Platelet and endothelial activation in catastrophic and quiescent antiphospholipid syndrome

2013 ◽  
Vol 109 (05) ◽  
pp. 901-908 ◽  
Author(s):  
Agnese Bontadi ◽  
Emanuela Falcinelli ◽  
Silvia Giannini ◽  
Alessandro Marturano ◽  
Marta Tonello ◽  
...  
Keyword(s):  
Antiphospholipid Syndrome ◽  
Ex Vivo ◽  
Endothelial Activation ◽  
Monocyte Chemoattractant Protein 1 ◽  
Glycoprotein I ◽  
In Vitro Effects ◽  
Von Willebrand ◽  
Cell Adhesion Molecule 1 ◽  
Willebrand Factor

SummaryAntiphospholipid antibodies (aPL) seem to induce a prothrombotic state by activating endothelium and platelets, but no studies have evaluated systematically the effects of aPL from patients with the antiphospholipid syndrome (APS) in quiescent versus catastrophic phase. Our aims were to evaluate the in vitro effects on platelet activation of anti-β2 glycoprotein I (anti-β2GPI) antibodies isolated from APS patient in either quiescent or catastrophic phase and to investigate ex vivo platelet and endothelial activation in patients with quiescent or catastrophic APS. Anti-β2GPI antibodies were isolated from plasma of a pregnant woman in two different stages of APS (quiescent and catastrophic, respectively). They were co-incubated with washed platelets from healthy controls that were then challenged with TRAP-6 (thrombin receptor activating peptide 6) and the expression of P-selectin (P-sel) on platelets was assessed by flow cytometry. Moreover, plasma samples from six patients with quiescent, four with catastrophic APS and 10 controls were assessed for several markers of platelet and endothelial activation. The results showed that purified anti-β2GPI antibodies co-incubated with platelets enhanced TRAP-6-induced platelet P-sel expression. Notably, anti-β2GPI antibodies isolated during the catastrophic phase enhanced platelet P-sel expression more than antibodies isolated from the same patient in the quiescent stage of disease. Moreover, APS patients had significantly higher plasma levels of soluble (s) Psel, sCD40 ligand, soluble vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1 than control subjects. In addition, sP-sel and von Willebrand factor activity were significantly higher during catastrophic than in quiescent phase.

Significance of Platelet β-Adrenoceptors for Platelet Responses In Vivo and In Vitro

1992 ◽  
Vol 68 (06) ◽  
pp. 687-693 ◽  
Author(s):  
P T Larsson ◽  
N H Wallén ◽  
A Martinsson ◽  
N Egberg ◽  
P Hjemdahl
Keyword(s):  
Ex Vivo ◽  
High Dose ◽  
Platelet Aggregability ◽  
Adrenoceptor Agonist ◽  
Dose Infusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThe significance of platelet β-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the β-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed α- and β-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma β-thromboglobulin levels. Adrenaline levels were 3–4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels β-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that β-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an α-adrenoceptor mediated proaggregatory action.

Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4486-4493 ◽  
Author(s):  
Gregor Theilmeier ◽  
Carine Michiels ◽  
Erik Spaepen ◽  
Ingrid Vreys ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

Toxico-Kinetics of Recombinant VWF In Non-Human Primates and Rabbits

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1414-1414
Author(s):  
Eva-Maria Muchitsch ◽  
Barbara Dietrich ◽  
Hanspeter Rottensteiner ◽  
Herbert Gritsch ◽  
Hartmut J. Ehrlich ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Dependent Manner ◽  
Cynomolgus Monkeys ◽  
Toxicological Effect ◽  
Safety Margins ◽  
Hemostatic Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Kinetics Of

Abstract Abstract 1414 Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. We showed previously by in vitro and ex vivo studies that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine and rat ADAMTS13, whereas ADAMTS13 from rabbits and cynomolgus monkeys is able to cleave human rVWF. Here we tested the pharmacological behavior of human rVWF in rabbits and cynomolgus monkeys. The animals were infused once with different doses of human rVWF. VWF antigen rose sharply in a dose-dependent manner (∼25 IU/ml VWF:Ag for the highest dose, 15 min after injection) and then declined gradually (∼7 IU/ml VWF:Ag for the highest dose, 18 hours after injection). By contrast, the ADAMTS13 activity did not show relevant changes throughout the entire test period in the rabbit or in the monkey samples, indicating that an excess of intravenously administered rVWF as the substrate does not exhaust its enzyme ADAMTS13. Most importantly, neither rabbits nor cynomolgus monkeys showed any exaggerated pharmacological or toxic effects upon rVWF administration. Even the administration of 14 daily doses of rVWF to cynomolgus monkeys did not lead to any toxicological effect. Our data indicate that rabbits and cynomolgus monkeys, two species with a sufficient rVWF cleavage capacity by endogenous ADAMTS13, are appropriate animal models for a meaningful preclinical evaluation of the rVWF product, which allows the therapeutic safety margins for human patients to be determined. Disclosures: Muchitsch: Baxter Innovations GmbH: Employment. Dietrich:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment.

