suggest that women do not obtain equal therapeutic benefit from antiplatelet therapy compared with men. The link between sex differences in platelet biology and response to antiplatelet therapies is unclear. We therefore investigated the role of sex differences in platelet reactivity in a cohort of outpatients with chest pain, in response to treatment with antiplatelet agents.

Methods: Platelet reactivity was measured in 382 randomly selected patients participating in the Myocardial Ischemia Detection by Circulating Biomarkers (MYOMARKER) study, an observational cohort study of outpatients suspected of myocardial ischemia. In all patients, blood was collected during diagnostic workup, and platelet reactivity was assessed with a flow cytometry-based platelet activation test that quantifies both platelet degranulation (P-selectin expression) and platelet aggregation (fibrinogen binding to integrin αIIbβ3) in whole blood.

Results: Platelet reactivity was higher in women compared with men when activated with protease activating receptor 1-activating peptide SFLLRN (PAR1-AP) and adenosine 5'-phosphate (ADP), independent of age, basal activation status, estimated glomerular filtration rate < 60, platelet count, statin use, the use of P2Y12 inhibitors, or the use of aspirin. P2Y12 inhibitor use strongly reduced fibrinogen binding after stimulation with PAR1-AP, but only slightly reduced platelet P-selectin expression. Calculation of the relative inhibition in P2Y12 users indicated 62% inhibition of the response toward ADP. Stratified analysis showed that women (n = 14) using P2Y12 inhibitors showed less inhibition of fibrinogen binding after PAR1-AP stimulation than men (n = 38) using P2Y12 inhibitors.

Conclusions: These findings call for further study of differential effects of P2Y12 inhibitors in women with suspected myocardial ischemia.
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http://dx.doi.org/10.1002/rth2.12344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354392PMC
July 2020

Schistosoma mansoni infection affects the proteome and lipidome of circulating extracellular vesicles in the host.

Mol Biochem Parasitol 2020 07 27;238:111296. Epub 2020 Jun 27.

Department of Medical Microbiology and Infectious Diseases, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands. Electronic address:

Eggs, schistosomula and adult Schistosoma worms are known to release extracellular vesicles (EV) during in vitro incubations and these EVs are postulated to affect the host responses. So far only those EVs released during in vitro incubations of schistosomes have been studied and it is unknown whether in blood of infected hosts the schistosomal EVs can be detected amidst all the circulating EVs of the host itself. In this study we analyzed the protein as well as the phospholipid composition of EVs circulating in blood plasma of S. mansoni infected hamsters and compared those with the EVs circulating in blood of non-infected hamsters. Although neither proteins nor lipids specific for schistosomes could be detected in the circulating EVs of the infected hamsters, the infection with schistosomes had a marked effect on the circulating EVs of the host, as the protein as well as the lipid composition of EVs circulating in infected hamsters were different from the EVs of uninfected hamsters. The observed changes in the EV lipid and protein content suggest that more EVs are released by the diseased liver, the affected erythrocytes and activated immune cells.
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http://dx.doi.org/10.1016/j.molbiopara.2020.111296DOI Listing
July 2020

Bleeding phenotype and diagnostic characterization of patients with congenital platelet defects.

Am J Hematol 2020 Jun 19. Epub 2020 Jun 19.

Van Creveldkliniek, University Medical Center Utrecht, University Utrecht, Utrecht, the Netherlands.

Phenotypic characterization of congenital platelet defects (CPDs) could help physicians recognize CPD subtypes and can inform on prognostic implications. We report the analyses of the bleeding phenotype and diagnostic characteristics of a large cohort of adult patients with a confirmed CPD. A total of 96 patients were analyzed and they were classified as Glanzmann thrombasthenia, Bernard-Soulier syndrome, dense granule deficiency, defects in the ADP or thromboxane A2 (TxA2) pathway, isolated thrombocytopenia or complex abnormalities. The median ISTH-BAT bleeding score was nine (IQR 5-13). Heavy menstrual bleeding (HMB) (80%), post-partum hemorrhage (74%), post-operative bleeds (64%) and post-dental extraction bleeds (57%) occurred most frequently. Rare bleeding symptoms were bleeds from the urinary tract (4%) and central nervous system (CNS) bleeds (2%). Domains with a large proportion of severe bleeds were CNS bleeding, HMB and post-dental extraction bleeding. Glanzmann thrombasthenia and female sex were associated with a more severe bleeding phenotype.
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http://dx.doi.org/10.1002/ajh.25910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540397PMC
June 2020

[Diagnostics on suspicion of a bleeding disorder].

