Comparison of effect between early and delayed in primary intramedullary nailing combined with locked plate fixation for the treatment of multi-segments tibial fractures of type / 中国骨伤

<p><b>OBJECTIVE</b>To compare the clinical results of early and delayed intramedullary nailing and locked plating for the treatment of multi-segments tibial fractures of type AO/ASIF-42C2.</p><p><b>METHODS</b>Between January 2010 and January 2013,45 patients with multi-segments closed tibial fractures of AO/ASIF-42C2 were treated by early primary intramedullary nailing and locked plating in 20 cases as early group and delayed in 25 cases as delayed group. In early group,20 cases included 13 males and 7 females with an average age of (37.9±14.3) years old ranging from 20 to 56 years;according to soft tissue injury Tscherne classification, 8 fractures were frade I,12 were grade II. In delayed group, 25 cases included 17 males and 8 females with an average age of (38.7±17.2) years old ranging from 24 to 55 years,4 fractures were grade I ,19 were grade II ,2 were grade III. The operative time, blood loss, hospital stay,fracture healing time and complications were recorded. At final follow-up, the Johner-Wruhs score were used to evaluate functional efficacy, and the posterior-anterior and lateral X-ray to evaluate fracture reduction and alignment.</p><p><b>RESULTS</b>All the patients were followed up for (12.5±2.5) months in early group and (13.2±2.8) months in delayed group (P>0.05). No wounds infections were happened. At the last follow-up, the mean range of knee joint was 10°-0°-120°. According to Johner-Wruhs scoring,there were 15 cases in excellent,3 in good,fair in 2 in early group; 21 in excellent,2 in good,2 in fair. The average operative time,blood loss had no significant differences between two groups (P>0.05), but hospital stay in early group was significantly shorter than those in delayed group(P<0.05). Average fracture healing time of early group and delayed group were (5.3±2.6) months and (6.0±2.9) months, respectively (P>0.05).</p><p><b>CONCLUSION</b>For multi-segments tibial fractures of type AO/ASIF-42C2 with preoperative minor soft tissue injuries lighter of Tscherne grade I or II, early primary intramedullary nailing and locked plating does not significantly increase the postoperative incidence of soft tissue complications for patients. The early and delayed primary intramedullary nailing and locked plating for treatment of AO/ASIF-42C2 proximal third tibial fractures can get similar curative effect.</p>

Study on T13254C polymorphism of the platelet membrane glycoprotein VI in Chinese Han population / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the T13254C polymorphism frequency in GPVI gene among Chinese Han population and its relevance to the arterial thrombotic diseases.</p><p><b>METHODS</b>The enrolled population in this study consisted of 314 healthy subjects and 274 patients with myocardial or cerebral infarctions. GPVI T13254C genotypes were determined by PCR amplification of a 355 bp fragment encompassing exon 5 of GPVI gene, followed by Msp I digestion of the product. The digested products were analyzed in 15% polyacrylamide gel electrophoresis (PAGE).</p><p><b>RESULTS</b>The frequencies of the T allele and C allele in the T13254C polymorphism were 0.9809 and 0.0191, respectively, with a frequency of heterozygous of 0.0319, which were significantly different from those reported in western population (P < 0.01). As compared with controls, no significant difference in T13254C genotype distribution was found in the arterial thrombotic diseases group.</p><p><b>CONCLUSION</b>The GPVI T13254C polymorphism appears in a low frequency in Chinese Han population. No relationship is found between T13254C polymorphism and the risk for thrombotic diseases.</p>

A2 domain of human von Willebrand factor expressed in E. coli and its biological activity / 中国实验血液学杂志

Von Willebrand factor (vWF) is the unique substrate for the metalloprotease, ADAMTS-13, and plays a pivotal role in the pathology of von Willebrand disease (vWD) and thrombotic thrombocytopenic purpure (TTP). To study the pathogenesis of TTP and to establish a method to diagnose TTP, the DNA fragment of vWF-A2 domain was amplified and inserted into expression vector with 6 x His tag (pQE-30), the recombinant expression vector was transformed into E. coli (strain M15) and induced by IPTG. The recombinant fragment comprising residues 718-905 of mature vWF was designated as rvWF-A2. It was purified by Ni-NTA resin column chromatography and refolded in Tris buffer containing GSH and GSSG. The results demonstrated that rvWF-A2 was expressed successfully in E. coli M15, amounting to 42% of total bacterial protein with the purity over 98%. It was identified that rvWF-A2 can be efficiently cleaved by the citrated normal plasma while no cleavage can be detected by the TTP plasma or plasma with EDTA. It is concluded that rvWF-A2 expressed efficiently in E. coli demonstrated excellent biological activity, which lays a solid foundation for establishment of method to measure quantatively the activity of ADAMTS-13.

