Shear-induced unfolding activates von Willebrand factor A2 domain for proteolysis.

BACKGROUND: To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13-mediated proteolysis in a shear flow dependent manner. OBJECTIVE AND METHODS: We examined whether tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. RESULTS: We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We find this mutant A2 (N1493C and C1670S) to feature ADAMTS13-resistant behavior in vitro. CONCLUSIONS: Our results yield molecular-detail evidence for the force-sensing function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional 'knotted' Rossmann fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we discuss the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage-activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases.

[The effect of the time interval between fractions on the result of a fractionated irradiation of the R1H tumor]. / Einfluss des Zeitintervalls zwischen den Fraktionen auf das Ergebnis einer fraktionierten Bestrahlung des R1H-Tumors.

BACKGROUND: The response of an experimental tumor to hyperfractionated irradiation with different time intervals between the two daily fractions has been investigated. MATERIAL AND METHODS: Tumors were exposed to irradiation five days per week over six weeks. A standard treatment of 30 fractions, i.e. one fraction per day (200 kVp X-rays) was compared with a hyperfractionated schedule of 60 fractions, i.e. two fractions per day, with time intervals of either one, two, three, five or six hours between the two daily fractions. RESULTS: Compared with standard treatment a significant reduction (p < 0.005) of net growth delay was observed for the tumors treated with two daily fractions separated by two hours. However, at a time interval of five and six hours between the two daily fractions net growth delay increased considerably (p < 0.0001 and p < 0.01) as compared with the standard treatment. CONCLUSION: Our results indicate the importance of the time interval between the daily fractions in hyperfractionated and accelerated radiotherapy.

Radiotherapy of the rhabdomyosarcoma R1H of the rat: influence of the time interval between two daily fractions during hyperfractionated radiotherapy.

The response of the rhabdomyosarcoma R1H of the rat to hyperfractionated irradiation with different time intervals between the two daily fractions has been investigated. All tumours were exposed to irradiation 5 days per week over 6 weeks. A standard treatment of 30 fractions, i.e. one fraction per day, of 1.83-2.75 Gy (200 kVp X-rays) was compared with a hyperfractionated schedule of 60 fractions, i.e. two fractions per day, of 0.92-1.38 Gy with time intervals of either 1, 2, 3, 5 or 6 h between the two daily fractions. Tumour response has been assessed by (a) net growth delay and (b) local tumour control. Compared with standard treatment (one fraction per day) significant reduction (p < 0.005) of net growth delay was observed for the tumours treated with two daily fractions separated by 2 h. However, at a time interval of 5 and 6 h between the two daily fractions net growth delay increased considerably (p < 0.0001 and p < 0.01) as compared with the standard treatment and an increased rate of local tumour control was observed. The major findings is that tumour response is not as would be predicted by repair if the interval between the two daily fractions exceeds 2 h. The competing or overriding mechanisms cannot be identified ultimately, but the data, though based on small animal numbers and collected over an extended time period, reflect a significant effect.