Ned Tijdschr Geneeskd 2020 03 3;164. Epub 2020 Mar 3.

UMC Utrecht, Van Creveldkliniek, Utrecht.

Bleeding symptoms occur frequently in the general population, but the possibility of an underlying bleeding disorder is not always recognised. Women with a bleeding disorder are disproportionally affected due to blood loss during menstruation and giving birth. Taking a thorough family history and a history of bleeding are most important in the workup to detect a potential underlying bleeding disorder. In patients with a bleeding disorder, potentially life-threatening complications due to bleeding can be prevented by compiling an individualized treatment plan and timely targeted blood coagulation treatment. If a bleeding disorder is suspected, initial diagnostic testing consists of determining the aPTT, PT, platelet count and von Willebrand factor activity; global tests for disorders of haemostasis, such as the coagulation time and platelet function are not of any added value. It cannot be excluded that a patient in whom test results are normal may still have a platelet function disorder or a rare bleeding disorder. If there is a strong suspicion of a bleeding disorder this should always be discussed with a coagulation specialist.
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March 2020

Congenital platelet disorders and health status-related quality of life.

Res Pract Thromb Haemost 2020 Jan 11;4(1):100-105. Epub 2019 Dec 11.

Van Creveldkliniek University Medical Center Utrecht University Utrecht Utrecht The Netherlands.

Background: Patients with congenital blood platelet disorders (CPDs) demonstrate a predominantly mucocutaneous bleeding tendency. Repeated bleeds throughout life can have a significant impact on health status-related quality of life (HR-QoL), but few studies have investigated HR-QoL in patients with CPDs.

Objectives: To determine HR-QoL in patients with suspected or confirmed CPDs as compared with the general Dutch population and to assess the association between bleeding phenotype and HR-QoL.

Methods: Data were derived from the Thrombocytopathy in the Netherlands (TiN) study, a cross-sectional study of individuals suspected for a congenital platelet defect. TiN patients with an increased ISTH Bleeding Assessment Tool (ISTH-BAT) score (>3 in men and > 5 in women) were included for analysis. HR-QoL was assessed with the Short Form (SF)-36 survey. Bleeding symptoms were evaluated with the ISTH-BAT, resulting in a bleeding score.

Results: One hundred fifty-six patients were analyzed, of whom 126 (81%) were women. Sixty-two patients (40%) had a confirmed CPD. Compared to the general Dutch population, patients with a suspected or confirmed CPD reported decreased physical functioning, limitations in daily activities due to physical health problems, limitations in social activities, decreased energy levels and fatigue, pain, and lower general health status. HR-QoL was not correlated with the ISTH-BAT score and was similar in patients with a confirmed CPD and those in whom a CPD could not be diagnosed.

Conclusion: A bleeding tendency in patients with a suspected or confirmed CPD significantly impacts HR-QoL, independent of a confirmed explanatory diagnosis.
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http://dx.doi.org/10.1002/rth2.12281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971322PMC
January 2020

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency.

J Thromb Haemost 2020 03 27;18(3):706-713. Epub 2019 Dec 27.

Van Creveld Laboratory, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: δ-storage pool disease (δ-SPD) is a bleeding disorder characterized by a reduced number of platelet-dense granules. The diagnosis of δ-SPD depends on the measurement of platelet ADP content, but this test is time consuming and requires a relatively large blood volume. Flow cytometric analysis of platelet mepacrine uptake is a potential alternative, but this approach lacks validation, which precludes its use in a diagnostic setting.

Objectives: To evaluate the performance of platelet mepacrine uptake as a diagnostic test for δ-SPD.

