Publications by authors named "Philip G de Groot"

204 Publications

Mechanisms of thrombosis in systemic lupus erythematosus and antiphospholipid syndrome.

Best Pract Res Clin Rheumatol 2017 06 16;31(3):334-341. Epub 2017 Nov 16.

Synapse Research Institute, Maastricht, The Netherlands.

The presence of antiphospholipid antibodies is one of the most common acquired risk factors for thrombosis. Antiphospholipid antibodies is a collective term for a set of autoantibodies with closely related but different specificity. Experiments in which isolated patient antibodies were injected into mice have shown that a specific subset of autoantibodies, those directed against the first domain of plasma protein β-glycoprotein I, can explain the increased risk of thrombosis. Experiments performed with these mice have shown that autoantibodies against β-glycoprotein I bind to and activate cells such as endothelial cells, monocytes, and platelets. Activation of these cells, all involved in the regulation of hemostasis, results in a shift towards a prothrombotic state. How this process is regulated, whether this is the only mechanism involved, and whether this is the only subpopulation responsible for the increased thrombotic risk is unknown. In this review, we will critically discuss what is known and what is debatable on the pathophysiology of antiphospholipid syndrome.
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http://dx.doi.org/10.1016/j.berh.2017.09.008DOI Listing
June 2017

Increased Platelet-Monocyte Interaction in Stable COPD in the Absence of Platelet Hyper-Reactivity.

Respiration 2018;95(1):35-43. Epub 2017 Oct 12.

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

Background: Chronic obstructive pulmonary disease (COPD) is well known for its cardiovascular co-morbidities. Increased platelet-monocyte interaction is found in COPD and may reflect altered platelet function and a potential role for anti-platelet therapy.

Objectives: The objectives were to investigate platelet-monocyte interaction, platelet activation and reactivity and plasmatic coagulation in stable COPD.

Methods: Platelet-monocyte interaction and platelet activation were determined by flow cytometry in 30 stable COPD patients and 25 controls. Platelet activation was measured by binding of fibrinogen to the activated fibrinogen receptor and platelet P-selectin expression at baseline and after platelet stimulation with platelet agonists. Plasmatic coagulation was measured by D-dimer and thrombin generation.

Results: Platelet-monocyte interaction was increased in stable COPD (median fluorescence intensity of platelet CD61 was 19.8 [IQR 14.0-33.2] vs. 10.0 [IQR 8.7-16.7], p = 0.002). In contrast, platelet activation and reactivity, reflected by fibrinogen binding and P-selectin expression, were the same in both groups. Plasma P-selectin and interleukin-6 were increased in COPD (p = 0.01 and p = 0.02, respectively), whereas soluble fibrinogen, D-dimer and thrombin generation were similar.

Conclusions: Increased platelet-monocyte interaction was found in the absence of platelet hyper-reactivity and activation of plasmatic coagulation in stable COPD. Future clinical evaluation of the effects of different anti-platelet drugs in COPD is warranted, as anti-platelet therapy may interfere with platelet-monocyte interaction.
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http://dx.doi.org/10.1159/000480457DOI Listing
October 2018

The effects of signal transducer and activator of transcription three mutations on human platelets.

Platelets 2018 Sep 29;29(6):602-609. Epub 2017 Sep 29.

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

Involvement of signal transducer and activator of transcription 3 (STAT3) in inflammation is well known. Recently, a role for STAT3 in platelet activation and platelet production has been suggested. Platelets exhibit important immune functions and engagement of STAT3 in platelet physiology may link inflammation and hemostasis. This study investigated the effects of STAT3 loss-of-function mutations and single nucleotide polymorphisms (SNPs) in STAT3 on glycoprotein VI (GPVI)-mediated platelet activation and platelet numbers in humans. Two cohorts were studied. The first cohort concerned patients with STAT3 loss-of-function mutations. Platelet numbers were investigated in eight patients and GPVI-mediated platelet activation was functionally tested in four patients. Additional experiments were performed to investigate underlying mechanisms. The second cohort concerned 334 healthy volunteers and investigated the consequences of SNPs in STAT3 on GPVI-mediated platelet activation and platelet numbers. Platelet activation was lower in STAT3 loss-of-function patients at baseline and after stimulation of the GPVI receptor, reflected by decreased P-selectin expression. This was independent of gene transcription. Blockade of the adenosine di-phosphate (ADP) pathway resulted in a further decrease of P-selectin expression, particularly in STAT3 loss-of-function patients. In contrast, the SNPs in STAT3 did not influence GPVI-mediated platelet activation. Also, platelet numbers were not affected by STAT3 loss-of-function mutations, nor was there an association with the SNPs. In conclusion, STAT3 signaling does not seem to play a major role in thrombopoiesis. We confirm that STAT3 is involved in GPVI-mediated platelet activation in humans, independent of gene transcription. GPVI-mediated platelet activation is highly dependent on secondary ADP release. Our findings suggest that STAT3 modulation may affect inflammation, hemostasis, and their interaction.
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http://dx.doi.org/10.1080/09537104.2017.1349309DOI Listing
September 2018

