Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor–treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.

Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a Cterminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS-NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. N itric oxide (NO) is a potent mediator in biological processes such as neurotransmission, inflammatory response, and vascular homeostasis (1). The prime source of NO in the cardiovascular system is endothelial NO synthase (eNOS), which is tightly regulated with respect to activity and localization. For example, coordinated phosphorylation contributes to activity control of eNOS because of activating and inhibiting phosphorylation at S1179 and T495, respectively (2-6). Myristoylation and dual palmitoylation at its extreme N terminus target eNOS to the cytoplasmic face of the Golgi complex and to the plasma membrane (7), where eNOS is thought to be fully capable of activation (8, 9). Misrouting of acylation-deficient eNOS impairs NO production (10, 11), indicating that correct subcellular targeting is critical for stimulus-dependent activation of the enzyme (8). Posttranslational modifications are efficiently complemented by multiple proteinprotein interactions that help regulate eNOS activity with respect to time and space. For instance, chaperone hsp90 bound to eNOS may mediate vascular endothelial growth factor-induced eNOS phosphorylation by promoting the interaction between eNOS and Akt (12, 13). At the plasma membrane, eNOS is complexed to and inhibited by the master components of caveolae, i.e., caveolin-1 in endothelial cells (9, 14) and caveolin-3 in cardiac myocytes (15). After stimulus-induced [Ca 2ϩ ] i increase, the Ca 2ϩ -calmodulin complex displaces eNOS from caveolin (16), stimulates eNOS to produce NO, and subsequently leads to the redistribution of eNOS from plasma membrane caveolae (17). The complexity...

Hodgkin's and Reed/Sternberg (HRS) cells, the tumour cells in classical Hodgkin's lymphoma (HL), represent transformed B cells in nearly all cases. The detection of destructive somatic mutations in the rearranged immunoglobulin (Ig) genes of HRS cells in classical HL indicated that they originate from preapoptotic germinal centre (GC) B cells that lost the capacity to express a highaffinity B-cell receptor (BCR). Several aberrantly activated signalling pathways and transcription factors have been identified that contribute to the rescue of HRS cells from apoptosis. Among the deregulated signalling pathways, activation of multiple receptor tyrosine kinases in HRS cells appears to be a specific feature of HL. In about 40% of cases of classical HL the HRS cells are infected by Epstein-Barr virus (EBV), indicating an important role of EBV in HL pathogenesis. Interestingly, nearly all cases of HL with destructive Ig gene mutations eliminating BCR expression (e.g. nonsense mutations) are EBV-positive, suggesting that EBV-encoded genes have a particular function to prevent apoptosis of HRS-cell precursors that acquired such crippling mutations. This idea is further supported by the recent demonstration that isolated human GC B cells harbouring crippled Ig genes can be rescued by EBV from cell death, giving rise to lymphoblastoid cell lines. The molecular analysis of composite Hodgkin's and nonHodgkin's lymphomas indicated that many cases develop from a common GC B-cell precursor in a multistep transformation process with both shared and distinct oncogenic events. 2-6 Nearly all cases of classical and lymphocyte-predominance HL carry somatically mutated V genes, suggesting an origin from (post) germinal centre (GC) B cells, as somatic hypermutation of Ig V genes specifically takes place in GC B cells.7 In lymphocyte-predominance HL, the detection of intraclonal V gene diversity and several phenotypic features suggest an origin of the L&H cells from mutating and selected GC B cells. 2,4,5 In classical HL, crippling mutations that destroyed the coding capacity of originally functional rearrangements were detected in about a quarter of cases.3,8 Such obviously crippling mutations happen in mutating GC B cells, but represent only a small fraction of all disadvantagous mutations that normally result in apoptosis of the respective GC B cells. In the GC, there is a stringent selection for B cells acquiring affinity-increasing mutations, so also many GC B cells that still express a B-cell receptor (BCR) but which fails to bind to the cognate antigen with improved affinity will undergo apoptosis.9,10 Thus, we speculated that the vast majority of HRS cells in classical HL are derived from preapoptotic GC B cells. 3 In rare cases (about 2%) HRS cells originate from T cells. 11,12 Although HRS cells are usually transformed B cells, global gene expression analysis of HRS cell lines using microarrays revealed that the HRS cells have lost the expression of most B-cell markers to an extent that is unique among B-cell lymphomas. 13 As t...

