Human placental ectonucleoside triphosphate diphosphohydrolase gene transfer via gelatin-coated stents prevents in-stent thrombosis.

BACKGROUND: In-stent thrombosis is mainly triggered by adenosine diphosphate (ADP)-dependent platelet aggregation after percutaneous coronary stent implantation. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) rapidly hydrolyzes ADP to adenosine monophosphate, inhibiting platelet aggregation. We tested the hypothesis that local delivery of human placental E-NTPDase (pE-NTPDase) gene into injured arteries via gene-eluting stent could prevent subacute in-stent thrombosis. METHODS AND RESULTS: We generated gene-eluting stents by coating bare metal stents with cationic gelatin hydrogel containing pE-NTPDase cDNA (pE-NTPDase stent), and implanted the stents into rabbit femoral arteries (FA) prone to production of platelet-rich thrombi due to repeated balloon injury at 4-week intervals. After the second injury, E-NTPDase gene expression was severely decreased; however, the implantation of pE-NTPDase stent increased E-NTPDase mRNA levels and NTPDase activity to higher level than normal FA. The FAs with pE-NTPDase stents maintained patency in all rabbits (P<0.01), whereas the stent-implanted FAs without pE-NTPDase gene showed low patency rates (17% to 25%). The occlusive platelet-rich thrombi, excessive neointimal growth, and infiltration of macrophages were inhibited in stent implanted FA with pE-NTPDase gene, but not without pE-NTPDase gene. CONCLUSIONS: Human pE-NTPDase gene transfer via cationic gelatin-coated stents inhibited subacute in-stent thrombosis and suppressed neointimal hyperplasia and inflammation without antiplatelet drugs.

Thrombotic thrombocytopenic purpura associated with pegylated-interferon alpha-2a by an ADAMTS13 inhibitor in a patient with chronic hepatitis C.

A deficiency of ADAMTS13 leads to platelet clumping and/or thrombi formation, finally resulting in thrombotic thrombocytopenic purpura (TTP). In this study, a 62-year-old male with chronic hepatitis C developed TTP a month after long-term pegylated-interferon (PEG-IFN) treatment. The observed low level of activity of plasma ADAMTS13 following PEG-IFN treatment was shown to gradually increase with the improvement of TTP, while the titer of an inhibitory anti- ADAMTS13 IgG antibody decreased concomitant with the increase in ADAMTS13 activity. Serial determination of ADAMTS13 activity and its inhibitor may provide useful information for the diagnosis and treatment of IFN-associated TTP, as well as its pathogenesis.

Von Willebrand factor-cleaving protease and Upshaw-Schulman syndrome.

Vascular endothelial cell (EC)-produced plasma von Willebrand factor (vWF) plays a critical role in primary hemostasis through its action of anchoring platelets onto the injured denuded subendothelial matrices under high shear stress. Unusually large vWF multimers (UL-vWFMs), present in plasma immediately after release from ECs, are most biologically active, but they are soon cleaved and degraded into smaller vWFMs by a specific plasma protease, termed vWF-cleaving protease (vWF-CPase), in normal circulation. Recent studies on the relationship between UL-vWFMs and vWF-CPase, together with its autoantibody (inhibitor) have brought about a clear discrimination between thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Furthermore, a congenital deficiency of this enzyme activity has been shown to cause Upshaw-Schulman syndrome, a complex constitutional bleeding diathesis. Successful purification of vWF-CPase revealed that this enzyme is composed of a single polypeptide with a molecular mass of approximately 190 kd, and its complementary DNA cloning unambiguously indicated that it is uniquely produced in the liver and its gene is located on chromosome 9q34. The messenger RNA of vWF-CPase had a span of 4.6 kb, and its enzyme was designated ADAMTS 13. The predicted complete amino acid sequence of this enzyme consisted of 1427 residues, including a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain, an ADAMTS spacer, 7 additional TSP1 repeats, and 2 CUB domains.

Two newborn-onset patients of Upshaw-Schulman syndrome with distinct subsequent clinical courses.

Upshaw-Schulman syndrome (USS) is caused by a congenital deficit in ADAMTS13 activity owing to genetic mutations. USS is characterized by severe neonatal jaundice with a negative Coombs test and repeated childhood episodes of thrombocytopenia reversible by fresh frozen plasma (FFP) infusions. We present two patients with USS, both of whom underwent exchange blood transfusions as newborns, although the disease subsequently developed along different clinical courses. USS-CC5 initially received a diagnosis of neonatal jaundice due to fetomaternal ABO incompatibility with an indirect positive Coombs test, which masked the diagnosis of USS. Before prophylactic FFP infusions were initiated, USS-CC5 had chronic thrombocytopenia. In contrast, thrombocytopenia developed in USS-HH4 only in response to infections and spontaneously normalized without FFP infusions. Analyses of the ADAMTS13 genes in USS-CC5 and USS-HH4 revealed compound heterozygotes of p.R398C/p.Q723K and p.Q449X/p.Q1374Sfs, respectively. Analysis of von Willebrand factor (VWF) multimers in plasma samples taken from both patients in remission showed single symmetrical multimer bands, which differ from the triplet structure of bands observed in normal samples. These data suggested that plasma VWF multimers in the patients had not been proteolytically modified. Our results indicate the presence of a previously unknown regulatory mechanism for VWF-dependent high-shear stress-induced platelet aggregation.

