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Von Willebrand Factor (VWF) and Factor VIII (FVIII) circulate in a noncovalent complex in blood and promote primary haemostasis and clotting respectively. A new VWF A1-domain binding aptamer, BT200, demonstrated good subcutaneous bioavailability and a long half-life in non-human primates. This first-in-human, randomised, placebo-controlled, double-blind trial tested the hypothesis that BT200 is well tolerated and has favourable pharmacokinetic and pharmacodynamic effects in 112 volunteers. Participants received one of the following: Single ascending dose of BT200 (0.18-48mg) subcutaneously, an intravenous dose, BT200 with concomitant desmopressin or multiple doses. Pharmacokinetics were characterised, and the pharmacodynamic effects were measured by VWF levels, FVIII clotting activity, ristocetin induced aggregation, platelet function under high shear rates, and thrombin generation. Mean half-lives ranged from 7-12 days and subcutaneous bioavailability increased dosedependently exceeding 55% for doses of 6-48 mg. By blocking free A1 domains, BT200 dose-dependently decreased ristocetin-induced aggregation, and prolonged collagenadenosine diphosphate and shear-induced platelet plug formation times. However, BT200 also increased VWF antigen and FVIII levels 4-fold (p

Type 2B von Willebrand disease (VWD) is characterised by an increased binding affinity of von Willebrand factor (VWF) to platelet glycoprotein Ib. This can lead to clearance of high molecular weight (HMW) multimers and thrombocytopenia with a resulting moderate-severe bleeding phenotype. Rondoraptivon pegol (BT200) is a pegylated aptamer binding to the A1 domain of VWF with a novel mechanism of action: it enhances VWF/FVIII levels by decreasing their clearance. To study the potential benefit of rondoraptivon pegol in type 2B VWD patients, we conducted a prospective phase II trial. Type 2B VWD patients received 3mg rondoraptivon pegol subcutaneously on study days 1, 4 and 7, followed by 6-9 mg every week until day 28. Five patients (M:F=3:2) were included (median age: 61 years [range: 24-72]. Rondoraptivon pegol rapidly increased platelet counts, which rose from median 60x10E9/L (41-167) to 179x10E9/L (89-264; p<0.001) and normalized in 3 of 4 thrombocytopenic patients. Circulating VWF antigen increased from median 64% (32-106%) to 143% (103-351%, p<0.001) which doubled FVIII activity levels from 67% (44-91%) to 134% (114-200%; p=0.002). In all thrombocytopenic patients, plasma levels of VWF:GPIbM normalized, VWF ristocetin cofactor and VWF collagen-binding activity increased (p<0.01), and HMW multimers appeared. These pronounced improvements reversed during wash-out of the drug, thus demonstrating causality. The A1 domain binding aptamer directly corrects the underlying defect of type 2B VWD, thus providing a novel potential option for prophylaxis and treatment of patients with this VWD type. These data provide the basis for a phase IIb/III trial in such patients. This trial is registered at www.clinicaltrials.gov as NCT04677803.

Von Willebrand factor (VWF) plays a major role in arterial thrombosis. Antiplatelet drugs induce only a moderate relative risk reduction after atherothrombosis, and their inhibitory effects are compromised under high shear rates when VWF levels are increased. Therefore, we investigated the ex vivo effects of a third-generation anti-VWF aptamer (BT200) before/after stimulated VWF release. We studied the concentration-effect curves BT200 had on VWF activity, platelet plug formation under high shear rates (PFA), and ristocetin-induced platelet aggregation (Multiplate) before and after desmopressin or endotoxin infusions in healthy volunteers. VWF levels increased > 2.5-fold after desmopressin or endotoxin infusion (p < 0.001) and both agents elevated circulating VWF activity. At baseline, 0.51 µg/ ml BT200 reduced VWF activity to 20% of normal, but 2.5-fold higher BT200 levels were required after desmopressin administration (p < 0.001). Similarly, twofold higher BT200 concentrations were needed after endotoxin infusion compared to baseline (p < 0.011). BT200 levels of 0.49 µg/ml prolonged collagen-ADP closure times to > 300 s at baseline, whereas 1.35 µg/ml BT200 were needed 2 h after desmopressin infusion. Similarly, twofold higher BT200 concentrations were necessary to inhibit ristocetin induced aggregation after desmopressin infusion compared to baseline (p < 0.001). Both stimuli elevated plasma VWF levels in a manner representative of thrombotic or pro-inflammatory conditions such as arterial thrombosis. Even under these conditions, BT200 potently inhibited VWF activity and VWF-dependent platelet function, but higher BT200 concentrations were required for comparable effects relative to the unstimulated state. Von Willebrand factor (VWF) is driving the first step and is a key component in platelet thrombus formation when vascular injury occurs under conditions of moderate to high shear force 1,2. High shear force is commonly found in stenotic arteries and it is known to cause myocardial infarction 3 or stroke 4,5. It has been shown previously that plasma levels of VWF are predicting major adverse cardiovascular events in patients with asymptomatic carotid stenosis 6 , as well as in those with acute coronary syndrome 7. At high shear rates, the main mediator of platelet plug formation is VWF, which is why inhibitors of GPIIb/IIIa, P2Y12 receptor or cyclooxygenase-1 are less potent in conditions where VWF levels are increased 8-10. Moreover, those antiplatelet drugs typically only produce a limited relative risk reduction in some patients groups such as those with acute coronary syndrome or patients undergoing percutaneous coronary intervention (PCI) 11. On one end VWF binds to platelets via GpIb, and on the other end it binds to collagen and forms a bridge between platelets and collagen. BT200 is a third-generation aptamer inhibitor of the A1 domain of VWF and prevents VWF from binding to platelet GPIb 12. BT200 has low nanomolar K d for VWF 12 and has previously been proven to be effective in human

Defibrotide is approved for the treatment of sinusoidal obstruction syndrome after allogeneic stem cell transplantation. The exact mode of action of defibrotide is unclear and human in vivo data are scarce. In this randomized, double blind, crossover trial we included 20 healthy volunteers. Four were randomized to receive placebo, while 16 received a 2 ng/kg bodyweight bolus of lipopolysaccharide (LPS). Infusion of 6.25 mg/kg defibrotide or placebo was started one hour before the injection of the LPS bolus. Plasma levels of prothrombin fragments F1 + 2, thrombin-antithrombin complexes, von Willebrand factor, E-selectin, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), plasmin-antiplasmin complexes (PAP), tumor necrosis factor-α, interleukin 6, and C-reactive protein were measured. Thromboelastometry was performed. Infusion of defibrotide did not reduce the LPS-induced activation of coagulation, the endothelium or the release of pro-inflammatory cytokines. However, defibrotide increased t-PA antigen levels by 31% (Quartiles: 2–49%, p = 0.026) and PAP concentrations by 13% (−4–41%, p = 0.039), while PAI-1 levels remained unaffected. Moreover, defibrotide reduced C-reactive protein levels by 13% (0–17%, p = 0.002). A transient increase in the clotting time in thromboelastometry and a decrease in F1 + 2 prothrombin fragments suggests modest anticoagulant properties. In conclusion, defibrotide infusion enhanced fibrinolysis and reduced C-reactive protein levels during experimental endotoxemia.