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

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

Human recombinant alkaline phosphatase inhibits ex vivo platelet activation in humans.

Thromb Haemost 2016 Nov 22;116(6):1111-1121. Epub 2016 Sep 22.

Peter Pickkers, Department of Intensive Care Medicine, Radboud university medical center, PO Box 9101, Internal Mailbag 710, 6500 HB Nijmegen, The Netherlands, Tel.: +31 24 36 15363, Fax: +31 24 36 68058, E-mail:

Sepsis-associated acute kidney injury (AKI) is associated with high morbidity and mortality. Excessive platelet activation contributes to AKI through the formation of microthrombi and amplification of systemic inflammation. Two phase II trials demonstrated that bovine-intestinal alkaline phosphatase (AP) improved renal function in critically ill patients with sepsis-associated AKI. In this study, we characterised the platelet-inhibiting effects of a human recombinant AP. Whole blood and platelet-rich plasma (PRP) of healthy volunteers (n=6) was pre-treated ex vivo with recAP, whereafter platelet reactivity to ADP, collagen-related peptide (CRP-XL) and Pam3CSK4 was determined by flow cytometry. RecAP (40 U/ml) reduced the platelet reactivity to ADP (inhibition with a median of 47 %, interquartile range 43-49 %; p<0.001) and tended to reduce platelet reactivity to CRP-XL (9 %, 2-25 %; p=0.08) in whole blood. The platelet-inhibiting effects of recAP were more pronounced in PRP both for ADP- (64 %, 54-68 %; p=0.002) and CRP-XL-stimulated samples (60 %, 46-71 %; p=0.002). RecAP rapidly converted ADP into adenosine, whereas antagonism of the A2A adenosine receptor partially reversed the platelet inhibitory effects of recAP. Platelets of septic shock patients (n=5) showed a 31% (22-34%; p=0.03) more pronounced reactivity compared to healthy volunteers, and this was completely reversed by recAP treatment. In conclusion, we demonstrate that recAP inhibits ex vivo human platelet activation through dephosphorylation of ADP and formation of adenosine as its turnover product. RecAP is able to reverse the platelet hyperreactivity present in septic shock patients. These effects may contribute to the beneficial effects of recAP as a new therapeutic candidate for sepsis-associated AKI.
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http://dx.doi.org/10.1160/TH16-03-0206DOI Listing
November 2016

Antiphospholipid Antibodies and the Risk of Stroke in Urban and Rural Tanzania: A Community-Based Case-Control Study.

Stroke 2016 10 13;47(10):2589-95. Epub 2016 Sep 13.

From the Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands (Q.d.M., A.J.v.d.V., P.G.d.G.); Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, The Netherlands (J.E.M., P.G.d.G., R.T.U.); North Tyneside General Hospital, North Shields (W.K.G., R.W.W.); Kilimanjaro Christian Medical Centre, Moshi, Tanzania (A.J.); Muhimbili University College Hospital, Dar-es-Salaam, Tanzania (F.M.); and Institute of Health and Society, Newcastle University, Newcastle-upon-Tyne, United Kingdom (R.W.W.).

Background And Purpose: The burden of stroke is high in sub-Saharan Africa, and improved knowledge of risk factors is needed. Antiphospholipid antibodies are a common acquired stroke risk factor in young individuals. Antiphospholipid antibodies may be induced by infectious diseases. Sub-Saharan Africa has a high infectious burden, and we analyzed the contribution of antiphospholipid antibodies to the risk of stroke in an incident population from rural and urban Tanzania.

