Patients referred for evaluation of bleeding symptoms occasionally have a prolonged platelet function analyser (PFA) closure time, without evidence for von Willebrand disease or impaired platelet aggregation. The aim of this study was to establish a shear-dependent platelet function defect in these patients. Patients were included based on high bleeding score and prior PFA prolongation. Common tests of von Willebrand factor (VWF) and platelet function and exome sequencing were performed. Microfluidic analysis of shear-dependent collagen-induced whole-blood thrombus formation was performed. In 14 PFA-only patients, compared to healthy volunteers, microfluidic tests showed significantly lower platelet adhesion and thrombus formation parameters. This was accompanied by lower integrin activation, phosphatidylserine exposure and P-selectin expression. Principal components analysis indicated VWF as primary explaining variable of PFA prolongation, whereas conventional platelet aggregation primarily explained the reduced thrombus parameters under shear. In five patients with severe microfluidic abnormalities, conventional platelet aggregation was in the lowest range of normal. No causal variants in Mendelian genes known to cause bleeding or platelet disorders were identified. Multiparameter assessment of whole-blood thrombus formation under shear indicates single or combined effects of low-normal VWF and low-normal platelet aggregation in these patients, suggesting a shear-dependent platelet function defect, not detected by static conventional haemostatic tests.