• 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)
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Amyloid β (Aβ) polypeptide plays a key role in determining the state of protein aggregation in Alzheimer's disease. The hydrophobic C-terminal part of the Aβ peptide is critical in triggering the transformation from α-helical to β- sheet structure. We hypothesized that phospholipase A2 (PLA2) may inhibit the aggregation of Aβ peptide by interacting with the peptide and keeping the two peptide chains apart. In order to examine the nature of interactions between PLA2 and Aβ peptide, we prepared and crystallized complex of Naja naja sagittifera PLA2 with the C-terminal hepta-peptide Val-Gly-Gly-Val-Val-Ile-Ala. The X-ray intensity data were collected to 2.04 A resolution and the structure was determined by molecular replacement and refined to the crystallographic R factor of 0.186. The structural analysis revealed that the peptide binds to PLA2 at the hydrophobic substrate binding cavity forming at least eight hydrogen bonds and approximately a two dozen Van der Waals interactions. The number and nature of interactions indicate that the affinity between PLA2 and the hepta-peptide is greater than the affinity between two Aβ peptide chains. Therefore, PLA2 is proposed as a probable ligand to prevent the aggregation of Aβ peptides.
Background Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder, resulting from autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). However, the mechanism underlying anti-ADAMTS13 autoantibody formation is not known, nor it is known how genetic aberrations contribute to the pathogenesis of iTTP. Methods Here we performed whole exome sequencing (WES) of DNA samples from 40 adult patients with iTTP and 15 local healthy subjects with no history of iTTP and other hematological disorders. Results WES revealed variations in the genes involved in protein glycosylation, including O-linked glycosylation, to be a major pathway affected in patients with iTTP. Moreover, variations in the ANKRD gene family, particularly ANKRD36C and its paralogs, were also more prevalent in patients with iTTP than in the healthy controls. The ANKRD36 family of proteins have been implicated in inflammation. Mass spectrometry revealed a dramatic alternation in plasma glycoprotein profile in patients with iTTP compared with the healthy controls. Conclusion Altered glycosylation may affect the disease onset and progression in various ways: it may predispose patients to produce ADAMTS13 autoantibodies or affect their binding properties; it may also alter clearance kinetics of hemostatic and inflammatory proteins. Together, our findings provide novel insights into plausible mechanisms underlying the pathogenesis of iTTP.
Alzheimer’s disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD’s neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer’s Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ–Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.
Human neutrophil peptides (HNPs) or alpha (α)-defensins are a family of small antimicrobial peptides which play important roles in innate immunity against invading bacteria, fungi, and viruses. HNPs contain 29-33 amino acid residues which share a distinct pattern of disulfide bonding. HNPs can be further subdivided into myeloid (HNPs 1-4) and enteric (HD5-6) forms. HNPs-1, -2, and -3 are structurally identical except for the first amino acid residue and are predominantly expressed in human neutrophils from which they are released at sites of infection or inflammation. Previous studies have demonstrated that HNP1 promotes thrombus formation. However, the mechanisms underlying its prothrombotic effects are not fully understood. In the present study, we demonstrate that HNP-1, -2, and -3 all inhibit plasma-derived and recombinant ADAMTS13 activity in a concentration-dependent manner as determined by the cleavage of FRETS-VWF73 (Fig. 1A & 1B) and multimeric VWF under denaturing conditions. At final concentrations of ~10-15 µM, purified and synthetic HNP-1, -2, and -3 completely abolish ADAMTS13Õs ability to cleave FRETS-VWF73 (Fig. 1A & 1B). The concentrations required for complete inhibition of proteolytic cleavage of pre-denatured VWF by ADAMTS13 using the urea dialysis method is higher, likely resulting from the removal of peptides from the reaction. HNP-1 binds to ultra large VWF released from endothelial cells, soluble multimeric VWF, GST-VWF73 peptide, and ADAMTS13 as determined by cultured endoethelial cells in a microfluidic channels and by surface plasmon resonance. The affinity (the dissociation constant, KD) for HNP-1 to bind VWF, GST-VWF73, and ADAMTS13 is 8.0 micro mol/L, 1.0 micro mol/L, and 3.2 micro mol/L, respectively. Sequence analysis reveals that the amino acid residues of HNP-1, -2, and -3 all contain a RRY motif that is also found in the spacer domain (i.e. 659RRYGEEY665) of ADAMTS13. We hypothesize that competition by HNPs with ADAMTS13 for binding to VWF-A2 domain mediates their inhibition. As shown, a deletion or alanine substitution of RRY within HNP1 nearly abolishes its ability to inhibit ADAMTS13 activity determined by the cleavage of FRETS-VWF73 (in Fig. 1C) and multimeric VWF under denaturing conditions. Similarly, HNP-beta and aliphatic (with no aromatic rings directly on the nitrogen atom) HNP-1 exhibit no inhibitory activity on ADAMTS13 (Fig. 1C). To further demonstrate the inhibitory activity of HNP1 towards ADAMTS13 under more physiological conditions, a BioFlux microfluidic system is employed. Addition of purified (native) HNP-1 (6-15 micro mol/L) to D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) anticoagulated whole blood dramatically augments the rate of platelet adhesion and aggregation to the fibrillar collagen-coated surface under arterial shear stress (approximately 100 dyne per square centimeter). These results indicate that HNP-1 plays a role in the inhibition of VWF proteolysis by ADAMTS13 under flow. We conclude that HNP-1, -2, and -3 released from activated neutrophils at sites of infection or inflammation could significantly augment thrombus formation by inhibiting the local or residual plasma ADAMTS13 activity, when the circulating ADAMTS13 activity is already at critically low levels as in cases of hereditary or acquired autoimmune TTP and HUS, thereby triggering the onset of thrombotic complications. (*authors contribute equally to this work). Figure 1. Figure 1. HNPs are potent inhibitors of ADAMTS13 metalloprotease. A. Purified HNPs inhibit ADAMTS13-mediated cleavage of FRETS-VWF73; B. HNP-1, -2, and -3 all inhibit the cleavage of FRETS-VWF73 by ADAMTS13; C. No inhibition of the ADAMTS13 dependent cleavage of FRETS-VWF73 by HNP1 mutants, HNP-beta, and aliphatic HNP1 compared with WT control. Disclosures No relevant conflicts of interest to declare.
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