• HNPs inhibit proteolytic cleavage of VWF by ADAMTS13 by physically blocking VWF-ADAMTS13 interactions.• Plasma levels of HNP1, HNP2, and HNP3 are markedly increased in patients with acquired autoimmune TTP.Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood.Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 mM and 45 mM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (K D 5 0.72 mM), soluble VWF (K D 5 0.58 mM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n 5 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n 5 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001).There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman r 5 0.7932, P < .0001).Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/ infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders. (Blood. 2016;128(1):110-119)

Neutral amino acid exchange by the alanine serine cysteine transporter (ASCT)2 was reported to be electroneutral and coupled to the cotransport of one Na+ ion. The cotransported sodium ion carries positive charge. Therefore, it is possible that amino acid exchange is voltage dependent. However, little information is available on the electrical properties of the ASCT2 amino acid transport process. Here, we have used a combination of experimental and computational approaches to determine the details of the amino acid exchange mechanism of ASCT2. The [Na+] dependence of ASCT2-associated currents indicates that the Na+/amino acid stoichiometry is at least 2:1, with at least one sodium ion binding to the amino acid–free apo form of the transporter. When the substrate and two Na+ ions are bound, the valence of the transport domain is +0.81. Consistently, voltage steps applied to ASCT2 in the fully loaded configuration elicit transient currents that decay on a millisecond time scale. Alanine concentration jumps at the extracellular side of the membrane are followed by inwardly directed transient currents, indicative of translocation of net positive charge during exchange. Molecular dynamics simulations are consistent with these results and point to a sequential binding process in which one or two modulatory Na+ ions bind with high affinity to the empty transporter, followed by binding of the amino acid substrate and the subsequent binding of a final Na+ ion. Overall, our results are consistent with voltage-dependent amino acid exchange occurring on a millisecond time scale, the kinetics of which we predict with simulations. Despite some differences, transport mechanism and interaction with Na+ appear to be highly conserved between ASCT2 and the other members of the solute carrier 1 family, which transport acidic amino acids.

Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a Paired basic Amino acid Cleaving Enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulate FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared to FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4 fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

Purpose of review ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity is accountable for a potentially fatal blood disorder thrombotic thrombocytopenic purpura (TTP). Understanding of ADAMTS13–VWF interaction is essential for developing novel treatments to this disorder. Recent findings Despite the proteolytic activity of ADAMTS13 being restricted to the metalloprotease domain, the ancillary proximal C-terminal domains including the disintegrin domain, first TSP-1 repeat, cysteine-rich region, and spacer domain are all required for cleavage of VWF and its analogs. Recent studies have added to our understandings of the role of the specific regions in the disintegrin domain, the cysteine-rich domain, and the spacer domain responsible for its interaction with VWF. Additionally, regulative functions of the distal portion of ADAMTS13 including the TSP-1 2–8 repeats and the CUB domains have been proposed. Finally, fine mapping of anti-ADAMTS13 antibody epitopes have provided further insight into the essential structural elements in ADAMTS13 for VWF binding and the mechanism of autoantibody-mediated TTP. Summary Significant progress has been made in our understandings of the structure–function relationship of ADAMTS13 in the past decade. To further investigate ADAMTS13–VWF interactions for medical applications, these interactions must be studied under physiological conditions in vivo.

Sodium-coupled neutral amino acid transporter 2 (SNAT21) belongs to the SLC38 family of solute transporters. Transport of 1 amino acid molecule into the cell is driven by the co-transport of 1 Na+ ion. The functional significance of the C-terminus of SNAT2, which is predicted to be located in the extracellular space, is currently unknown. Here, we removed 13 amino acid residues from the SNAT2 C-terminus and studied the effect of the deletion on transporter function. The truncation abolished amino acid transport currents at negative membrane potentials (< 0 mV), as well as substrate uptake. However, transport currents were observed at positive membrane potentials, demonstrating that transport was accelerated while the driving force decreased. Membrane expression levels were normal in the truncated transporter. SNAT2Del C-ter showed 3-fold higher apparent affinity for alanine, and 2-fold higher Na+ affinity compared to SNAT2WT, suggesting that the C-terminus is not required for high-affinity substrate and Na+ interaction with SNAT2. pH sensitivity of amino acid transport was partially retained after the truncation. In contrast to the truncation after the final trans-membrane domain, TM11, deletion of TM11 resulted in an inactive transporter, most likely due to a defect in cell surface expression. Together, the results demonstrate that the C-terminal domain of SNAT2 is an important voltage regulator that is required for a normal amino acid translocation process at physiological membrane potentials. However, the C-terminus appears not to be involved in regulation of membrane expression.