Influence of bioresorbable, unsintered hydroxyapatite/poly-L-lactide composite films on spinal cord, nerve roots, and epidural space.

The effect of forged unsintered hydroxyapatite/poly-L-lactide (u-HA/PLLA) composite films on spinal cord and nerve roots and its degradation behavior and osteoconductivity in epidural space were compared with those of calcined HA (c-HA)/PLLA and unfilled PLLA films. Partial laminectomy was performed on 20 rabbits, and u-HA/PLLA and PLLA films were implanted in the intervertebral space. Total laminectomy was performed on 30 rabbits to implant u-HA/PLLA, c-HA/PLLA, and PLLA films in both epidural and subcutaneous spaces. For up to 50 weeks, there were no histological changes in the spinal cord or nerve root, and no inflammatory cell infiltration into the epidural space around the films. The rate of decrease in viscosity average molecular weight of both composite films was initially higher than that of PLLA but eventually became lower, although there was no difference in the degradation behavior of the three films in either the epidural or subcutaneous spaces after 50 weeks. Scanning electron microscopic and energy-dispersive X-ray analysis indicated calcium phosphate deposits on the surface of composite films with new bone formation from 4 weeks. The u-HA/PLLA composite film therefore has good biocompatibility, osteoconductivity, and fast primary degradation rate, which may prove compatible with application to spinal surgery.

Comparative biomechanical analysis of a cervical cage made of an unsintered hydroxyapatite particle and poly-L-lactide composite in a cadaver model.

STUDY DESIGN: A new cage made from a forged composite of unsintered hydroxyapatite particles and poly-L-lactide (F-u-HA/PLLA) is compared biomechanically with the Ray threaded fusion cage. OBJECTIVES: To compare the stability imparted to the human cadaveric spine by two different threaded cervical cages and the effect of cyclic loading on construct stability. SUMMARY OF BACKGROUND DATA: Threaded cages have been developed for use in anterior cervical interbody fusions to provide initial stability during the fusion process. However, metallic instrumentation has several limitations. Recently, totally bioresorbable bone fixation devices made of F-u-HA/PLLA have been developed, including a cage for spinal interbody fusion. However, no biomechanical study has compared the F-u-HA/poly-L-lactide (PLLA) cage with metallic cages. METHODS: For this study, 12 fresh ligamentous human cervical spines (C4-C7) were used. After anterior discectomy across C5-C6, stabilization was achieved with the F-u-HA/PLLA cage in six spines and with the Ray threaded fusion cage in the remaining six spines. Biomechanical testing of the spines was performed with six degrees of freedom before and after stabilization, and after cyclic loading of the stabilized spines (5000 cycles of flexion-extension at 0.5 Nm). RESULTS: The specimens stabilized with either the F-u-HA/PLLA cage or the Ray cage were significantly more stable than the discectomy case in all directions except in extension. In extension, both groups were stiffer, although not at a significant level (P > 0.05). After fatigue, the stiffness, as compared with that in the prefatigue case, decreased in both groups, although not at a significant level. The Ray cage group exhibited better stability than the F-u-HA/PLLA cage group in all directions, although a significant difference was found only in right axial rotation. CONCLUSIONS: The F-u-HA/PLLA cage has the possibility to supplant the use of metallic devices in interbody fusions of the cervical spine.

Hydroxyapatite block for use in posterolateral lumbar fusion: a report of four cases.

Autologous bone grafts for posterolateral lumbar fusion are harvested from the iliac crests. Recently, several alternatives to autologous bone have been evaluated (such as graft substitutes, graft extenders, or both) with variable results. However, no clinical long-term studies have validated the efficacy of these techniques in posterolateral lumbar fusion. This study evaluated radiographic and histologic findings in four patients (mean age, 66 years) during the first 5 years after posterolateral fusion with an hydroxyapatite block. The mean followup was 7 years 1 month. Radiologic evaluation was by plain radiographs and computed tomography scans. Histologic evaluation was done in one patient. Capillaries extended into the porous structure of the hydroxyapatite substrate, and some of the pores were replaced by newly formed bone tissue. The long-term results of graft substitutes were stable and hydroxyapatite appeared to have some potential to achieve union in posterolateral lumbar fusion. However, hydroxyapatite block alone has not functioned effectively as a complete graft substitute in posterolateral lumbar fusion. Thus, a suitable osteogenic material is required to induce the formation of new bone and achieve a solid union.