Methods: Stroke cases and age- and sex-matched community-acquired controls from the rural Hai district and urban Dar-es-Salaam areas of Tanzania were recruited in a wider study of stroke incidence between June 2003 and June 2006. Lupus anticoagulant, anticardiolipin, anti-β2-glycoprotein I, and antiphosphatidylserine/prothrombin antibodies were determined in stored plasma, as well as IgG antibodies against Treponema pallidum.

Results: Data from 158 stroke cases and 369 controls were analyzed. Thirty cases (19%) and 4 controls (1%) had a lupus anticoagulant (odds ratio, 20.8; 95% confidence interval, 7.2-60.5). Anticardiolipin IgG was the only other antiphospholipid antibody subtype associated with increased stroke risk (odds ratio, 2.1; 95% confidence interval, 1.0-4.3), but this association disappeared when corrected for IgG antibodies against Treponema pallidum results. The prevalence of anti-β2-glycoprotein I IgG antibodies in the Tanzanian healthy population was high when Dutch cutoff values were applied (67%), whereas presence of anti-β2-glycoprotein I IgM was associated with a reduced stroke risk (odds ratio 0.3; 95% confidence interval, 0.1-1.1).

Conclusions: The presence of lupus anticoagulant is a strong, and to date unrecognized, risk factor for stroke in Tanzania, especially in young and middle-aged individuals.
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http://dx.doi.org/10.1161/STROKEAHA.116.013760DOI Listing
October 2016

In vitro uptake of recombinant factor VIIa by megakaryocytes with subsequent production of platelets containing functionally active drug.

Br J Haematol 2017 08 12;178(3):482-486. Epub 2016 Jun 12.

Surgical Research Laboratory, Department of Surgery, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands.

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http://dx.doi.org/10.1111/bjh.14149DOI Listing
August 2017

A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation.

Sci Rep 2016 05 23;6:26306. Epub 2016 May 23.

Institut National de la Santé et de la Recherche Médicale, UMR_S 1176, Univ. Paris-Sud, Université Paris-Saclay, 94276 Le Kremlin-Bicêtre, France.

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.
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http://dx.doi.org/10.1038/srep26306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876317PMC
May 2016

Plasmin is a natural trigger for bradykinin production in patients with hereditary angioedema with factor XII mutations.

J Allergy Clin Immunol 2016 11 6;138(5):1414-1423.e9. Epub 2016 Apr 6.

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

Background: Patients with angioedema experience unpredictable attacks of tissue swelling in which bradykinin is implicated. Several distinct mutations in Factor XII (FXII) are associated with hereditary angioedema (HAE) in the presence of normal C1 esterase inhibitor activity (FXII-HAE). The underlying disease mechanisms are unclear, which complicates diagnosis and treatment.

Objective: We sought to identify the natural trigger for FXII activation, which causes uncontrolled bradykinin production in patients with FXII-HAE.

Methods: We generated recombinant variants of FXII, representing health and disease, and studied their behavior in functional studies. We investigated bradykinin-forming pathways in blood plasma with newly developed nanobody-based analytic methods.

Results: We here report that FXII-HAE mutations collectively introduce new sites that are sensitive to enzymatic cleavage by plasmin. These FXII mutants rapidly activate after cleavage by plasmin, escape from inhibition through C1 esterase inhibitor, and elicit excessive bradykinin formation. Furthermore, our findings indicate that plasmin modulates disease activity in patients with FXII-HAE. Finally, we show that soluble lysine analogs attenuate this mechanism, explaining their therapeutic value in patients with HAE.

Conclusion: Our findings indicate a new pathway for bradykinin formation in patients with HAE, in which FXII is cleaved and activated by plasmin. This should lead to the identification of new markers for diagnosis and targets for treatment.
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http://dx.doi.org/10.1016/j.jaci.2016.02.021DOI Listing
November 2016

The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding.

Thromb Haemost 2016 07 7;116(1):87-95. Epub 2016 Apr 7.

Catherine P. M. Hayward, McMaster University Medical Centre, HSC 2N29A, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5, Tel.: +1 905 521 2100 Ext. 76274, Fax: +1 905 521 2338, E-mail:

Multimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbα binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.
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http://dx.doi.org/10.1160/TH15-09-0700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5175582PMC
July 2016

Strenuous exercise induces a hyperreactive rebalanced haemostatic state that is more pronounced in men.