Patients/methods: Mepacrine fluorescence was determined with flow cytometry before and after platelet activation in 156 patients with a suspected platelet function disorder and compared with platelet ADP content as a reference test. Performance was analyzed with a receiver operating characteristic (ROC) curve.

Results: Eleven of 156 patients had δ-SPD based on platelet ADP content. Mepacrine fluorescence was inferior to platelet ADP content in identifying patients with δ-SPD, but both mepacrine uptake (area under the ROC curve [AUC] 0.87) and mepacrine release after platelet activation (AUC 0.80) had good discriminative ability. In our tertiary reference center, mepacrine uptake showed high negative predicitive value (97%) with low positive predictive value (35%). Combined with a negative likelihood ratio of 0.1, these data indicate that mepacrine uptake can be used to exclude δ-SPD in patients with a bleeding tendency.

Conclusion: Mepacrine fluorescence can be used as a screening tool to exclude δ-SPD in a large number of patients with a suspected platelet function disorder.
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http://dx.doi.org/10.1111/jth.14698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065135PMC
March 2020

Heparin Forms Polymers with Cell-free DNA Which Elongate Under Shear in Flowing Blood.

Sci Rep 2019 12 4;9(1):18316. Epub 2019 Dec 4.

Department of Clinical Chemistry & Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Heparin is a widely used anticoagulant which inhibits factor Xa and thrombin through potentiation of antithrombin. We recently identified that the nucleic acid stain SYTOX reacts with platelet polyphosphate due to molecular similarities, some of which are shared by heparin. We attempted to study heparin in flowing blood by live-cell fluorescence microscopy, using SYTOX for heparin visualisation. Immunostaining was performed with monoclonal antibodies directed against various heparin-binding proteins. In addition, we studied modulation of heparin activity in coagulation assays, as well its effects on fibrin formation under flow in recalcified whole blood. We found that SYTOX-positive polymers appear in heparinised blood under flow. These polymers typically associate with platelet aggregates and their length (reversibly) increases with shear rate. Immunostaining revealed that of the heparin-binding proteins assessed, they only contain histones. In coagulation assays and flow studies on fibrin formation, we found that addition of exogenous histones reverses the anticoagulant effects of heparin. Furthermore, the polymers do not appear in the presence of DNase I, heparinase I/III, or the heparin antidote protamine. These findings suggest that heparin forms polymeric complexes with cell-free DNA in whole blood through a currently unidentified mechanism.
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http://dx.doi.org/10.1038/s41598-019-54818-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892814PMC
December 2019

Platelet RNA modules point to coronary calcification in asymptomatic women with former preeclampsia.

Atherosclerosis 2019 12 11;291:114-121. Epub 2019 Oct 11.

Laboratory of Experimental Cardiology, UMC Utrecht, Utrecht University, Utrecht, the Netherlands. Electronic address:

Background And Aims: Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia.

Methods: Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS).

Results: We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, p = 0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (p = 3.9 × 10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (p = 0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (p = 1.6 × 10 to 1.7 × 10).

Conclusions: We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.
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http://dx.doi.org/10.1016/j.atherosclerosis.2019.10.009DOI Listing
December 2019

Transforming growth factor-beta profiles correlate with clinical symptoms and parameters of haemostasis and inflammation in a controlled human malaria infection.

Cytokine 2020 01 13;125:154838. Epub 2019 Sep 13.

Department of Medical Microbiology & Infectious Diseases, Erasmus MC University Medical Center, Rotterdam 3015 GD, the Netherlands. Electronic address:

Background: After a controlled human malaria infection (CHMI), presentation of clinical signs and symptoms and host responses is heterogeneous. Transforming growth factor-beta (TGF-β) is the first serum cytokine that changes in malaria-naïve volunteers after CHMI. We studied a possible relation between TGF-β changes, pro-inflammatory cytokines, activation of haemostasis and endothelial cells and clinical symptoms.

Methods: A panel of cytokines including TGF-β, and markers of activation of haemostasis and endothelial cells were measured in blood samples of 15 volunteers at baseline before CHMI and during CHMI at day of treatment. The change of the parameters on the day of treatment was examined for a significant alteration during infection.

