Anti-β2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies interfere with cleavage of factor V(a) by activated protein C

BACKGROUND: The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against β2-glycoprotein I (β2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against β2GPI and prothrombin cause APC resistance is currently unclear. OBJECTIVES: To investigate how anti-β2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance. METHODS: The effects of anti-β2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies. RESULTS: APC resistance was observed in LA-positive patients with anti-β2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-β2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-β2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-β2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306. CONCLUSION: Anti-β2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage

Conservation and Divergence of Regulatory Strategies at Hox Loci and the Origin of Tetrapod Digits

The evolution of tetrapod limbs from fish fins enabled the conquest of land by vertebrates and thus represents a key step in evolution. Despite the use of comparative gene expression analyses, critical aspects of this transformation remain controversial, in particular the origin of digits. Hoxa and Hoxd genes are essential for the specification of the different limb segments and their functional abrogation leads to large truncations of the appendages. Here we show that the selective transcription of mouse Hoxa genes in proximal and distal limbs is related to a bimodal higher order chromatin structure, similar to that reported for Hoxd genes, thus revealing a generic regulatory strategy implemented by both gene clusters during limb development. We found the same bimodal chromatin architecture in fish embryos, indicating that the regulatory mechanism used to pattern tetrapod limbs may predate the divergence between fish and tetrapods. However, when assessed in mice, both fish regulatory landscapes triggered transcription in proximal rather than distal limb territories, supporting an evolutionary scenario whereby digits arose as tetrapod novelties through genetic retrofitting of preexisting regulatory landscapes. We discuss the possibility to consider regulatory circuitries, rather than expression patterns, as essential parameters to define evolutionary synapomorphies

Regulatory potential of the fish <i>HoxA</i> and <i>HoxD</i> landscapes in mouse transgenic limbs.

<p>(A) Scheme of the <i>HoxAa</i> BAC used for transgenesis in the mouse with the expression of the fish <i>Hoxa11a</i>, <i>Hoxa13a</i>, and <i>Evx1</i> genes in mouse embryonic limbs. All genes assayed showed expression in a proximal domain, yet not in the presumptive digit domain. Note that <i>Hoxa11a</i> expression was not observed in forelimb buds. (B) Scheme of the <i>HoxAb</i> BAC (bottom) with the expression of several genes. The fish <i>Hoxa10b</i>, <i>Hoxa11b</i>, and <i>Hoxa13b</i> genes are expressed in a proximal domain, and transcripts are absent from the presumptive digit domain. Likewise, the 5′ flanking genes <i>HIBADHb</i>, <i>TAX1BP1b</i>, and <i>JAZF1b</i> respond to the same proximal regulation. A comparison with the endogenous <i>Hoxd11</i> expression (mmu<i>Hoxd11</i>) shows that limb expression of the transgenes is confined to the distal zeugopod and mesopod. (C) Two BAC clones containing either the entire 5′ (top) or 3′ (bottom) landscape flanking the <i>HoxDa</i> cluster with their corresponding expression patterns. Here again, expression is observed in a proximal domain but is absent from developing digits. In the various schemes, genes analyzed are shown in black. All samples are right hind limbs, dorsal views with anterior to the left, except for the endogenous mouse gene “mmu<i>Hoxd11</i>” (B), which is a mirror image of the left hind limb of the limb bud stained for <i>Hoxa11b</i> to its right, in order to facilitate the comparison of transcription domains. The anterior-to-posterior polarity is indicated with arrows. (D, digits; F and T, distal parts of the femur and tibia, respectively).</p

The <i>Tetraodon Hoxa13b</i> expression domain in mice: from “distal” to “proximal.”

<p>(A) <i>In situ</i> hybridization of a <i>Tetraodon Hoxa13b</i> probe using E9.5 to E12.5 fetuses transgenic for the <i>Tetraodon HoxAb</i> cluster. Top panels are dorsal views of forelimbs (anterior to the left), and bottom panels are whole mount pictures. <i>Hoxa13b</i> is expressed in limb buds and posterior trunk, whereas the staining in the head vesicles at E10.5 and E11.5 is a routinely observed artifact. At day E10.5, before the appearance of digits, expression initiates in the distal limb bud (arrowhead). In subsequent stages, however, this domain becomes increasingly “proximal” due to the distal expansion of the digit domain (arrows in E11.5 and E12.5 specimen). The distal expression of <i>Hoxa13b</i> at E10.5 is strikingly similar to the distal expression of <i>Hoxa13b</i> in the fish fin <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Ahn1" target="_blank">[24]</a>. The anterior-to-posterior polarity is indicated with arrows. (B) Scheme illustrating the difficulty in using relative parameters such as “proximal” or “distal” to assign homologies. Due to the developmental expansion of the autopod, the zeugopod domain becomes relatively more proximally positioned within the limb bud, along with time. During digit evolution, a similar process may have occurred and structures that are distal in the fin apparently shifted to a more proximal position in the limb, due to the distal growth of the autopod. The fish <i>Hoxa13b</i> expression in mouse limb buds (purple color in the left scheme) in fact illustrates that distal fish fin tissues correspond to proximal limb structures after the evolution into limbs (right scheme). The fin bud scheme only depicts the endoskeletal part of the fin and not the exoskeleton, which derives from a distinct developmental lineage.</p

Interaction profiles of mouse <i>Hoxa</i> genes.