Thromb Haemost 2016 06 11;115(6):1109-19. Epub 2016 Feb 11.

Dana Huskens, Oxfordlaan 70, Maastricht 6229EV, The Netherlands, Tel.: +31 43 388 58 96, Fax: +31 43 388 45 70, E-mail:

Physical exercise is recommended for a healthy lifestyle. Strenuous exercise, however, may trigger the haemostatic system, increasing the risk of vascular thrombotic events and the incidence of primary cardiac arrest. Our goal was to study the effects of strenuous exercise on risk factors of cardiovascular disease. Blood was collected from 92 healthy volunteers who participated in the amateur version of the pro-tour Amstel Gold cycling race, before and directly after the race. Thrombin generation showed a shortening of the lag time and time to peak and an increase of the velocity index. Interestingly, the endogenous thrombin potential measured in plasma decreased due to reduced prothrombin conversion. Platelet reactivity increased and this effect was stronger in men than in women. Lower fibrinogen and higher D-dimer levels after exercise indicated higher fibrin formation. On the other hand, fibrinolysis was also elevated as indicated by a shortening of the clot lysis time. Exercise activated the endothelium (von Willebrand factor (VWF) and active VWF levels were elevated) and the immune system (concentrations IL-6, IL-8, MCP-1, RANTES and PDGF increased). Additionally, an increased cardiac troponin T level was measured post-exercise. Strenuous exercise induces a temporary hyperreactive state in the body with enhanced pro- and anticoagulant responses. As strenuous exercise has a more pronounced effect on platelet function in male subjects, this gives a possible explanation for the higher incidence of sudden cardiac death during exercise compared to women. This trial is registered at www.clinicaltrials.gov as NCT02048462.
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http://dx.doi.org/10.1160/TH15-10-0821DOI Listing
June 2016

Shear-Resistant Binding to von Willebrand Factor Allows Staphylococcus lugdunensis to Adhere to the Cardiac Valves and Initiate Endocarditis.

J Infect Dis 2016 Apr 6;213(7):1148-56. Epub 2016 Jan 6.

Center for Molecular and Vascular Biology, KU Leuven, Belgium.

Background: Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow.

Methods: S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis.

Results: Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves.

Conclusions: S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci.
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http://dx.doi.org/10.1093/infdis/jiv773DOI Listing
April 2016

Keeping von Willebrand Factor under Control: Alternatives for ADAMTS13.

Semin Thromb Hemost 2016 Feb 23;42(1):9-17. Epub 2015 Nov 23.

Laboratory of Clinical Chemistry and Hematology, UMC Utrecht, Utrecht, The Netherlands.

Von Willebrand factor (VWF) is one of the most important proteins of the hemostatic system. Its multimeric state is essential for its natural function to guide platelets to sites of injury. ADAMTS13 is the key protease that regulates the multimeric state of VWF. Without ADAMTS13, VWF multimers can grow to pathologically large sizes. This is a risk factor for the life-threatening condition thrombotic thrombocytopenic purpura (TTP). In this condition, VWF-rich thrombi occlude the microvasculature of various tissues. Intriguingly, a complete ADAMTS13 deficiency does not cause continuous TTP, either in patients or genetically targeted mice. Instead, TTP occurs in episodes of disease, separated by extended periods of remission. This indicates that regulating factors beyond ADAMTS13 are likely involved in this pathologic cascade of events. This raises the question of what really happens when ADAMTS13 is (temporarily) unavailable. In this review, we explore the possible role of complementary mechanisms that are capable of modifying the thrombogenic potential of VWF.
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http://dx.doi.org/10.1055/s-0035-1564838DOI Listing
February 2016

Matrix metalloproteinase-2 enhances platelet deposition on collagen under flow conditions.

Thromb Haemost 2016 Jan 29;115(2):333-43. Epub 2015 Oct 29.