scholarly journals Ex Vivo Evaluation of the Effect of Plasma-Derived Factor VIII/Von Willebrand Factor in Patients with Severe Hemophilia_A on Prophylaxis with Emicizumab By Thrombin Generation Assay

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4233-4233
Author(s):  
Maria-Isabel Bravo ◽  
Aida Raventós ◽  
Alba Pérez ◽  
Elena G Arias-Salgado ◽  
María Teresa Alvarez Román ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Research Funding ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Concomitant Treatment ◽  
Healthy Controls ◽  
Normal Range ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Introduction: Hemophilia A (HA) patients under emicizumab prophylaxis treatment may require the concomitant use of procoagulant factors for breakthrough bleedings or immune tolerance induction. Thromboembolic events have been described with the concomitant use of emicizumab and activated prothrombin complex concentrate (aPCC), but not with recombinant activated factor VII (rFVIIa). Previous studies showed that the in vitro combination of emicizumab and plasma-derived Factor VIII/Von Willebrand Factor (pdFVIII/VWF) had a non-additive effect on thrombin generation (TG)(Bravo M-I, et al J Thromb Haemost. 2020;18:1934-39). The aim of this study was to evaluate the TG resulting from ex vivo combination of plasma samples from HA patients treated with emicizumab, with a pdFVIII/VWF concentrate (Fanhdi ®, Grifols). Methods: Twelve adult patients with severe HA without inhibitors on prophylaxis with emicizumab and nine healthy controls were included in the study. Blood samples were drawn in citrate plus corn trypsin inhibitor tubes. Then, platelet poor plasma (PPP) was collected for the TG assay, which measures the whole kinetics of TG. Thrombin peak (TP) and endogenous thrombin potential (ETP) were calculated using calibrated automated thrombogram (Thrombinoscope ™ software, Stago) after in vitro activation of coagulation by trigger solution, PPP Reagent LOW TM (4 μM phospholipids/1 pM tissue factor), fluorogenic substrate and CaCl 2 (FLUKAkit TM) reagents (Diagnostica Stago). Fluorescence was read in a Fluoroskan Ascent reader (Thermo) equipped with a 390/460 filter set. Samples were spiked with increasing concentrations of pdFVIII/VWF (10 to 400 IU/dL), rFVIIa (0.9 µg/mL) or aPCC (0.5 U/mL). Results: TG from healthy control samples was measured to establish TP and ETP normal ranges. TP and ETP results obtained from HA plasma with emicizumab were lower than in healthy controls. The addition of pdFVIII/VWF as of 25 IU/kg (prophylaxis dose in HA w/o inhibitors) to samples from HA patients concomitantly treated with emicizumab restored TP and ETP levels within healthy controls normal range (Table 1). Increasing ex vivo concentrations of pdFVIII/VWF maintained TP and ETP similar to healthy controls. The highest concentration of concomitant treatment with pdFVIII/VWF (200 IU/kg) and emicizumab did not result in excessive TP and, importantly, ETP levels were always within the normal range. The combination with the bypassing agent rFVIIa moderately increased TP and ETP values up to normal range. However, when HA plasma was spiked with aPCC in the presence of emicizumab, TP and ETP dramatically increased above normal range resulting in a synergistic procoagulant profile. Conclusions: The concomitant use of pdFVIII/VWF in patients with prophylaxis with emicizumab did not trigger a multiplying effect on TG. These results were aligned with previous in vitro data and suggested the low risk of overdose and thrombotic events of concomitant treatment emicizumab with the pdFVIII/VWF concentrate in HA patients. Figure 1 Figure 1. Disclosures Bravo: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Raventós: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Pérez: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Butta: CSL-Behring: Research Funding; Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novo-Nordisk: Speakers Bureau. Jiménez-Yuste: Bayer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Costa: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Willis: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®.

Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

1995 ◽  
Vol 73 (02) ◽  
pp. 318-323 ◽  
Author(s):  
K Azzam ◽  
L I Garfinkel ◽  
C Bal dit Sollier ◽  
M Cisse Thiam ◽  
L Drouet
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Mesenteric Arteries ◽  
Antithrombotic Effects ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

Decreased beta2-Glycoprotein I Plasma Levels as a Risk Factor for Myocardial Infarction in Men.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1813-1813
Author(s):  
Bas De Laat ◽  
Philip G. de Groot ◽  
Ronald H.W.M. Derksen ◽  
Rolf T. Urbanus ◽  
Koen Mertens ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Confidence Interval ◽  
Von Willebrand Factor ◽  
Plasma Levels ◽  
Odds Ratio ◽  
Arterial Thrombosis ◽  
Glycoprotein I ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: Several factors influence the occurrence of acute myocardial infarction. One of these factors is thought to be Von Willebrand Factor which serves as adhesive surface for platelets to adhere to the vessel wall. We have recently found that beta2- glycoprotein I is able to inhibit platelet binding to von Willebrand Factor by binding to the A1 domain of Von Willebrand Factor 1. This could indicate that beta2-glycoprotein I possesses antithrombotic properties with respect to arterial thrombosis. In the present study we investigated whether differences in beta2-glycoprotein I plasma levels influence the risk of myocardial infarction. Methods and Results: We have measured beta2-glycoprotein I and Von Willebrand Factor antigen levels in 539 men with a first myocardial infarction and in 611 control subjects who participated in the case-control Study of Myocardial Infarction Leiden (SMILE). Although we did not find a profound effect of beta2-glycoprotein I plasma levels on myocardial infarction in the overall population (odds ratio 0.93, 95% confidence interval 0.65–1.33), there appeared to be a dose-dependent protective effect of increasing beta2-glycoprotein I plasma levels on myocardial infarction in men of 60 years and older. In this age group we found an odds Ratio of 0.44 (95% confidence interval 0.25–0.77) for high beta2-glycoprotein I levels compared to low levels. Furthermore, high plasma levels of beta2-glycoprotein I remained protective for myocardial infarction despite high levels of Von Willebrand Factor. In addition, we studied a possible association between age and Von Willebrand Factor and beta2-glycoprotein I plasma levels. It appeared that both Von Willebrand Factor and beta2-glycoprotein I plasma levels increased with age, but a larger increase in Von Willebrand Factor plasma levels was observed than in beta2-glycoprotein I plasma levels (13.7 % every 10 years versus 5.7% every 10 years). Conclusions: In this study high circulating levels of beta2-glycoprotein I appeared to be associated with a lower risk of myocardial infarction in men over 60 years. In addition we observed a larger increase in Von Willebrand Factor levels with age than beta2- glycoprotein I levels. As beta2-glycoprotein I possesses antithrombotic properties by inhibiting the activity of Von Willebrand Factor in-vitro, this might indicate that during aging the haemostatic balance slowly shifts to a more prothrombotic state 1. Future in-vivo experiments are needed to investigate the exact contribution of beta2-glycoprotein I on the pathophysiology of myocardial infarction and arterial thrombosis in general.

scholarly journals Acute von Willebrand Factor Secretion from the Endothelium In Vivo: Assessment through Plasma Propeptide (vWf:AgII) Levels

1997 ◽  
Vol 77 (02) ◽  
pp. 387-393 ◽  
Author(s):  
Ulrich M Vischer ◽  
Jørgen Ingerslev ◽  
Claes B Wollheim ◽  
Jean-Claude Mestries ◽  
Dimitrios A Tsakiris ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Time Course ◽  
Plasma Concentrations ◽  
Vascular Wall ◽  
Endothelial Activation ◽  
Relative Increase ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryElevated plasma concentrations of von Willebrand factor (vWf) are increasingly recognized as a cardiovascular risk factor, and are used as a marker of endothelial activation. However, the factors which determine the rate of vWf release from the endothelium in vivo have not been defined clearly. In addition, vWf plasma levels may also be influenced by adhesion of vWf to the vascular wall or to platelets, and by its rate of degradation. The propeptide of vWf (also called vWf:AgII) is stored and released in equimolar amounts with vWf. In the present study we attempted to determine whether this propeptide could be a more reliable marker of endothelial secretion than vWf itself. To accomplish this we developed an ELISA based on monoclonal antibodies. The propeptide levels in normal plasma were found to be 0.7 µg/ml, more than 10 times lower than vWf itself. Administration of desmopressin (DDAVP) induced a rapid relative increase in propeptide (from 106 to 879%) and in vWf (from 112 to 272%). However, the increases in vWf and propeptide were equivalent when expressed in molar units. A time course study indicated a half-life of the propeptide of 3 h or less. In a baboon model of disseminated intravascular coagulation (DIC) induced by FXa, vWf increased by less than 100%, whereas the propeptide concentrations increased by up to 450%. In view of the massive thrombin generation (as assessed by fibrinogen depletion), the increases in vWf are small, compared to the strong secretory response to thrombin and fibrin previously observed in vitro. Our results suggest that due to its rapid turnover, the propeptide could provide a sensitive plasma marker of acute endothelial secretion.