Results: Nine of 15 volunteers showed a significant decrease in TGF-β compared to baseline, with concomitant increased concentrations of D-dimer (p = 0.012), Von Willebrand factor (p = 0.017), IL-6 (p = 0.012) and IFN-γ (0.028) and a significantly decreased platelet count (p = 0.011). In contrast, 6 of 15 volunteers showed sustained or increased TGF-β concentrations without change in the aforementioned parameters. The sustained responders presented with less moderate and severe clinical symptoms than the negative responders (p = 0.036) and had a higher baseline lymphocyte count (p = 0.026). TGF-β concentrations did not correlate with the parasitaemia on day of treatment.

Conclusion: Early decreases of serum TGF-β might function a marker for a pro-inflammatory host response and downstream clinical symptoms and pathology during CHMI.
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http://dx.doi.org/10.1016/j.cyto.2019.154838DOI Listing
January 2020

A head-to-head comparison of conjugation methods for VHHs: Random maleimide-thiol coupling versus controlled click chemistry.

Int J Pharm X 2019 Dec 21;1:100020. Epub 2019 Jun 21.

Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Targeted delivery of therapeutics is an attractive strategy for vascular diseases. Recently, variable domains of heavy-chain-only antibodies (VHHs) have gained momentum as targeting ligands to achieve this. Targeting ligands need adequate conjugation to the preferred delivery platform. When choosing a conjugation method, two features are critical: a fixed and specified stoichiometry and an orientation of the conjugated targeting ligand that preserves its functional binding capacity. We here describe a comparison of popular maleimide-thiol conjugation with state-of-the-art "click chemistry" for conjugating VHHs. First, we demonstrate the modification of VHHs with azide via Sortase A mediated transpeptidation. Subsequently, optimal clicking conditions were found at a temperature of 50 °C, using a 3:1 M ratio of DBCO-PEG:VHH-azide and an incubation time of 18 h. Second, we show that stoichiometry was controllable with click chemistry and produced defined conjugates, whereas maleimide-thiol conjugation resulted in diverse reaction products. In addition, we show that all VHHs - independent of the conjugation method or conjugated residue - still specifically bind their cognate antigen. Yet, VHH's functional binding capacities after click chemistry were at least equal or better than maleimide thiol conjugates. Together these data underline that click chemistry is superior to maleimide-thiol conjugation for conjugating targeting ligands.
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http://dx.doi.org/10.1016/j.ijpx.2019.100020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733300PMC
December 2019

Desialylation of platelets induced by Von Willebrand Factor is a novel mechanism of platelet clearance in dengue.

PLoS Pathog 2019 03 8;15(3):e1007500. Epub 2019 Mar 8.

Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Thrombocytopenia and platelet dysfunction are commonly observed in patients with dengue virus (DENV) infection and may contribute to complications such as bleeding and plasma leakage. The etiology of dengue-associated thrombocytopenia is multifactorial and includes increased platelet clearance. The binding of the coagulation protein von Willebrand factor (VWF) to the platelet membrane and removal of sialic acid (desialylation) are two well-known mechanisms of platelet clearance, but whether these conditions also contribute to thrombocytopenia in dengue infection is unknown. In two observational cohort studies in Bandung and Jepara, Indonesia, we show that adult patients with dengue not only had higher plasma concentrations of plasma VWF antigen and active VWF, but that circulating platelets had also bound more VWF to their membrane. The amount of platelet-VWF binding correlated well with platelet count. Furthermore, sialic acid levels in dengue patients were significantly reduced as assessed by the binding of Sambucus nigra lectin (SNA) and Maackia amurensis lectin II (MAL-II) to platelets. Sialic acid on the platelet membrane is neuraminidase-labile, but dengue virus has no known neuraminidase activity. Indeed, no detectable activity of neuraminidase was present in plasma of dengue patients and no desialylation was found of plasma transferrin. Platelet sialylation was also not altered by in vitro exposure of platelets to DENV nonstructural protein 1 or cultured DENV. In contrast, induction of binding of VWF to glycoprotein 1b on platelets using the VWF-activating protein ristocetin resulted in the removal of platelet sialic acid by translocation of platelet neuraminidase to the platelet surface. The neuraminidase inhibitor oseltamivir reduced VWF-induced platelet desialylation. Our data demonstrate that excessive binding of VWF to platelets in dengue results in neuraminidase-mediated platelet desialylation and platelet clearance. Oseltamivir might be a novel treatment option for severe thrombocytopenia in dengue infection.
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http://dx.doi.org/10.1371/journal.ppat.1007500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426266PMC
March 2019

