VWF multimer analysis in patients without CALI who were not treated with bevacizumab.

<p>Results of VWF multimer analysis in 4 representative patients out of 11 patients without CALI not treated with bevacizumab are shown. UL-VWFMs were found in Patients 10, 14, and 15, who did not develop CALI. In Patient 10, decreased levels of H-VWFMs were observed during months 1 and 4. VWF von Willebrand factor, CALI chemotherapy-associated liver injury, UL-VWFMs unusually-large VWF multimers, H-VWFM high molecular weight VWF multimers, AST aspartate transaminase, T-Bil total bilirubin, VWF:Ag VWF antigen, VWF:CB VWF collagen binding activity.</p

Immunohistochemical analysis of liver specimens in patients with SOS.

<p>Histological findings in the liver with hematoxylin and eosin (H. E.) staining in Patient 4 included extensive Grade 2 sinusoidal dilatation (A) and platelet thrombi in the liver sinusoids, as indicated with white arrows (B). Many of these thrombi were positive for both platelet-specific anti-IIb/IIIa (C, D) and anti-VWF (E), which showed that they are platelet thrombi, as indicated with arrows. Some thrombi were positive for fibrinogen (F), which indicated that they are fibrinogen thrombi, but there were much less frequently observed than platelet thrombi.</p

Comparison of platelet count, VWF:Ag, ADAMTS13:AC, and AST between patients with splenomegaly and those without splenomegaly.

<p>(A) Platelet counts decreased as the number of chemotherapy cycles increased in patients with and without splenomegaly. (B) Plasma levels of VWF antigen (:Ag) increased as the number of chemotherapy cycles increased in patients with splenomegaly. (C) Plasma levels of ADAMTS13:AC were unchanged in both groups. (D) Plasma levels of aspartate transaminase (AST) increased as the number of chemotherapy cycles increased in patients with splenomegaly, but not in patients without splenomegaly. VWF:Ag von Willebrand factor antigen, ADAMTS13:AC ADAMTS13 activity, AST Aspartate transaminase.</p

Clinical characteristics, chemotherapy regimen, response of chemotherapy and spleen size ratio.

<p>^# tumor response was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST). (response)</p><p>^## the spleen size ratio was the ratio of spleen size after chemotherapy to those before chemotherapy (spleen size ratio)</p><p>* unresectable metastatic colorectal cancer (UMCRC)</p><p>** recurrence (rec.)</p><p>Patients 18 through 23 were treated without bevacizumab.</p><p>Clinical characteristics, chemotherapy regimen, response of chemotherapy and spleen size ratio.</p

Comparison of platelet count, VWF:Ag, ADAMTS13:AC, AST, and T-Bil between patients treated with and not treated with bevacizumab.

<p>(A) Platelet counts decreased until months 3 as the number of chemotherapy cycles increased in both patients who received and did not receive bevacizumab. However, platelet counts in patients not treated with bevacizumab decreased much less in patients who received bevacizumab in months 5. (B) Plasma levels of VWF:Ag increased as the number of chemotherapy cycles increased in patients not treated with bevacizumab, but did not change in patients treated with bevacizumab. (C) Plasma levels of ADAMTS13:AC were unchanged in both groups. (D) Serum AST levels increased as the number of chemotherapy cycles in patients who did not receive bevacizumab, but they were unchanged in patients with bevacizumab. (E) Plasma levels of T-Bil did not change significantly in either group. VWF:Ag von Willebrand factor antigen, ADAMTS13:AC ADAMTS13 activity, AST Aspartate transaminase, T-Bil total bilirubin.</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

VWF multimer analysis in patients with CALI who were not treated with bevacizumab.

<p>VWF multimer analysis was performed in 4 representative patients out of 6 patients with CALI not treated with bevacizumab. These patients developed CALI during month 3 or 4. UL-VWFMs were found before and during CALI in all patients. Decreased levels of H-VWFMs were found in Patient 4 at months 0 and 1, and in Patient 17 at month 2. VWF von Willebrand factor, CALI chemotherapy-associated liver injury, UL-VWFMs unusually-large VWF multimers, H-VWFM high molecular weight VWF multimers, AST aspartate transaminase, T-Bil total bilirubin, VWF:Ag VWF antigen, VWF:CB VWF collagen binding activity.</p

Family 2A with a C3-p.I1157T mutation.

<p>(A) Patient 2A1 (male) had six bouts of atypical hemolytic uremic syndrome (aHUS), at the age of 9, 15, 18, 22 and 29 (twice). Gray squares or circle indicate individuals who were not analyzed in this study. (B) The hemolytic assay showed that neither patient (P) nor his parents (father: F and mother: M) had appreciable hemolysis. However, Patient 2A1 and his father carried the same predisposing mutation p.I1157T in C3, confirmed by restriction fragment length polymorphism (RFLP) analysis (C) and direct DNA sequencing.</p

Relationship between hemolytic activity and complement abnormalities in 45 patients with atypical hemolytic uremic syndrome (aHUS).

<p>The results of the hemolytic assay for 45 aHUS patients are shown according to the predisposing (bold type) or potentially predisposing mutations and/or the acquired abnormalities. The patients carrying two mutations were classified according to the predisposing mutation, and the other mutations were described alongside the predisposing mutations or black bars. Two patients (2V1 and H2) were categorized into the C3 group, although they had two predisposing mutations (C3-p.I1157T and THBD-p.D486Y). In two patients (2P1 and 3G1), the hemolysis induced by serum was represented by gray bars. The normal range of hemolysis (5.4±1.8%: mean ± standard deviation) obtained from 20 healthy individuals (10 males and 10 females) is shown at the bottom of Fig 3. The results of the restriction fragment length polymorphism (RFLP) analysis of C3-p.I1157T are summarized to the right of the hemolytic assay results. Regarding the RFLP analysis of C3-p.I1157T (c.3470T>C), the heterozygous mutation (T/C) showed two bands (278 bp, 314 bp) and the homozygous mutation (C/C) showed one band (314 bp). † H2 had a homozygous mutation in C3-p.I1157T.</p

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