Differentiation of Patients with Symptomatic Low von Willebrand Factor from Those with Asymptomatic Low von Willebrand Factor.

BACKGROUND: Accurate diagnosis of symptomatic low von Willebrand factor (VWF) remains a major challenge in von Willebrand disease (VWD). However, present tests do not adequately take into account flow forces that, at very high shear rates, reveal a weakness in the VWF-platelet glycoprotein glycoprotein Ib bond in normal subjects. The degree of this weakness is greater in symptomatic, but not asymptomatic, low VWF. OBJECTIVE: The aim of this study is to distinguish patients with symptomatic low VWF (levels in the 30-50 IU/dL range) from those with asymptomatic low VWF and normal subjects. METHODS: We measured platelet adhesion (PA)/aggregation in our novel microfluidic flow system that permits real-time assessment of PA (surface coverage) and PA/aggregation (V, aggregate volume) using epifluorescence digital videomicroscopy in flowing noncitrated whole blood at 4,000 second-1. Blood samples from 24 low VWF patients and 15 normal subjects were collected into plastic tubes containing 4 U/mL enoxaparin. MetaMorph software was used to quantify rates of PA and V increase. RESULTS: Rates of PA increase showed a bimodal distribution, with values for 16/24 patients (Group I) all below the 2.5th percentile of normal, and values for 8/24 patients (Group II) similar to controls. Bleeding scores (mean ± standard error) were 5.50 ± 0.45 versus 2.75 ± 0.45 (p = 0.00077), and 10 clinically significant bleeding events were observed in seven versus zero (p = 0.0295) Group I and Group II subjects, respectively. CONCLUSION: The present approach may offer a definitive means to distinguish symptomatic low VWF from either asymptomatic low VWF or normal controls.

Human mannose-binding lectin inhibitor prevents Shiga toxin-induced renal injury.

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2-expressing mice (MBL2 KI) that lack murine Mbls (MBL2(+/+)Mbl1(-/-)Mbl2(-/-)), a novel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, but a significantly decreased glomerular filtration rate compared with control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition in glomeruli in Stx-2-injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels and significantly attenuated Stx-2 induced-renal injury, free serum hemoglobin levels, renal C3d, and fibrin deposition and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can be used to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2-induced renal injury.

Genetic analysis of Escherichia coli†RadA: functional motifs and genetic interactions.

The RadA/Sms protein is a RecA-related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double-strand break repair for YejH.