Delimiting the autoinhibitory module of von Willebrand factor.

Essentials The self-inhibitory mechanism of von Willebrand factor (VWF) remains unclear. Residues flanking the A1 domain of VWF form a discontinuous autoinhibitory module (AIM). rVWF1238-1493 exhibited greater thermostability and inactivity than its shorter counterparts. The cooperative coupling between the N- and C- AIM regions are required for inhibiting A1. SUMMARY: Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF1238-1493 ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF1238-1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF1238-1461 , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF1271-1493 , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

Multiple vertebral osteotomies in the treatment of rigid adult spine deformities.

STUDY DESIGN: Retrospective review of the clinical and radiographic results in adult revision spine deformity surgery using the techniques of osteotomies to effect spine balance and curve correction. OBJECTIVES: To assess the efficacy of multiple vertebral osteotomies in correction of rigid spine deformities in adult patients undergoing revision surgery. METHOD: The records and radiographs of 27 adult patients with idiopathic scoliosis who underwent revision surgery requiring anterior release (discectomy and/or osteotomy) and posterior osteotomy to correct rigid spinal deformities were retrospectively reviewed. RESULTS: All 27 patients were available for follow-up evaluation. Fifteen patients had anterior discectomies followed by posterior osteotomies, whereas 12 had anterior and posterior osteotomies in staged or sequential (same day) fashion. Diagnosis was idiopathic scoliosis for the index operation. At revision, the primary deformity was flatback deformity in 10 patients and pseudarthrosis with progressive deformity in 17 patients. Eleven patients had predominant sagittal decompensation, 11 patients had multiplanar decompensation, and five patients were balanced. The average number of osteotomies performed anteriorly was 4.3 levels (range, 1-8) and the average number of osteotomies posteriorly was 4.6 levels (range, 1-10). There were a total of nine complications in eight patients including three pseudarthroses (11%), five hardware failures (19%), and one transient neurologic deficit (4%). There were no deep wound infections, deep vein thromboses, pulmonary emboli, or deaths. The average scoliosis correction was 40% (range, 5-81%), whereas the average sagittal balance was corrected 6.5 cm (range, -5-29.5 cm), on average, and coronal balance was corrected 2.5 cm (range, 1-6 cm), on average. CONCLUSION: This study demonstrates multiple vertebral osteotomies (anterior and/or posterior) in the management of rigid adult spine deformities and deformity correction with an acceptable complication rate. Use of vertebral osteotomies for patients undergoing revision spine surgery is a safe and reasonable approach to obtain an arthrodesis.

Use of a tobramycin-impregnated polymethylmethacrylate pin sleeve for the prevention of pin-tract infection in goats.

OBJECTIVE: To test the hypothesis that antibiotic-laden polymethylmethacrylate (PMMA) pin sleeves prevent infection around skeletal external fixation pins. DESIGN: An experimental study using an animal model was conducted. ANIMALS: In each of five goats, three four-millimeter half-pins were placed in the left and right iliac crests, for a total of thirty half-pins. The pins were infected with one milliliter of broth containing 7.6 x 10(5) colony-forming units per milliliter of Staphylococcus aureus (ATCC 25923). INTERVENTION: The pins in the right iliac crest were treated with the tobramycin-impregnated pin sleeves, and the pins in the left iliac crest (control) were left untreated. RESULTS: The results showed clinical evidence of infection (i.e., looseness and gross pus) and heavy bacterial growth (average 6.8 x 10(10) colony-forming units per milliliter) for the untreated pins, but no clinical evidence of infection and no bacterial growth at forty-eight hours for the treated pins. CONCLUSION: The present results indicate that the use of the antibiotic-impregnated PMMA pin sleeve can prevent the development of pin-tract infection and appears to prevent colonization of the external fixation pins.

Antibiotic-leaching from polymethylmethacrylate beads.

Aminoglycoside-impregnated polymethylmethacrylate beads, which are used to deliver antibiotic directly to infected sites in the musculoskeletal system, are available as a manufactured product or they can be mold-made by a pharmacy or hand-rolled by the orthopaedist in the operating suite. We investigated the leaching of antibiotic from each of these types of beads. Our hypothesis was that the elutions of antibiotic from the three types of beads are similar. Three study groups (hand-made, mold-made, and manufactured beads), each composed of four five-bead subsets, were formed so that twenty beads of each type were tested. Each bead was leached daily in a two-milliliter aliquot of normal saline solution throughout a sixty-day period, and the aminoglycoside concentration in twenty of these aliquots was determined. Analysis of variance showed no statistically significant differences when the antibiotic elutions within each subset, between the different subsets, and between the three groups were compared. The clinically important finding of this investigation is that the leaching characteristics of the three types of aminoglycoside-impregnated beads are equivalent when the beads have been fabricated out of comparable materials.

The use of antibiotic-impregnated polymethylmethacrylate beads to prevent the evolution of localized infection.

The effect of antibiotic bead chains on the evolution of infection cannot be studied entirely in man due to the ethical problems of obtaining valid controls. Therefore, a model of musculoskeletal injury was devised in rabbits by making a paraspinal wound, fracturing a spinous process, and contaminating the wound with 1 x 10(4) colony-forming units/ml of Staphylococcus aureus. These contaminated wounds were treated with tobramycin-containing polymethylmethacrylate (PMMA) beads. Control rabbits were either treated with PMMA beads that did not contain antibiotic, treated with IM tobramycin, or left untreated. At 5 days, six of eight animals treated with antibiotic-impregnated beads had no recoverable organisms. Six of eight rabbits receiving IM tobramycin had wound infections, and five of five in whom non-antibiotic-containing beads had been implanted had significant wound infections, with one of the five dying of sepsis on the 3rd day of the experiment. The clinical course of infected controls was the same as the course of those animals receiving IM antibiotics and the same as those in whom beads without antibiotics were implanted. That is, the rabbits had grossly infected wounds and the organisms recovered were of the same type as those implanted. This research shows a highly statistically significant effect of tobramycin-containing antibiotic beads in retarding the evolution of an experimental Staphylococcus infection in rabbits.