Elucidation of Mild Bleeding Disorders Reported Under Ibrutinib (Imbruvica(R)) Therapy: Implications for Optimal Clinical Management

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3296-3296 ◽  
Author(s):  
Loic Ysebaert ◽  
Marie Levade ◽  
Garcia Cedric ◽  
Anne-Sophie Michallet ◽  
Constantine Tam ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Ex Vivo ◽  
Bleeding Risk ◽  
High Shear ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Platelet Functions

Abstract Introduction Ibrutinib is the first-in-class covalent inhibitor of Bruton's Tyrosine Kinase (BTK), now approved for the therapy of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Mild bleeding disorders (grade 1-2) have been reported in 44-60% of patients across clinical trials, with <5% grade 3 hemorrhages after trauma. After vascular injury, platelets adhere onto von Willebrand factor (vWF, through GPIb-IX-V complex) and collagen (through a2b1 and GPVI receptor), and activate phospholipase Cg2 (PLCg2) through BTK phosphorylation. In this study, we sought to examine phosphorylation pathways and platelet functions in vitro and ex vivo from ibrutinib-treated patients. Patients and Methods Within the compassionate access program of ibrutinib in France (started Feb 2014), we investigated whether ibrutinib could impact on platelet functions in vitro and ex vivo, as measured at day 0 and day 15-30 by: aggregometry using various agonists, measurement of intra-cellular levels of phosphorylation of BTK and PLCg2 phosphorylations, monitoring adhesion onto vWF matrix under high shear rate. We next assess how in vitro tests could help identify bleeding risk in a larger cohort of patients from three institutions. Results First, we demonstrated that in healthy donors' platelets, ibrutinib inhibits collagen and collagen related peptide (CRP) -induced platelet aggregation in a dose-dependent manner (mean EC50=250nM, a dose achievable in patients). This effect was paralleled by the inhibition of PLCg2 phosphorylation on the Btk-dependent phosphorylation site Tyr753, and of the auto-phosphorylation Tyr223 site of BTK itself, suggesting a specific targeting by ibrutinib. Of note, adhesion on vWF under high shear rate was dramatically decreased. In parallel, in 7/14 patients had bleeding symptoms (5/7 with grade 1-2 bleedings) and they all presented a strong inhibition of platelet aggregation in response to collagen and a significant decrease in adhesion onto vWF. Thus, the easy-to-use collagen-induced platelet aggregation test in platelet rich plasma could help physicians to decide when to perform surgical procedures without haemostasis concerns. Moreover, we show that addition of 50% untreated platelets is sufficient to efficiently reverse the effects of ibrutinib, and that platelet functions recover following treatment interruption as physiological platelet renewal occurs, supporting the in vitro data. On the other hand, patients who received aspirin (n=6) had no cases of severe bleeding and no significant impact on collagen/CRP-induced platelet aggregation. Because aspirin+P2Y12 inhibitors (such as clopidogrel, Plavix®) is widely used in the elderly population, ibrutinib therapy should be given very cautiously to these patients (who receive then three major pathway platelet activation pathway inhibitor), as recommended for vitamin K antagonists drugs. Aggregometry tests may provide important information to physicians to predict the bleeding risk as observed in our cohort of >30 patients (as of June 2014, recruitment still ongoing). Two last points should be emphasized when considering bleeding risk of ibrutinib: (i) from our study, some patients had no anti-platelet detectable effect ex vivo under ibrutinib therapy, the mechanism of which still remains unclear, and (ii) in patients with mild bleedings, platelet functions recovery and cessation of symptoms occured in virtually all patients (except those on aspirin therapy) after 3-6 months despite ongoing lymphoma responses, suggesting a potential adaptative process in platelets. Summary and Conclusion We identified that ibrutinib affects collagen and Von Willebrand Factor-mediated platelet activation in vitro and ex vivo. The mild bleeding diathesis observed in a subgroup of ibrutinib-treated patients correlates with defects in collagen-induced platelet aggregation and platelet adhesion on von Willebrand Factor at high shear rate. Based on in vitro analyses and in vivo platelet turnover, 2-3 days ibrutinib cessation appears to be enough for effective aggregation response recovery, and reintroduction of the drug should be rapid to avoid disease recurrence. Our study also suggests that platelet transfusion at a dose sufficient to get 50 % of fresh platelets may correct haemostasis in emergency, provided it was given after elimination of ibrutinib from blood (4-6h). Disclosures Tam: Pharmacyclics and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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