Activated protein C (APC) is a serine protease with potent anticoagulant properties, which is formed in blood on the endothelium from an inactive precursor. During normal haemostasis, APC limits clot formation by proteolytic inactivation of factors Va and VIIIa (ref. 2). To do this efficiently the enzyme needs a nonenzymatic cofactor, protein S (ref. 3). Recently it was found that the anticoagulant response to APC (APC resistance) was very weak in the plasma of 21% of unselected consecutive patients with thrombosis and about 50% of selected patients with a personal or family history of thrombosis; moreover, 5% of healthy individuals show APC resistance, which is associated with a sevenfold increase in the risk for deep vein thrombosis. Here we demonstrate that the phenotype of APC resistance is associated with heterozygosity or homozygosity for a single point mutation in the factor V gene (at nucleotide position 1,691, G-->A substitution) which predicts the synthesis of a factor V molecule (FV Q506, or FV Leiden) that is not properly inactivated by APC. The allelic frequency of the mutation in the Dutch population is approximately 2% and is at least tenfold higher than that of all other known genetic risk factors for thrombosis (protein C (ref. 8), protein S (ref. 9), antithrombin10 deficiency) together.

Key Points VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios define the pathophysiological mechanisms that play a role in VWD and various VWF mutations. A high VWFpp/VWF:Ag ratio indicates increased clearance of VWF and a high FVIII:C/VWF:Ag ratio decreased synthesis of VWF.

Surgery for locoregionally advanced head and neck squamous cell carcinoma (HNSCC) results in 30‒50% five-year overall survival. In IMCISION (NCT03003637), a non-randomized phase Ib/IIa trial, 32 HNSCC patients are treated with 2 doses (in weeks 1 and 3) of immune checkpoint blockade (ICB) using nivolumab (NIVO MONO, n = 6, phase Ib arm A) or nivolumab plus a single dose of ipilimumab (COMBO, n = 26, 6 in phase Ib arm B, and 20 in phase IIa) prior to surgery. Primary endpoints are feasibility to resect no later than week 6 (phase Ib) and primary tumor pathological response (phase IIa). Surgery is not delayed or suspended for any patient in phase Ib, meeting the primary endpoint. Grade 3‒4 immune-related adverse events are seen in 2 of 6 (33%) NIVO MONO and 10 of 26 (38%) total COMBO patients. Pathological response, defined as the %-change in primary tumor viable tumor cell percentage from baseline biopsy to on-treatment resection, is evaluable in 17/20 phase IIa patients and 29/32 total trial patients (6/6 NIVO MONO, 23/26 COMBO). We observe a major pathological response (MPR, 90‒100% response) in 35% of patients after COMBO ICB, both in phase IIa (6/17) and in the whole trial (8/23), meeting the phase IIa primary endpoint threshold of 10%. NIVO MONO’s MPR rate is 17% (1/6). None of the MPR patients develop recurrent HSNCC during 24.0 months median postsurgical follow-up. FDG-PET-based total lesion glycolysis identifies MPR patients prior to surgery. A baseline AID/APOBEC-associated mutational profile and an on-treatment decrease in hypoxia RNA signature are observed in MPR patients. Our data indicate that neoadjuvant COMBO ICB is feasible and encouragingly efficacious in HNSCC.

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Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF. The hemostatic protein von Willebrand factor (VWF)2 plays important roles in hemostasis by facilitating platelet adhesion to injured endothelium and by carrying coagulation factor VIII (FVIII) to protect it from rapid proteolytic inactivation (1). A defect in VWF leads to the most common inherited human bleeding disorder, von Willebrand disease (VWD) (2). VWD is classified into 3 types: types 1 and 3 are quantitative VWD characterized by defects that result in a partial (type 1) or virtually complete (type 3) deficiency of VWF in plasma; type 2 VWD is caused by defects that result in qualitatively different VWF with abnormal function (2). Among all the index cases, type 1 VWD is the most common form. Missense mutations compose the majority of mutations causing type 1 VWD (up to 75%), but only a minority of the mutations causing type 3 VWD (3-6). In the latter cases nonsense mutations and deletions predominate.VWF is synthesized as a precursor protein containing a signal peptide (22-amino acids), an N-terminal propeptide (D1 and D2 domains, 741 amino acids) and a mature peptide comprising multiple domains (DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK, 2050 amino acids) (7). Following its synthesis in the endoplasmic reticulum (ER) proVWF (after cleavage of the signal peptide) dimerizes via formation of intermolecular disulfide bridges in the cysteine knot (CK) domain (1). ProVWF dimers are transported to the Golgi-apparatus; in this compartment propeptide-mediated assembly of multimers occurs through formation of intermolecular disulfide bridges within the DЈD3 domains (1). Processing of proVWF presumably by furin occurs in the trans-Golgi network. In the same compartment, VWF is either secreted constitutively or tu...