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von Willebrand factor (VWF) is a key player in hemostasis, acting as a carrier for factor VIII and capturing platelets at sites of vascular damage. To capture platelets, it must undergo conformational changes, both within its A1 domain and at the macromolecular level through A2 domain unfolding. Its size and this function are regulated by the metalloproteinase ADAMTS‐13. Recently, it has been shown that ADAMTS‐13 undergoes a conformational change upon interaction with VWF, and that this enhances its activity towards its substrate. This review summarizes recent work on these conformational transitions, describing how they are controlled. It points to their importance in hemostasis, bleeding disorders, and the developing field of therapeutic application of ADAMTS‐13 as an antithrombotic agent in obstructive microvascular thrombosis and in cardiovascular disease.

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Sera from dogs rendered aplastic by total-body irradiation stimulate human bone marrow megakaryocyte progenitors to form megakaryocyte colonies in plasma clot cultures. In this investigation, we evaluated the effects of varying concentrations of such sera on both the mitotic and endomitotic phases of human megakaryocyte development in vitro. When low concentrations of aplastic canine sera (2.5% to 5.0% [vol/vol]) were added to cultures of human peripheral blood mononuclear cells in place of normal AB serum, megakaryocyte colony formation was augmented fivefold, cell numbers per colony increased approximately 2.5- fold, and the geometric mean megakaryocyte ploidy almost doubled. Further increasing the aplastic canine serum concentration from 10% to 30% (vol/vol) stimulated no additional colony formation. However, there was a further augmentation of cell numbers per colony associated with a progressive decrease in the mean megakaryocyte ploidy. Megakaryocyte cultures were harvested after 7, 12, 15, and 19 days of incubation, and these demonstrated that the lower mean ploidy values found at the higher concentrations of aplastic canine serum did not result from delayed endoreduplication. At all aplastic serum concentrations evaluated, there existed a strong correlation between nuclear ploidy and cell diameter. We conclude that both the mitotic and endomitotic events in human megakaryocytopoiesis may be influenced by a factor or factors present in aplastic canine serum. At lower in vitro concentrations, such sera stimulate both mitosis and endomitosis, which promotes the development of megakaryocyte colonies composed of larger cells with a higher mean ploidy. With increasing aplastic serum concentrations, colony formation plateaus and mitosis is favored over endomitosis. This results in colonies composed of more numerous but smaller megakaryocytes with a lower mean ploidy. Our data suggest that the size and extent of polyploidization that can be achieved by a developing megakaryocyte may be influenced by the mitotic prior history of its immediate precursor cell.

IntroductionChronic thromboembolic pulmonary hypertension (CTEPH) Results from failure of thrombus resolution following acute pulmonary embolism. Abnormalities in haemostasis are implicated in the pathobiology, including elevated levels of von Willebrand factor (VWF), which is normally regulated by ADAMTS13. Interim analysis of a genome-wide association study (GWAS) identified a significant association in CTEPH with the ADAMTS13andABOgene loci. We aimed to determine if ADAMTS13 protein levels are altered in CTEPH.MethodsADAMTS13 and VWF plasma antigen levels were measured by ELISA in 208 individuals with CTEPH and compared to 68 healthy controls. Levels were also measured in subjects with chronic thromboembolic disease but without pulmonary hypertension (CTED), and other disease comparator groups summarised in figure 1. In 22 CTEPH individuals ADAMTS13 and VWF levels were measured pre-operatively and at least 3 months post-pulmonary endarterectomy (PEA).ResultsADAMTS13 levels were decreased in CTEPH (median ±IQR: 0.88±0.40 µg/ml; p=5.7x10-09) and CTED (0.83±0.22 µg/ml; p=2.1x10-06) patients compared to healthy controls (1.15±0.30 µg/ml) (figure 1). ADAMTS13 levels remained low in CTEPH patients following PEA (pre: 0.78±0.27 µg/ml vs. post: 0.83±0.29 µg/ml; p=0.92) even in those with normalised mean pulmonary arterial pressures (<25 mmHg) after PEA. Furthermore, ADAMTS13 levels were lowest in the CTEPH and CTED groups when covariates (age, gender and batch) were included in multivariate rank regression models. VWF levels were increased in CTEPH (16.7±15.2 µg/ml; p=4.0x10-12) and CTED (17.0±10.1 µg/ml; p=3.9x10-06) compared to healthy controls (8.5±8.8 µg/ml). There was no change post-PEA (pre: 22.2±17.3 µg/ml vs. post: 19.6±14.2 µg/ml; p=0.24).ConclusionsPlasma ADAMTS13 antigen levels are markedly decreased in CTEPH. This is not secondary to pulmonary hypertension, as demonstrated by the similarly low levels in CTED, and individuals with normal pulmonary artery pressures post-PEA. Thus, the VWF/ADAMTS13 axis is implicated in the underlying disease pathophysiology. Ongoing work will clarify if there is a causal link by defining whether genetic variation at the ADAMTS13locus contributes to reduced ADAMTS13 protein levels and CTEPH.[Figure]

Decreased hemoglobin concentration occurs at six weeks of age. Its mechanism is unclear. We studied six infants at six weeks of age and their parents, with the in vitro plasma clot assay for erythroid progenitor cells CFU-E and BFU-E. Serum samples and peripheral blood (PB) mononuclear cells (MNC) were obtained by venipuncture. Bone marrow (BM) aspirate MNC were obtained from a normal adult volunteer. MNC were fractionated into T-cells and non-T-cells by sheep erythrocyte rosetting. Some PB-MNC from each baby were pre-incubated with: a) fresh plasma (FP), b) rabbit complement (RC), c) FP and baby's serum (BS) and d) RC and BS, for one hour at 37°C. All cultures contained 5 × 104cells per micro-well. 1 × 104T-cells were added to some experiments. The baby's PB-MNC produced a mean of 7±2 BFU-E colonies per clot, and 9.5±5 BFU-E when cultured in the presence of BS. Pre-incubation with FP, RC, FP+BS and RC+BS produced 5±1, 4±1, 5.5±2 and 4±2 BFU-E respectively. Adding baby's T-cells to his/her parents' PB-MNC increased the number of BFU-E from 11 to 19. BM-MNC produced a control of 250±10 CFU-E and 30±6 BFU-E per clot. In the presence of BS there were 219±30 CFU-E and 26±8 BFU-E. The addition of BS to cultures of baby's PB-MNC or the pre-incubation of these cells with BS and complement showed no significant inhibition of PB-BFU-E. The baby's T-cells increased the number of PB-BFU-E. Similarly, BM-CFU-E and BM-BFU-E were not inhibited by BS. T-cell suppression of erythropoiesis or serum inhibitors of erythropoiesis, either complement mediated or not, do not explain the physiologic anemia of the newborn.