Expression of the metalloproteinase domain of von Willebrand factor-cleaving protease and preparation of its McAb / 中国实验血液学杂志

The von Willebrand factor-cleaving protease (vWF-cp) is a newly identified plasma metalloproteinase and plays an important role in the pathogenesis of thrombotic microangiopathy. In the present study, the metalloproteinase domain of vWF-cp was expressed by IPTG-induced the recombinant engineered E.coli strain harbouring pET28a (+)-vWF-cp/MD and purified using chromatography on Ni-NTA column. Then the BALB/c mice were immunized with the recombinant protein to prepare the monoclonal antibodies (McAb) against vWF-cp and the obtained McAbs were characterized. Furthermore, the expression panels of vWF-cp in human normal tissues were investigated using immunohistochemistry. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 28% of total bacteria protein. Three monoclonal antibodies against the metalloproteinase domain of vWF-cp were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belong to IgG(1). The concentration of them in ascites was 4 mg/ml, and their titers were as high as 1 x 10(-5). The data of ELISA showed that SZ-112 and SZ-113 recognized different epitopes of recombinant protein. The Western blot and immunoprecipitation data showed that the two monoclonal antibodies reacted not only with the recombinant protein, but also with a 200 kD protein in platelet lysate. Moreover, the vWF-cp was found to be present in the cytoplasm of many human tissues such as liver, prostate, ovary, etc. However, the protease could not be detected in brain tissue. In conclusion, the above-mentioned research work contributed not only to the further study of the structure and function of this protease, but also to the establishment of the method for quantifying the vWF-cp antigen in plasma.

Residual collagen binding assay for von Willebrand factor-cleaving protease activity and its clinical application / 中国实验血液学杂志

von Willebrand factor-cleaving protease (vWF-cp) is a newly identified metalloproteinase. The activity of vWF-cp would vary in different physiological or pathological states. To explore activity detectron of von Willebrand factor-cleaving protease and its clinical application, the vWF-cp activity was measured by a sensitive enzyme-linked immunoadsorbent assay to detect the residual collagen binding activity (R-CBA) of von Willebrand factor before and after digestion with vWF-cp. Moreover, its activity deficiency in patients with thrombotic thrombocytopenic purpura (TTP) and solid tumors was also investigated. The results showed that the residual collagen binding assay was sensitive enough to measure the serum or plasma vWF-cp activity in 87 health individuals, 79 patients suffering from TTP and solid tumors. The coefficient of variation within and between the batches was were 3.60% and 8.35%, respectively. The serum and plasma vWF-cp activity in health individuals was (79.47 +/- 10.78)% (n = 53) and (78.79 +/- 9.17)% (n = 30), respectively, whereas the vWF-cp activity in patients with TTP, benign and malignant tumors was significantly decreased (P values were less than 0.001, 0.03 and 0.001, respectively). It is concluded that the vWF-cp activity in plasma or serum of patients with TTP and solid tumors markedly decrease, especially in patients with TTP. Assay of the vWF-cp activity using R-CBA is a simple method.

Study of C1423T polymorphism of the von Willebrand factor-cleaving protease gene in Chinese Han population / 中华血液学杂志

<p><b>OBJECTIVE</b>The C1423T polymorphism in von Willebrand factor-cleaving protease (vWF-cp) gene affects its enzyme activity. The present study was to investigate the polymorphism frequency among Chinese Han population and its relevance to arterial thrombotic disorders.</p><p><b>METHODS</b>An amplified 366 bp fragment of human vWF-cp gene was analyzed by Rsa I restriction assay in 400 unrelated individuals including 150 with acute ischemic stroke (AIS), 103 with acute myocardial infarction (AMI) and 147 age- and gender-matched healthy controls. The resulting products were analyzed by 12% polyacrylamide gels electrophoresis and stained with ethidium bromide.</p><p><b>RESULTS</b>Twelve cases were C1423T heterozygous, the C1423T frequencies were 98.5% and 1.5%, and the heterozygosity and allele frequency were 3% and 1.5%, respectively, which were remarkably lower than those reported in Japanese population. No 1423T/T homozygote was found. Besides, there was no significant difference between healthy controls and patients with thrombotic disorders.</p><p><b>CONCLUSION</b>C1423T polymorphism is low frequency in both controls and patients of Han population.</p>

Evaluation of von Willebrand factor-cleaving protease activity in patients with thrombotic thrombocytopenic purpura / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.</p><p><b>METHODS</b>The plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover, the serum vWF-cp activities were compared between the patients with TTP and those with tumors.</p><p><b>RESULTS</b>The coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79 +/- 9.17)% (n = 30) and (79.47 +/- 10.78)% (n = 53), respectively, while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83 +/- 19.98)%, P < 0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased (P < 0.03 and P < 0.001, respectively), they were relatively high in comparison with that of TTP patients (P < 0.001).</p><p><b>CONCLUSION</b>Measurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.</p>