The integrin-linked kinase-PINCH-parvin complex supports integrin αIIbβ3 activation.

Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (αIIbα6Bβ3) and mutant CHO cells expressing inactive αIIbα6Bβ3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and α-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and α-parvin, resulting in the restoration of αIIbα6Bβ3 activation. In the parental cells expressing active αIIbα6Bβ3, ILK, PINCH, and α-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both α- and β-parvin mRNA in the parent cells impaired the αIIbα6Bβ3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore αIIbα6Bβ3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bβ3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbβ3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbβ3 activation and the THD-induced αIIbβ3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the αIIbβ3-expressing CHO cells further augmented THD-induced αIIbβ3 activation, whereas they did not induce αIIbβ3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation

A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan.

For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients

Characterization of ILK-deficient mutant cells expressing inactive αIIbα6Bβ3.

<p>(A) Immunoblotting for ILK, PINCH, α-parvin, talin, and kindlin-2. Cell lysates obtained from parental cells with constitutively active αIIbα6Bβ3, ILK-deficient mutant cells with inactive αIIbα6Bβ3, and mutant cells transiently transfected with rat ILK cDNA were electrophoresed on SDS-PAGE gels and immunoblotted with indicated Abs. GAPDH shows an internal loading control. (B) Flow cytometry analysis showing PAC-1 (an activation-specific mAb for αIIbβ3) binding to mutant cells transiently transfected with either ILK plasmid or empty plasmid. Bound PAC-1 was detected with a PE-conjugated secondary mAb. </p

Knockdown effects of PINCH, parvins, and kindlin-2 in αIIbα6Bβ3-active parental cells.

<p>αIIbα6Bβ3-active parental cells were transiently transfected with PINCH siRNAs (p157 and p755) (A), α-parvin siRNAs (pa503 and pa761) (C), β-parvin siRNAs (pb900 and pb1011) (C), kindlin-2 siRNAs (k770 and k1733) (E), negative control siRNAs, and scrambled siRNAs. Cell lysates were electrophoresed on SDS-PAGE gels, and the separated proteins were immunoblotted with the indicated Abs. GAPDH and β-actin are shown as internal loading controls. The activation indexes of transfected cells (B, D, F) were calculated using the formula shown in Materials and Methods. A value of 100% implies the maximum PAC-1 binding to the cells treated with dithiothreitol (DTT). Data represent means ± standard deviation (SD) of three (B, F) or four (D) independent experiments. ** indicates <i>P</i> < 0.01.</p

Detection of IPP complex proteins in αIIbα6Bβ3-active parental cells.

<p>Cell lysates obtained from αIIbα6Bβ3-active parental cells were immunoprecipitated with Abs against PINCH (A), α-parvin (B, C), and ILK (D). The co-precipitates were detected by Abs for α-parvin (A), ILK (B), and PINCH (C, D). IgG means immunoprecipitation (IP) using non-immune control IgG. IB stands for immunoblotting. Arrows indicate the predicted sizes of the indicated proteins. Arrowheads (D) indicate the antibody heavy chains used in the IP. Different mobilities between those of the two IgG antibodies are probably caused by differences in the amino acid compositions of them. </p

Correction: A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan.

[This corrects the article DOI: 10.1371/journal.pone.0124655.]

Effects of ILK mutants with defects in either PINCH or parvin binding.

<p>The activation indexes of transfected cells (A, C, E). ILK-deficient mutant cells were transiently transfected with GFP cDNA, GFP-fused wild-type ILK (GFPILK-WT) cDNA, GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) cDNA (A, E), or GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) cDNA (C, E). After transfection, the binding of either PAC-1 (A, C) or fibrinogen (E) to the cells was analyzed by flow cytometry. The activation index was determined by the formula shown in Materials and Methods. A value of 100% represents the maximal binding of PAC-1 or fibrinogen to the cells treated with dithiothreitol. Data represent means ± SD of three independent experiments. ** indicates <i>P</i> < 0.01. Immunoblotting showing protein expression of GFP (B, D), GFP-fused wild-type ILK (GFPILK-WT) (B, D), GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) (B), and GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) (D) in ILK-deficient mutant cells. Cell lysates were electrophoresed and immunoblotted with indicated Abs. </p

A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan.

For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients.博士（医学）・甲第638号・平成27年7月31日© 2015 Yoshida et al. This is an open access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.identifier:PloS one Vol.10 No.5 Article No.e0124655 (2015 May 7)identifier:19326203identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/3076identifier:PloS one, 10(5): Article No.e012465

Family 2K with no predisposing or potentially predisposing mutations.

<p>(A) Patient 2K1 (female) developed atypical hemolytic uremic syndrome (aHUS) at the age of 35. Her husband (gray square) was not analyzed in this study. (B) The hemolytic assay showed that plasma from the patient (P), her father (F), and two daughters (D1 and D2) induced severe hemolysis, which was not observed in her mother (M). (C) The enhanced hemolysis detected in these four family members was corrected by the addition of purified complement factor H (CFH). However, no predisposing or potentially predisposing mutations were detected in Patient 2K1.</p

Comparison of genetic or acquired abnormalities among Western countries and Japan.

<p>The frequency of each genetic or acquired abnormality in our aHUS cohort in Japan was compared to that in three other cohorts (Italy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124655#pone.0124655.ref032" target="_blank">32</a>], USA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124655#pone.0124655.ref033" target="_blank">33</a>], France [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124655#pone.0124655.ref034" target="_blank">34</a>]). The data reported by Noris et al. included 47 secondary aHUS patients. Sixty-percent of aHUS patients in our cohort were enrolled from West Japan. In the data of Japan, both predisposing and potentially predisposing mutations were counted. Although two patients (2V1 and H2) had two predisposing mutations (C3-p.I1157T and THBD-p.D486Y), they were counted as C3 group.</p><p>CFH: complement factor H, Ab: antibody, C3: complement component C3, MCP: membrane cofactor protein, THBD: thrombomodulin, CFB: complement factor B, CFI: complement factor I, ND: no data</p><p>Comparison of genetic or acquired abnormalities among Western countries and Japan.</p