Paolo Gresele, MD, PhD, Section of Internal and Cardiovascular Medicine, Department of Medicine, University of Perugia, Via E. Dal Pozzo, 06126 Perugia, Italy, Tel.: +39 075 5783989, Fax: +39 075 5716083, E-mail:

Platelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O'Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i.e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, p<0.05) and increased retained platelets (from 72.3 ± 2.3% to 81.1 ± 1.8%, p<0.05) in the O'Brien system, an effect prevented by a specific MMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec⁻¹) (maximum +47.0 ± 11.9%, p<0.05, with 50 ng/ml), while pre-incubation with a MMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0% vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.
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http://dx.doi.org/10.1160/TH15-04-0300DOI Listing
January 2016

ApoE Receptor 2 Mediation of Trophoblast Dysfunction and Pregnancy Complications Induced by Antiphospholipid Antibodies in Mice.

Arthritis Rheumatol 2016 Mar;68(3):730-739

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.

Objective: Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine whether the low-density lipoprotein receptor family member apolipoprotein E receptor 2 (ApoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL.

Methods: Placental and trophoblast ApoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG and aPL were purified from healthy individuals and APS patients, respectively. The role of ApoER2 in aPL-induced changes in trophoblast proliferation and migration and in kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of ApoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant ApoER2(+/+) and ApoER2(-/-) mice injected with aPL or normal human IgG.

Results: We found that ApoER2 is abundant in human and mouse placental trophoblasts and in multiple trophoblast-derived cell lines, including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2 -glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by epidermal growth factor in trophoblasts was mediated by ApoER2. Furthermore, in a murine passive-transfer model of pregnancy complications of APS, ApoER2(-/-) mice were protected from both aPL-induced fetal loss and aPL-induced IUGR.

Conclusion: ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS.
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http://dx.doi.org/10.1002/art.39453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767551PMC
March 2016

Higher and lower active circulating VWF levels: different facets of von Willebrand disease.

Br J Haematol 2015 Dec 12;171(5):845-53. Epub 2015 Oct 12.

Department of Translational Research, Stem Cells Unit, IRCCS, C.R.O., Aviano, Italy.

Most circulating von Willebrand factor (VWF) is normally inactive and incapable of binding platelets, but numerous disorders may modify the proportion of active VWF. We explored active VWF levels in patients with von Willebrand disease (VWD) whose VWF had a higher affinity for platelet glycoprotein (GP)Ib, but different susceptibilities to ADAMTS13 and multimer patterns (9 patients lacking large multimers, 10 with a normal pattern); 12 patients with VWF C2362F and R1819_C1948delinsS mutations, which make VWF resistant to ADAMTS13 were also studied. Type 2B patients with abnormal or normal multimers had significantly more active VWF (3·33 ± 1·6 and 3·74 ± 0·74, respectively; normal 0·99 ± 0·23). The type of VWF mutation influenced VWF activation: V1316M was associated with the highest levels in patients with abnormal multimers, and R1341W in those with normal multimers. Pregnancy induced gradually rising active VWF levels and declining platelet counts in one type 2B VWD patient without large multimers. Active VWF levels dropped significantly in patients homozygous for the C2362F mutation or heterozygous for R1819_C1948delinsS mutations (0·2 ± 0·03 and 0·23 ± 0·1, respectively), and less in cases heterozygous for the VWF C2362F mutation (0·55 ± 0·17). We demonstrate that VWF may be more or less activated, with or without any direct involvement of the A1 domain, and regardless of ADAMTS13.
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http://dx.doi.org/10.1111/bjh.13785DOI Listing
December 2015

Platelet density per monocyte predicts adverse events in patients after percutaneous coronary intervention.

Thromb Haemost 2016 Jan 1;115(2):353-60. Epub 2015 Oct 1.

Dr. Bert Rutten, Department of Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, the Netherlands, E-mail:

Monocyte recruitment to damaged endothelium is enhanced by platelet binding to monocytes and contributes to vascular repair. Therefore, we studied whether the number of platelets per monocyte affects the recurrence of adverse events in patients after percutaneous coronary intervention (PCI). Platelet-monocytes complexes with high and low median fluorescence intensities (MFI) of the platelet marker CD42b were isolated using cell sorting. Microscopic analysis revealed that a high platelet marker MFI on monocytes corresponded with a high platelet density per monocyte while a low platelet marker MFI corresponded with a low platelet density per monocyte (3.4 ± 0.7 vs 1.4 ± 0.1 platelets per monocyte, P=0.01). Using real-time video microscopy, we observed increased recruitment of high platelet density monocytes to endothelial cells as compared with low platelet density monocytes (P=0.01). Next, we classified PCI scheduled patients (N=263) into groups with high, medium and low platelet densities per monocyte and assessed the recurrence of adverse events. After multivariate adjustment for potential confounders, we observed a 2.5-fold reduction in the recurrence of adverse events in patients with a high platelet density per monocyte as compared with a low platelet density per monocyte [hazard ratio=0.4 (95% confidence interval, 0.2-0.8), P=0.01]. We show that a high platelet density per monocyte increases monocyte recruitment to endothelial cells and predicts a reduction in the recurrence of adverse events in patients after PCI. These findings may imply that a high platelet density per monocyte protects against recurrence of adverse events.
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http://dx.doi.org/10.1160/TH15-03-0227DOI Listing
January 2016