Detection of Anti-Cardiolipin and Anti-β2glycoprotein I Antibodies Differs between Platforms without Influence on Association with Clinical Symptoms.

Thromb Haemost 2019 May 1;119(5):797-806. Epub 2019 Mar 1.

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

Background:  The anti-phospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with persistent presence of anti-phospholipid antibodies (aPL). Laboratory criteria include aPL detection by coagulation tests for lupus anticoagulant (LAC) or solid phase assays measuring anti-β2 glycoprotein I (aβ2GPI) or anti-cardiolipin (aCL) immunoglobulin (Ig) G/IgM antibodies. External quality control programs illustrate that commercially available aPL assays produce variable results.

Objective:  We aimed to investigate the agreement and diagnostic accuracy of solid phase assays.

Materials And Methods:  In this multi-centre study, 1,168 patient samples were tested on one site for aCL and aβ2GPI IgG/IgM antibodies by four solid phase test systems. Samples included APS patients, controls and monoclonal antibodies (MoAB) against different epitopes of β2GPI. LAC was determined by the local centre.

Results:  aCL IgM assays resulted in the most discrepancies (60%), while aCL IgG and aβ2GPI IgM assays resulted in lower discrepancies (36%), suggesting better agreement. Discrepant samples displayed lower median aPL titers. Dependent on the solid phase test system, odds ratios (ORs) for thrombosis and pregnancy morbidity ranged from 1.98 to 2.56 and 3.42 to 4.78, respectively. Three platforms showed lower sensitivity for MoAB directed against the glycine (Gly) 40-arginine (Arg) 43 epitope of domain I of β2GPI.

Conclusion:  Poor agreement was observed between different commercially available aCL and aβ2GPI IgG/IgM assays, hampering uniformity in the identification of aPL-positive patients. Clinical association was globally concordant between solid phase test systems considering results of the four aPL together. An assay sensitive in detecting the MoAB against Gly40-Arg43 of domain I of β2GPI reached the highest OR for thrombosis.
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http://dx.doi.org/10.1055/s-0039-1679901DOI Listing
May 2019

The influence of hypoxia on platelet function and plasmatic coagulation during systemic inflammation in humans .

Platelets 2019 25;30(7):927-930. Epub 2018 Dec 25.

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

Systemic inflammation and hypoxia frequently occur simultaneously in critically ill patients, and are both associated with platelet activation and coagulopathy. However, human data on the effects of hypoxia on platelet function and plasmatic coagulation under systemic inflammatory conditions are lacking. In the present study, 20 healthy male volunteers were randomized to either 3.5 h of hypoxia (peripheral saturation 80-85%) or normoxia (room air), and systemic inflammation was elicited by intravenous administration of 2 ng/kg endotoxin. Various parameters of platelet function and plasmatic coagulation were determined serially. Endotoxemia resulted in increased circulating platelet-monocyte complexes and enhanced platelet reactivity, effects which were attenuated by hypoxia. Furthermore, endotoxin administration resulted in decreased plasma levels of platelet factor-4 levels and increased concentrations of von Willebrand factor. These endotoxemia-induced effects were not influenced by hypoxia. Neither endotoxemia nor hypoxia affected thrombin generation. In conclusion, our data reveal that hypoxia attenuates the endotoxemia-induced increases in platelet-monocyte formation and platelet reactivity, while leaving parameters of plasmatic coagulation unaffected.
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http://dx.doi.org/10.1080/09537104.2018.1557617DOI Listing
January 2020

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Arterioscler Thromb Vasc Biol 2018 08;38(8):1748-1760

From the Biomedical Engineering, School of Medicine (J.Z.-R., S.E.R., C.P., R.A.R., E.I.T., A.G., O.J.T.M.).