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The single nucleotide polymorphism 118A>G of the human -opioid receptor gene OPRM1, which leads to an exchange of the amino acid asparagine (N) to aspartic acid (D) at position 40 of the extracellular receptor region, alters the in vivo effects of opioids to different degrees in pain-processing brain regions. The most pronounced N40D effects were found in brain regions involved in the sensory processing of pain intensity. Using the -opioid receptor-specific agonist DAMGO, we analyzed the -opioid receptor signaling, expression, and binding affinity in human brain tissue sampled postmortem from the secondary somatosensory area (S II ) and from the ventral posterior part of the lateral thalamus, two regions involved in the sensory processing and transmission of nociceptive information. We show that the main effect of the N40D -opioid receptor variant is a reduction of the agonist-induced receptor signaling efficacy. In the S II region of homo-and heterozygous carriers of the variant 118G allele (n ‫؍‬ 18), DAMGO was only 62% as efficient (p ‫؍‬ 0.002) as in homozygous carriers of the wild-type 118A allele (n ‫؍‬ 15). In contrast, the number of [ 3 H]DAMGO binding sites was unaffected. Hence, the -opioid receptor G-protein coupling efficacy in S II of carriers of the 118G variant was only 58% as efficient as in homozygous carriers of the 118A allele (p < 0.001). The thalamus was unaffected by the OPRM1 118A>G SNP. In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of -opioid receptor variant N40D.The human -opioid receptor variant N40D coded by the single nucleotide polymorphism (SNP) 2 118AϾG of the -opioid receptor gene, OPRM1 (dbSNP rs1799971; allelic frequency 8.2-17%) has been found to be associated with diminished opioid effects in experimental (1-5) and clinical (6 -9) settings. However, in vitro experiments trying to elucidate the underlying molecular mechanisms provided inconsistent results, falling short to support the comparatively strong clinical evidence of decreased opioid effects in carriers of the variant 118G allele.Among the reported molecular consequences of the 118AϾG polymorphism is a three times higher binding affinity of ␤-endorphin at the N40D -opioid receptors expressed in transfected Syrian hamster adenovirus-12-induced tumor cells (AV-12), whereas the affinity of other exogenous opioids was unaffected (10). In contrast, a leftward shift of the potencies of DAMGO and morphine-mediated Ca 2ϩ channel inhibition has been shown in N40D variant -opioid receptor expressed in rat sympathetic superior cervical ganglion (SCG) neurons (11). However, both findings of a higher ligand affinity and potency were not reproduced in three attempts using N40D variant -opioid receptors transfected Cercopithecus aethiops kidney cells (COS), human 293 embryonic kidney cells (HEK293), and again AV-12 cells (12-14). Moreover, they do not explain the decreased clinical opioid potency. As an alternative mechanism, decreased -opioid receptor expression caused...

IntroductionSTAT transcription factors are constitutively activated in several malignancies, often resulting from genomic alterations that deregulate STAT-activating kinases or interfere with termination of STAT signaling. 1,2 SOCS1, whose expression is induced by activated STATs, terminates JAK/STAT signaling by binding to and marking phosphorylated JAK for proteasomal degradation. 1,3 Function-impairing mutations in SOCS1 have recently been observed in primary mediastinal B-cell lymphoma (PMBL) and classic and nodular lymphocyte-predominant Hodgkin lymphoma (HL) and were associated with accumulation of several phosphorylated STATs. [4][5][6][7][8][9] Analysis of the mutation pattern of SOCS1 in nodular lymphocyte-predominant HL indicated a somatic hypermutation (SHM) origin. 7 The SHM process is largely restricted to rearranged immunoglobulin V-genes in germinal center (GC) B cells. 10 However, SHM occasionally also targets other genes. Rare BCL6 and FAS mutations present in GC but not naive B cells were ascribed as byproducts of "normal" SHM, 11,12 whereas mutations in PIM1, c-MYC, RHOH, and PAX5, present in several B-cell lymphomas but not in normal GC B cells, were ascribed to "aberrant" SHM. [12][13][14][15][16][17][18] Given the indications that SOCS1 is a target of SHM, SOCS1 inactivation by SHM could, in addition to HL and PMBL, also contribute to lymphomagenesis in other GC or post-GC B cellderived lymphomas. We thus analyzed the SOCS1 mutation status of other B-cell lymphomas, and, to clarify whether SOCS1 mutations are the result of (aberrant) SHM, also in normal GC and naive B cells, T-cell lymphomas, and solid tumors with constitutive activated STATs. Methods Tissue samplesTissue samples were from the files of the Senckenberg Institute of Pathology and originally submitted for diagnostic purposes. Approval for this study was obtained from the University of Frankfurt School of Medicine Institutional Review Board. Informed consent was obtained in accordance with the Declaration of Helsinki. Isolation of single tonsillar GC and naive B cellsSingle GC and naive tonsillar B cells were sorted by fluorescence-activated cell sorter as CD20 ϩ CD38 intermediate IgD Ϫ and IgD ϩ CD23 ϩ CD27 Ϫ CD38 Ϫ cells, respectively, in 10 L of 1ϫ polymerase chain reaction (PCR) buffer (supplemental data, available on the Blood website; see the Supplemental Materials link at the top of the online article). Sequence analysis of SOCS1 and V gene rearrangementsGenomic DNA was extracted from sections of frozen samples and various cell lines. DNA (10-100 ng) was used as template for amplification of the complete open reading frame of SOCS1 in a single-round PCR. PCR products were purified and directly sequenced. In cases with several mutations, genomic DNA was diluted and aliquots of various dilutions were amplified in a 2-round nested PCR. PCR products obtained from dilutions For personal use only. on May 11, 2018. by guest www.bloodjournal.org From where 30% to 50% of PCRs yielded PCR products were directly sequenced. For ampl...

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4 -helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CDloop/N-terminal D-helix).A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.