Is asymptomatic malaria really asymptomatic? Hematological, vascular and inflammatory effects of asymptomatic malaria parasitemia.

J Infect 2015 Nov 21;71(5):587-96. Epub 2015 Aug 21.

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

Asymptomatic malaria infections are highly prevalent in malaria endemic regions and most of these infections remain undiagnosed and untreated. Whereas conventional malaria symptoms are by definition absent, little is known on the more subtle health consequences of these infections. The aim of our study was to analyze the hematologic, vascular and inflammatory effects of patent and subpatent asymptomatic malaria parasitemia in children and adults on the Indonesian island Sumba. Both children and adults with parasitemia had increased high-sensitive C-reactive protein levels compared to aparasitemic individuals. In addition, children, but not adults with parasitemia also had lower platelet counts and Hb levels and higher levels of von Willebrand factor and platelet factor-4, markers of endothelial and platelet activation, respectively. These findings suggest that asymptomatic malaria infections have subtle health consequences, especially in children, and should be regarded as potentially harmful.
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http://dx.doi.org/10.1016/j.jinf.2015.08.005DOI Listing
November 2015

Antiphospholipid Syndrome--Not a Noninflammatory Disease.

Semin Thromb Hemost 2015 Sep 15;41(6):607-14. Epub 2015 Aug 15.

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

The autoimmune disease antiphospholipid syndrome (APS) is characterized by thrombosis or pregnancy morbidity in patients with persistent antiphospholipid antibodies (aPLs). Although inflammation is not a key feature of the clinical presentation of the syndrome, there are indications that the inflammatory response plays an important role in APS. The major antigen of aPLs, the plasma protein β2-glycoprotein I, is involved in clearance of microparticles and in the innate immune response. In light of these physiological functions, the formation of antibodies against the protein is easily understood, as antibodies might augment the clearance reaction. In addition, inflammatory mediators are thought to play a role in the activation of leukocytes and the induction of endothelial dysfunction in APS. Moreover, evidence for a role of complement activation in the pathogenesis of the syndrome is accumulating. This review will provide an overview of current knowledge on the physiological function of β2-glycoprotein I, the formation of autoantibodies against β2-glycoprotein I and will explore the contribution of inflammation to the clinical manifestations of APS.
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http://dx.doi.org/10.1055/s-0035-1556725DOI Listing
September 2015

Differential effects of platelets and platelet inhibition by ticagrelor on TLR2- and TLR4-mediated inflammatory responses.

Thromb Haemost 2015 May 26;113(5):1035-45. Epub 2015 Feb 26.

Rahajeng Tunjungputri, MD, Department of Internal Medicine (463), Radboud university medical center, PO Box 9101, 6500 HB Nijmegen, The Netherlands, Tel: +31 24 3618822, Fax: +31 24 3566336, E-mail:

Platelets and platelet-monocyte interaction play an important role in inflammation. Both pro- and anti-inflammatory effects of platelet inhibition have been reported in animal models. This study aimed to investigate the effect of platelets and platelet inhibition by the new P2Y12 receptor antagonist ticagrelor on monocyte function, as assessed by cytokine responses to Toll-like Receptor (TLR) ligands. In a set of in vitro experiments, peripheral blood mononuclear cells (PBMC) incubated with the TLR2 ligand Pam3CSK4 produced less cytokines in the presence of platelets, whereas platelets increased the production of cytokines when PBMC were exposed to TLR4 ligand lipopolysaccharide (LPS). These effects of platelets were dependent on direct platelet-leukocyte aggregation and for the Pam3CSK4-induced response, on phagocytosis of platelets by monocytes. In a double blind, placebo-controlled crossover trial in healthy volunteers, a single oral dosage of 180 mg ticagrelor reduced platelet-monocyte complex (PMC) formation. This was associated with an increase in pro-inflammatory cytokines in blood exposed to Pam3CSK4, but a decrease in these cytokines in blood exposed to LPS. These findings show that platelets differentially modulate TLR2- and TLR4-mediated cytokine responses of PBMC. Through inhibition of platelet-leukocyte interaction, P2Y12 receptor antagonists may either exert a pro- or anti-inflammatory effect during infections depending on the TLR primarily involved.
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http://dx.doi.org/10.1160/TH14-07-0579DOI Listing
May 2015