<p>Circular chromosome conformation capture (4C) analysis of either distal (A) or proximal (B) E12.5 dissected limb bud (schematized in the left) or forebrain (C). The proximal or distal fates of these cells are illustrated by adult skeletons (left) with the same colors as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio-1001773-g001" target="_blank">Figure 1</a>. Dark grey squares indicate regions of local interactions excluded from the analysis. Four interaction profiles are shown after using <i>Hoxa4</i>, <i>Hoxa9</i>, <i>Hoxa11</i>, and <i>Hoxa13</i> as viewpoints. The genomic orientation of <i>HoxA</i> is inverted with respect to <i>HoxD</i>. The percentage of contacts for each viewpoint is given, either in 5′ or in 3′ of the gene cluster. In both samples, <i>Hoxa4</i> mostly interacts with the 3′ landscape, whereas <i>Hoxa13</i> is biased toward the 5′ landscape. Both <i>Hoxa9</i> and <i>Hoxa11</i> change their bias from increased contacts in 3′, in the proximal limb bud sample (B), to contacts in 5′ in the distal sample (A), thus resembling <i>Hoxd</i> genes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773.s002" target="_blank">Figure S2</a>) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Andrey1" target="_blank">[31]</a>. Note that the interaction profiles obtained when using either autopod, proximal limb, or brain (C) tissues are quite similar to one another, indicating a constitutive chromatin organization at the <i>HoxA</i> locus. The size of the displayed DNA interval is of ca. 3 Mb. (D and E) Summaries of the directional 4C signals using bar diagrams in the 3′ and 5′ flanking regions of both <i>HoxA</i> and <i>HoxD</i> clusters. The colored bars represent 100% of the signal for each of the three tissues (color code at the bottom) and for three genes in either the <i>HoxD</i> (D) or the <i>HoxA</i> (E) clusters. The position of each bar with respect to the central black line (0) represents the balance between the contacts scored either in 5′ (left in D; right in E) or in the 3′ (right in D; left in E) landscapes. The <i>HoxA</i> and <i>HoxD</i> clusters are shown in opposite orientation regarding 3′ and 5′ directions to reflect their inverse locations on chromosomes 2 and 6. The four displayed topological domains were extracted from the Hi-C ES cell dataset of Dixon et al. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Dixon1" target="_blank">[32]</a>.</p

<i>Hoxa</i> gene expression in limb buds.

<p>(A) Expression of <i>Hoxa4</i>, <i>Hoxa9</i>, <i>Hoxa10</i>, <i>Hoxa11</i>, <i>Hoxa11</i> antisense (<i>Hoxa11as</i>), and <i>Hoxa13</i> in E12.5 limb buds. The <i>Hoxa11as</i> transcript <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-HsiehLi1" target="_blank">[37]</a> originates from a promoter within the intron of <i>Hoxa11</i> (upper panel) and is expressed like <i>Hoxa13</i>. (B) In control (WT) embryos, <i>Hoxa11</i> and <i>Hoxa13</i> are expressed in mutually exclusive domains, with <i>Hoxa13</i> in the autopod and <i>Hoxa11</i> in the distal zeugopod. In <i>Hoxa13</i> homozygous mutants embryos, the <i>Hoxa11</i> expression domain shifts into the proximal autopod, partly overlapping with <i>Hoxa13</i>. In <i>Hoxa13</i>^−/−/<i>Hoxd13^+/</i>⁻ double mutant animals, <i>Hoxa11</i>-expressing cells spread further distally. The <i>Hoxa13</i> probe is within the 3′ UTR and thus detects <i>Hoxa13</i> transcripts in mice carrying a loss of function for this gene. Although HOX13 proteins repress <i>Hoxa11</i> transcription, this latter gene has the capacity to respond to global distal enhancers, much like its <i>Hoxa9</i>, <i>Hoxa10</i>, <i>Hoxa11</i>, and <i>Hoxa13</i> neighbors (fl, forelimb; hl, hindlimb). The anterior-to-posterior polarity of the limb buds is indicated with arrows.</p

Regulatory mechanisms and the homology conundrum between fins and limbs.