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.
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http://dx.doi.org/10.1161/ATVBAHA.118.311193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205188PMC
August 2018

A switch to a raltegravir containing regimen does not lower platelet reactivity in HIV-infected individuals.

AIDS 2018 11;32(17):2469-2475

Department of Internal Medicine, Radboud Centre for Infectious Diseases, Radboud University Medical Centre, Nijmegen.

Objective: Platelet hyperreactivity and increased platelet-monocyte aggregation (PMA) are associated with increased cardiovascular risk and inflammation. In a previous cross-sectional study, individuals using a raltegravir (RAL)-based regimen were found to have reduced platelet reactivity and PMA compared with other antiretroviral regimens. Our aim was to investigate whether switching from a nonintegrase inhibitor regimen to a RAL-based regimen reduces platelet reactivity or PMA.

Design: An investigator initiated, single-centre, prospective randomized, open-label, blinded endpoint trial.

Methods: Forty HIV-infected adults using a nonintegrase inhibitor containing regimen with undetectable viral load were randomized to either continue their regimen or switch to a RAL-based regimen for 10 weeks, continuing the same backbone. The primary outcome was the change in platelet reactivity at week 10, which was determined as the expression of the platelet activation marker P-selectin and binding of fibrinogen before and after ex-vivo stimulation with different platelet agonists. Secondary outcomes included PMA, plasma markers of platelet activation and markers of inflammation and immune cell activation.

Results: Twenty-one participants were enrolled in the continuation group and 19 in the RAL group. Baseline characteristics were comparable between groups. There were no differences in the change in platelet reactivity to either platelet agonist at week 10, nor in plasma markers of platelet activation. PMA, C-reactive protein, T-cell activation (CD38HLA-DR) and monocyte (CD14CD16) subsets.

Conclusion: Switching a nonintegrase inhibitor containing regimen to a RAL-based regimen does not reduce platelet reactivity, platelet-leukocyte aggregation, inflammation and immune activation in virologically suppressed HIV-infected individuals.

Clinical Trial Number: NCT02383355.
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http://dx.doi.org/10.1097/QAD.0000000000001993DOI Listing
November 2018

Recent Developments in Antiphospholipid Antibodies and the Antiphospholipid Syndrome.

Authors:
Rolf T Urbanus

Semin Thromb Hemost 2018 07 25;44(5):417-418. Epub 2018 Jun 25.

Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

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http://dx.doi.org/10.1055/s-0038-1657761DOI Listing
July 2018

Toward Flow Cytometry Based Platelet Function Diagnostics.

Semin Thromb Hemost 2018 Apr 19;44(3):197-205. Epub 2018 Mar 19.

Department of Clinical Chemistry and Haematology, Center for Circulatory Health, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

The laboratory diagnostics of (inherited) platelet function disorders mainly comprises aggregation and secretion assays, which may be suitable for diagnosing some specific severe platelet function disorders, but are not reliable enough for diagnosing mild platelet function disorders or disorders associated with low platelet count. Flow cytometric assessment of platelet reactivity will expectedly provide additional value during the diagnostic work-up of platelet function disorders because it only requires a small volume of whole blood and allows the measurement of platelet function in thrombocytopenic samples. Flow cytometry has frequently been used to evaluate platelet function in the research setting, and therefore, these assays will require clinical validation before they can be used as routine diagnostic tools. The main challenge in the validation of innovative platelet function diagnostic tests is the lack of a gold standard test for mild platelet function disorders. This review aims to address the many applications of flow cytometry in the current diagnostic work-up of platelet function testing and to discuss the challenges in introducing new tools for diagnosing platelet function disorders.
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http://dx.doi.org/10.1055/s-0038-1636901DOI Listing
April 2018

Truncation of ADAMTS13 by Plasmin Enhances Its Activity in Plasma.

Thromb Haemost 2018 03 13;118(3):471-479. Epub 2018 Mar 13.