The role of cell surfaces and cellular receptors in the mode of action of recombinant factor VIIa.

Blood Rev 2015 Jul 23;29(4):223-9. Epub 2014 Dec 23.

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

Recombinant factor VIIa (rFVIIa) has been developed to treat bleeding episodes in patients with inhibitor-complicated hemophilia. More recently, it has become apparent that rFVIIa is also useful in a prophylactic setting, although its prophylactic effect with a once-daily administration is difficult to explain given its half life of ~2h. The prohemostatic effects of rFVIIa have been ascribed to enhancement of thrombin generation and various downstream effects thereof. There is an ongoing debate on the tissue factor-dependency of rFVIIa, but accumulating evidence is in favor of a TF-independent mechanism. rFVIIa interacts with cellular surfaces and with various receptors on vascular cells. These interactions may contribute significantly to its mode of action in patients with hemophilia. In this review we will summarize laboratory and clinical evidence on the mode of action of rFVIIa in hemophilia with an emphasis on recent insights regarding the interactions of rFVIIa with cellular receptors.
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http://dx.doi.org/10.1016/j.blre.2014.12.004DOI Listing
July 2015

Coagulation activation during air travel is not initiated via the extrinsic pathway.

Br J Haematol 2015 Jun 17;169(6):903-5. Epub 2014 Dec 17.

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

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http://dx.doi.org/10.1111/bjh.13257DOI Listing
June 2015

Annexin A5 haplotypes in the antiphospholipid syndrome.

Thromb Res 2015 Feb 5;135(2):417-9. Epub 2014 Dec 5.

Department of Laboratory Medicine, Laboratory of Hematology, Radboudumc, Nijmegen, the Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.thromres.2014.12.004DOI Listing
February 2015

Contact system activation on endothelial cells.

Semin Thromb Hemost 2014 Nov 11;40(8):887-94. Epub 2014 Nov 11.

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

When the contact system assembles and activates on negatively charged surface materials, plasma coagulation rapidly follows. This mechanism is redundant for hemostasis but mediates pathological thrombus formation, as was reported in a multitude of in vivo studies. The epidemiological data are presently scarce to firmly support a role for the contact system in human thrombotic disease, while its physiological function and mode of activation remains mysterious. Besides its role in blood coagulation in vitro, the contact system is responsible for the production of bradykinin. This inflammatory peptide is involved in episodes of pathological tissue swelling in (hereditary) angioedema, but potentially also in the physiological regulation of vascular permeability. A body of evidence indicates that contact system factors are recruited to the surface of activated endothelial cells, where proteins that are locally released can activate them. Furthermore, clinical and biochemical studies indicate that plasmin, the effector enzyme of the fibrinolytic system, can evoke contact system activation. This auxiliary role for plasmin may so far not have been fully appreciated in pathophysiology. To conclude this review, we propose a complementary model for contact system activation on the endothelial cell surface that is initiated by plasmin activity.
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http://dx.doi.org/10.1055/s-0034-1395159DOI Listing
November 2014

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network.

J Biol Chem 2014 Dec 7;289(52):35979-86. Epub 2014 Nov 7.

From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands, Synapse BV, 6229 EV, Maastricht, The Netherlands.

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.
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http://dx.doi.org/10.1074/jbc.M114.591677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276865PMC
December 2014

Endocannabinoids control platelet activation and limit aggregate formation under flow.

PLoS One 2014 29;9(9):e108282. Epub 2014 Sep 29.

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

Background: The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.

Objectives: Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.

Methods And Results: We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.

Conclusions: Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108282PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180465PMC
November 2015