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®.

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.

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.

scholarly journals The Low Von Willebrand Factor in Ireland Cohort Study - Defining Optimal Management for Procedures in Patients with Low VWF (30-50 IU/dL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1178-1178
Author(s):  
Dearbhla Doherty ◽  
Anjali Patel ◽  
Margaret Nolan ◽  
Mary Byrne ◽  
Sonia Aguila ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Research Funding ◽  
Clotting Factor ◽  
Bleeding Complications ◽  
Major Surgery ◽  
Normal Range ◽  
Normal Ranges ◽  
Significant Difference ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Recent international consensus guidelines recommend that patients with significant bleeding phenotypes and plasma VWF levels in the 30-50 IU/dL range should be classified as Low von Willebrand factor (Low VWF). Critically however, evidence-based guidelines regarding optimal clinical management strategies for this cohort have not been defined. In particular, data regarding the need for hemostatic cover for procedures in Low VWF are limited. To address this deficit, as part of the Low Von Willebrand in Ireland Cohort (LoVIC) study we conducted a systematic retrospective review of all procedures in a large cohort of well characterised patients with Low VWF. Methods: Following written informed consent, we collated data on all procedures performed in LoVIC patients over a 17 year period (1/1/00-12/31/17). Case notes were reviewed and data collected in a standard proforma - age at intervention, choice of hemostatic cover, perioperative VWF, haemoglobin (Hgb) levels and reported bleeding. Procedures were categorised as dental extractions (DE), minor (e.g. endoscopies, joint injections) or major surgery. Tranexamic acid (TA) was prescribed 1g TDS. All desmopressin (DDAVP) was administered intravenously (IV, 0.3mg/kg). Results: 165 procedures in 65 LoVIC participants were identified. The procedural group was significantly older (median 45 vs 38.8 years, p<0.0001) and with a longer duration of Low VWF (9.0 vs 6.5 years, p=0.02) compared to the rest of the cohort. The majority of procedures were either minor (69.1%) or DEs (24.2%), with only 11 major procedures. Overall, 41 (24.8%) were covered with TA alone; 103 (62.4%) with DDAVP+/- TA; clotting factor concentrate (CFC) was used in only 4 procedures. Dental extractions: DDAVP+/-TA was used in 72.5% of DEs with no excess bleeding. Of the 10 DE with only TA, 4 developed bleeding requiring treatment (DDAVP/suturing/TA). On comparison of the DDAVP/TA and TA groups, no significant difference in ISTH BAT (median 9 vs 7, p=0.3) or plasma VWF levels (Table 1) at time of procedure was seen. However, the TA only cohort were significantly older (median 55.1 vs 29.3 years, y, p=0.0002), influencing treatment choice. Of interest, in the 4/10 with TA only who bled, pre-DE plasma VWF levels were close to or within the normal range (VWF:RCo 47- 79 IU/dL). Minor procedures: Of the 114 minor procedures, 45.6% (52) were endoscopies, 31 with biopsy. The other 62 procedures were dermatological (19), orthopaedic (19), gynecological (8) or other (e.g. minor biopsies, 14). 17 procedures with low bleeding risk were performed with no hemostatic cover independent of the coagulation service. Excessive bruising was reported in 2/17 procedures, both patients were older (54 and 70 years) with pre-op plasma VWF levels within the normal range. For the remaining 97 procedures, 3 patients had CFC cover (unsuitable for DDAVP); 68 DDAVP/TA and 26 received only TA. Age contributed to prophylaxis choice, with TA cohort significantly older than the DDAVP/TA group (median 65.6 vs 42.5y, p=0.0004). However, phenotype also influenced with ISTH BAT scores significantly lower in the TA only cohort (median 3 vs 10.5, p<0.0001). No bleeding or significant change in perioperative Hgb levels were reported in any procedure covered with prophylaxis. Major : Data on major procedures were limited to 11 procedures covered with TA or DDAVP/TA. Plasma VWF levels were maintained following a single dose of DDAVP in all cases. A significant reduction in post-op Hgb levels was observed in TA only patients but no significant bleeding occurred (Table 1). The TA group were significantly older than DDAVP/TA patients (54.8 vs 33.6,p=0.03) but no significant difference in ISTH BAT score, pre-operative plasma VWF or Hgb levels or inpatient stay was seen, although numbers are limited. Conclusion: This study represents the largest analysis of procedural outcomes in patients with Low VWF to date. Our data suggest that age and ISTH BAT score significantly influence choice of hemostatic prophylaxis. Bleeding complications were observed only in LoVIC patients undergoing DE covered by TA, or with minor procedures with no hemostatic cover. Interestingly, patients who developed bleeding were typically older (median 59.7y) and, critically in this subgroup, bleeding occurred despite that plasma VWF levels had corrected to within the normal range, clearly raising the possibility that age-corrected normal ranges may need to be considered. Table Table. Disclosures Lavin: Shire: Honoraria, Research Funding, Speakers Bureau. O'Donnell:Bayer: Research Funding, Speakers Bureau; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Leo Pharma: Speakers Bureau; Octapharma: Speakers Bureau; CSL Behring: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Research Funding.

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.

Abnormal Structure of von Willebrand Factor in Myeloproliferative Syndrome Is Associated to Either Thrombotic or Bleeding Diathesis

1987 ◽  
Vol 58 (02) ◽  
pp. 753-757 ◽  
Author(s):  
M F López-Fernández ◽  
C López-Berges ◽  
R Martín ◽  
A Pardo ◽  
F J Ramos ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Relative Proportion ◽  
Variable Degree ◽  
Bleeding Diathesis ◽  
Myeloproliferative Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the “in vivo” proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed a relative increase in the 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of approximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearance of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase of the 176 kDa fragment. These data point to some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF occurring “in vivo” rather than “in vitro”, and which may be associated to either a thrombotic or a bleeding diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent to what is described in normal individuals.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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