Department for Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system. We next investigated how plasmin affects both VWF and ADAMTS13 under static conditions in plasma by western blotting. We found that globular VWF is largely protected from plasmin cleavage. However, ADAMTS13 is rapidly cleaved under these conditions, suggesting inactivation. Surprisingly, we observed that plasmin enhances ADAMTS13 activity in a modified two-stage FRETS-VWF73 assay that protects FRETS-VWF73 substrate from degradation. In direct binding studies under the same conditions, we found that plasmin generates multiple C-terminally truncated forms of ADAMTS13 with VWF-binding capacity. In an effort to seek evidence for this mechanism in vivo, we analysed plasma from patients with systemic amyloidosis, which is hallmarked by a hyperfibrinolytic state. We found that their plasma contained increased levels of C-terminally truncated forms of ADAMTS13, which correlated with their hyperfibrinolytic state.We propose that truncation of ADAMTS13 by plasmin abolishes intramolecular self-association, which improves interaction with unfolded VWF.
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http://dx.doi.org/10.1055/s-0038-1627460DOI Listing
March 2018

Fibrinogen and fibrin are novel substrates for Fasciola hepatica cathepsin L peptidases.

Mol Biochem Parasitol 2018 04 4;221:10-13. Epub 2018 Feb 4.

Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address:

Cathepsin peptidases form a major component of the secreted proteins of the blood-feeding trematodes Fasciola hepatica and Schistosoma mansoni. These peptidases fulfill many functions, from facilitating infection to feeding and immune evasion. In this study, we examined the Fasciola cathepsin L peptidases FhCL1, FhCL2, and FhCL3 and the schistosomal cathepsin peptidases SmCB1 and SmCL3 for their anticoagulant properties. Although no direct anticoagulant effect of these peptidases was observed, we discovered that cathepsin peptidases from Fasciola, but not from Schistosoma, were able to degrade purified fibrinogen, with FhCL1 having the highest fibrinogenolytic activity. Additionally, FhCL1 and FhCL2 both efficiently degraded fibrin. The lack of a direct anticoagulant or fibrinolytic effect of these peptidases is explained by their inhibition by plasma components. However, within the parasite gut, high concentrations of these peptidases could induce an anticoagulant environment, facilitating blood-feeding for extended periods.
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http://dx.doi.org/10.1016/j.molbiopara.2018.02.001DOI Listing
April 2018

Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis.

Sci Signal 2017 Nov 28;10(507). Epub 2017 Nov 28.

Systems Immunity Research Institute, Cardiff University, Heath Park, Cardiff CF14 4XN, UK.

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein β2GP1 (β2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.
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http://dx.doi.org/10.1126/scisignal.aan2787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5720345PMC
November 2017

Platelet dysfunction contributes to bleeding complications in patients with probable leptospirosis.

PLoS Negl Trop Dis 2017 Sep 21;11(9):e0005915. Epub 2017 Sep 21.

Department of Internal Medicine, Radboud university medical center, Nijmegen, The Netherlands.

Background: Severe leptospirosis is frequently complicated by a hemorrhagic diathesis, of which the pathogenesis is still largely unknown. Thrombocytopenia is common, but often not to the degree that spontaneous bleeding is expected. We hypothesized that the hemorrhagic complications are not only related to thrombocytopenia, but also to platelet dysfunction, and that increased binding of von Willebrand factor (VWF) to platelets is involved in both platelet dysfunction and increased platelet clearance.