<p>(A) The evolutionary changes that occurred during the transition from fins to limbs are mostly unresolved, in particular concerning the most distal segment of tetrapod limbs: the digits. Mammalian proximal and distal limb regions develop along with independent phases of <i>Hoxd</i> expression [indicated in red (arm) and blue (digits)] and fish fin buds have been probed for the existence of similar <i>Hoxd</i> expression patterns. A single expression domain of 5′ <i>Hoxd</i> genes along the distal fin margin (in grey) was interpreted either as corresponding to the distal phase in tetrapods or, alternatively, as homologous to the proximal phase <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Woltering1" target="_blank">[1]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Schneider1" target="_blank">[15]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Davis1" target="_blank">[16]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Sordino2" target="_blank">[20]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Shubin2" target="_blank">[25]</a>. Accordingly, radials (in grey) could be homologous with digits or this homology may not exist, in which case digits are tetrapod novelties. (B) Proximal (red) and distal (blue) <i>Hoxd</i> gene expression domains in the developing mouse limb are derived from enhancers located within distinct 3′- (red) and 5′- (blue) regulatory landscapes. The enhancer–promoter interaction profiles within these two landscapes were shown to precisely match two topological domains <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Andrey1" target="_blank">[31]</a> as determined by Hi-C using ES cells <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Dixon1" target="_blank">[32]</a>. This bimodal regulatory organization in tetrapods suggests distinct evolutionary trajectories for proximal and distal limbs. The presence or absence of such a modular regulatory strategy in fish would help clarify the origin of this mechanism and the homology between fins and limbs. The DNA domain shown is approximately 3 mb large.</p

Regulatory evolution and the fin-to-limb transition.

<p>Fish and tetrapod <i>HoxA</i> and <i>HoxD</i> clusters are regulated by 3′ and 5′ regulatory landscapes, represented here as triangles due to their correspondence to topological domains <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Andrey1" target="_blank">[31]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Dixon1" target="_blank">[32]</a>. Enhancer (indicated with colored shapes) interactions within these domains (indicated by arrows) occur with the neighboring parts of the <i>Hox</i> clusters, resulting in a regulatory partition between 3′ and 5′ parts of the clusters. In fishes, this mechanism may be used for patterning the fin proximal (red) to distal (orange) (P-D) polarity, through the potential function of these two landscapes in slightly different fin domains. Variation in the regulatory balance between these 3′ and 5′ landscapes through the acquisition of novel enhancers potentially explains interspecies differences in P-D fin morphology, as for instance between zebrafish and species such as coelacanth, which possesses a more elaborate fin skeleton. Although these regulatory landscapes may underlie the P-D patterning of fin skeletons, they both elicit a proximal response when assessed in transgenic mice, and hence the fish 5′ landscape is indicated as “proximal” (orange). In tetrapods, the 5′ domain (blue) has acquired new enhancers or modified existing ones, thereby evolving a novel, more distal autopodial identity, perhaps as a response to preexisting signals emanating from the apical ectoderm.</p

Zebrafish <i>Hox</i> clusters are partitioned into 3′ and 5′ interaction domains.

<p>4C analysis of zebrafish whole embryos (5 dpf, including well-developed fin buds) using as viewpoints (left) several genes within the <i>HoxAa</i> (A and B), <i>HoxAb</i> (C and D), and <i>HoxDa</i> (E and F) gene clusters (for <i>HoxAa</i>, see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773.s004" target="_blank">Figure S4</a>). The <i>HoxDa</i> cluster has a reversed chromosomal orientation when compared to both <i>HoxA</i> clusters. The percentages of interactions between the viewpoints and either the 5′ or the 3′ landscapes are indicated above each profile. Bar diagrams in (B, D, and F) give a summary of the signal directionality per viewpoint in the 3′ and 5′ flanking regions (compare <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio-1001773-g003" target="_blank">Figure 3C,D</a>). The blue bars are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio-1001773-g003" target="_blank">Figure 3</a>. Genes located at either extremity of their clusters display a strong bias toward the flanking landscape, such as <i>Hoxa4a</i> (B), <i>Hoxd4</i>a (F), <i>Hoxa13a</i> (B), or <i>Hoxd13a</i> (F). Genes located at more central positions in the clusters [e.g., <i>Hoxa11a</i> (B) or <i>Hoxd11a</i>, (F)] show more balanced interaction profiles, like for the mouse <i>HoxA</i> and <i>HoxD</i> clusters. Dark grey squares are regions of local interactions excluded from the analysis.</p

Coordinated changes in the expression of Wnt pathway genes following human and rat peripheral nerve injury

A human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood. Here we show that the expression of multiple genes of the Wnt family, including Wnt5a, is changed in NIC tissue from patients that underwent reconstructive surgery. The role of Wnt ligands in NIC pathology and nerve regeneration is of interest because Wnt ligands are implicated in tissue regeneration, fibrosis, axon repulsion and guidance. The observations in NIC prompted us to investigate the expression of Wnt ligands in the injured rat sciatic nerve and in the dorsal root ganglia (DRG). In the injured nerve, four gene clusters were identified with temporal expression profiles corresponding to particular phases of the regeneration process. In the DRG up- and down regulation of certain Wnt receptors suggests that nerve injury has an impact on the responsiveness of injured sensory neurons to Wnt ligands in the nerve. Immunohistochemistry showed that Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons traversing the NIC. Taken together, these observations suggest a central role for Wnt signalling in peripheral nerve regeneration