Methodology/principal Findings: A prospective study was carried out in Semarang, Indonesia, enrolling 33 hospitalized patients with probable leptospirosis, of whom 15 developed clinical bleeding, and 25 healthy controls. Platelet activation and reactivity were determined using flow cytometry by measuring the expression of P-selectin and activation of the αIIbβ3 integrin by the binding of fibrinogen in unstimulated samples and after ex vivo stimulation by the platelet agonists adenosine-diphosphate (ADP) and thrombin-receptor activating peptide (TRAP). Platelet-VWF binding, before and after VWF stimulation by ristocetin, as well as plasma levels of VWF, active VWF, the VWF-inactivating enzyme ADAMTS13, thrombin-antithrombin complexes (TAT) and P-selectin were also measured. Bleeding complications were graded using the WHO bleeding scale. Our study revealed that platelet activation, with a secondary platelet dysfunction, is a feature of patients with probable leptospirosis, especially in those with bleeding manifestations. There was a significant inverse correlation of bleeding score with TRAP-stimulated P-selectin and platelet-fibrinogen binding (R = -0.72, P = 0.003 and R = -0.66, P = 0.01, respectively) but not with platelet count. Patients with bleeding also had a significantly higher platelet-VWF binding. Platelet counts were inversely correlated with platelet-VWF binding (R = -0.74; P = 0.0009. There were no correlations between platelet-VWF binding and the degree of platelet dysfunction, suggesting that increased platelet-VWF binding does not directly interfere with the platelet αIIbβ3 signaling pathway in patients with probable leptospirosis.

Conclusion/significance: Platelet dysfunction is common in probable leptospirosis patients with manifest bleeding. Increased VWF-platelet binding may contribute to the activation and clearance of platelets.
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http://dx.doi.org/10.1371/journal.pntd.0005915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626517PMC
September 2017

The Lupus Anticoagulant Paradox.

Semin Thromb Hemost 2018 Jul 12;44(5):445-452. Epub 2017 Sep 12.

Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.

Lupus anticoagulant (LA) represents the most enigmatic antibody population in patients with antiphospholipid syndrome and represents a paradox that is still unsolved. This class of antiphospholipid antibody causes a phospholipid-dependent prolongation of the clotting time but is associated with an increased risk of thrombosis and pregnancy morbidity. In this review, we will provide an overview of the different antibodies that have been associated with LA activity, their importance based on clinical studies, and address the question why this prolongation of the clotting time is associated with thrombosis rather than a bleeding tendency.
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http://dx.doi.org/10.1055/s-0037-1606190DOI Listing
July 2018

Swarm Intelligence-Enhanced Detection of Non-Small-Cell Lung Cancer Using Tumor-Educated Platelets.

Cancer Cell 2017 08;32(2):238-252.e9

Department of Neurosurgery, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands; Brain Tumor Center Amsterdam, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands; Department of Neurology, Massachusetts General Hospital and Neuroscience Program, Harvard Medical School, 149 13(th) Street, Charlestown, MA 02129, USA. Electronic address:

Blood-based liquid biopsies, including tumor-educated blood platelets (TEPs), have emerged as promising biomarker sources for non-invasive detection of cancer. Here we demonstrate that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels from platelet RNA-sequencing libraries (n = 779). This resulted in accurate TEP-based detection of early- and late-stage non-small-cell lung cancer (n = 518 late-stage validation cohort, accuracy, 88%; AUC, 0.94; 95% CI, 0.92-0.96; p < 0.001; n = 106 early-stage validation cohort, accuracy, 81%; AUC, 0.89; 95% CI, 0.83-0.95; p < 0.001), independent of age of the individuals, smoking habits, whole-blood storage time, and various inflammatory conditions. PSO enabled selection of gene panels to diagnose cancer from TEPs, suggesting that swarm intelligence may also benefit the optimization of diagnostics readout of other liquid biopsy biosources.
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http://dx.doi.org/10.1016/j.ccell.2017.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381325PMC
August 2017

Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis.

Platelets 2017 Sep 29;28(6):595-601. Epub 2016 Dec 29.

a Department of Internal Medicine , Radboud University Medical Center , Nijmegen , The Netherlands.

Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, β-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications.
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http://dx.doi.org/10.1080/09537104.2016.1252837DOI Listing
September 2017

The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays.

Platelets 2017 Sep 25;28(6):567-575. Epub 2016 Nov 25.

a Department of Clinical Chemistry and Hematology , University Medical Center Utrecht , The Netherlands.

Patients on P2Y inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y inhibitors. The effect of in vitro and in vivo P2Y inhibition on αIIbβ3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y inhibitors was compared to VerifyNow results. P2Y inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition. Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.
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http://dx.doi.org/10.1080/09537104.2016.1246713DOI Listing
September 2017