Main Factors Influencing the Platelet Spreading / 中国实验血液学杂志

OBJECTIVE@#To explore the main factors of platelet spreading and provide the foundation for related research.@*METHODS@#Platelets (2×107/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×107/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists.@*RESULTS@#Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing.@*CONCLUSION@#The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.

Tratamento percutâneo de pseudoaneurismas por injeção de trombina guiada por ultrassom / Percutaneous treatment of pseudoaneurysms by ultrasound-guide thrombin injection

Pseudoaneurisma iatrogênico ocorre em 0,5% a 2% dos procedimentos percutâneos diagnósticos e em até 7% a 8% dos procedimentos percutâneos terapêuticos. A oclusão do pseudoaneurisma pela injeção de trombina guiada por ultrassom diretamente no interior do saco aneurismático é técnica pouco invasiva, de execução rápida e segura, e com alto índice de sucesso. A proposta deste trabalho é relatar nossa experiência no tratamento do pseudoaneurisma iatrogênico por injeção de trombina guiada por ultrassom. Método: Entre setembro de 2006 e fevereiro de 2010 realizamos 1.811 procedimentos cardíacos percutâneos com ocorrência de 15 pseudoaneurismas (0,8%), dos quais 13 foram tratados com injeção de trombina guiada por ultrassom de forma consecutiva. Características clínicas, tipo de procedimento, medicações coadjuvantes, tamanho e morfologia dos pseudoaneurismas, dose de trombina utilizada, resultado da terapia e ocorrência de complicações foram documentados e armazenados prospectivamente em banco de dados e, então, analisados retrospectivamente. Resultados: A taxa de sucesso primário foi de 100%, com apenas uma recidiva sete dias após a abordagem inicial, tratada com sucesso pela mesma técnica. Não foi observada ocorrência de complicações relacionadas à injeção de trombina guiada por ultrassom e todos os pacientes apresentavam-se assintomáticos aos 30 dias de acompanhamento. Conclusão: A injeção de trombina guiada por ultrassom realizada por profissionais com experiência em ultrassom vascular e em punções transparietais de cistos e coleções é uma opção terapêutica viável, prática e segura no tratamento do pseudoaneurisma iatrogênico.

Background: Iatrogenic pseudoaneurysm is observed in 0.5% to 2% of diagnostic percutaneous procedures and in up to 7% to 8% of therapeutic percutaneous procedures.Pseudoaneurysm occlusion by ultrasound-guided thrombin injection directly in the aneurysm sac is a minimally invasive, quick and safe procedure with a high success rate. The aim of our study is to report our initial experience in treatment of iatrogenic pseudoaneurysms by ultrasound-guidedthrombin injection. Method: Between September 2006 and February 2010, 1,811 percutaneous coronary interventions were carried out with an occurrence of 15 pseudoaneurysms(0.8%), of which 13 were consecutively treated by ultrasound-guided thrombin injection. Clinical characteristics, type of percutaneous procedure, adjuvant therapy, size and morphology of pseudoaneurysms, thrombin dose, success and complication rate were prospectively documented and later retrospectively analyzed. Results: Primary success rate was 100% with just one recurrence seven days after the initial approach, which was successfully treated using the same technique. There were no complicationsrelated to the ultrasound-guided thrombin injection and all of the patients were asymptomatic 30 days after the procedure. Conclusion: Ultrasound-guided thrombin injectionperformed by professionals experienced in intravascular ultrasound and transparietal punctures of cysts and collections is a feasible, practical and safe option in the treatment of iatrogenic pseudoaneurysms.

Expression and characterization of a mutant recombinant blood coagulation factor VIII (rFVIII (m))

In an earlier study, a site directed mutant rFVIII (rFVIII(m), Arg(336) -> Gln(336)) expressed in baculovirus-insect cell (Sf9) system was found to sustain high level activity during incubation at 37 for 24 h while the cofactor activity of normal plasma was declined steadily. In this study, a mutant B-domain deleted rFVIII(m), Arg(336) -> Gln(336) expressed in baculovirus-insect cell (Sf9) system was characterized for its enzymatic and chemical properties. The expressed rFVIII(m) and plasma FVIII (pFVIII) were purified by immunoaffinity column chromatography and identified by Western blot analysis. The partially purified rFVIII(m) exhibited cofactor specific activity of 2.01 X 10(3)units/mg protein. The molecular weight of rFVIII(m) ranged between 40 to 150 kDa with a major band at 150 kDa. Treatment of both rFVIII(m) and pFVIII with thrombin increased their cofactor activity in a similar pattern. Treatment of both the activated rFVIII(m) and native FVIII with APC decreased their cofactor activities, however, the former exhibited a slower decrease than the latter, although no significant difference was present. rFVIII(m) formed a complex with vWF, resulting in a stabilized form, and the lag period of thrombin-mediated activating was extended by vWF association. These results implicated that rFVIII(m) expressed in baculovirus-insect cell system had a comparable capacity as FVIII cofactor activity and might be a good candidate for the FVIII replacement therapy for hemophilia A patients.

Organizaçäo do citoesqueleto e distribuiçäo da integrina 'ALFA' Ýb ß3 em plaquetas estimuladas por trombina: aplicaçöes no estudo de pacientes portadores de hipertensäo arterial pulmonar / Organization of cytoskeleton and distribution of integrin 'ALFA' Ýb ß3 in the study of pulmonary hypertensive patients

Os mecanismos envolvidos na ativaçäo e agregaçäo plaquetária em condiçöes fisiológicas e patológicas, têm sido amplamente investigados. O propósito deste estudo foi: 1. Estabelecer alguns métodos para análise da organizaçäo de elementos contráteis e distribuiçäo da integrina 'ALFA' Ýb ß3 em plaquetas normais estimuladas por trombina. Os experimentos foram realizados utilizando-se lisado total e preparaçöes de citoesqueleto e membrana plaquetária. Em todos os ensaios, a integrina foi identificada por "Western immunoblotting" com anticorpo anti-cadeia ß3; 2. O mesmo protocolo foi empregado para analisar os resultados em pacientes portadores de hipertensäo pulmonar, nas quais ativaçäo e agregaçäo de plaquetas ocorrem in vivo como já demonstrado; 3. Finalmente, os efeitos do ácido acetilsalicílico na organizaçäo do citoesqueleto e na retençäo da integrina 'ALFA' Ýb ß3 foram estudados em plaquetas normais utilizando-se a mesma metodologia...

Physiology and function of platelets from patients with Alzheimer's disease.

The discovery that intact Alzheimer amyloid precursor protein is present in platelet granules, has created a great interest in the biochemistry, physiology and function of platelets of patients with Alzheimer disease (AD). In this study we monitored various biochemical and physiological parameters, such as serotonin and adenine nucleotide levels, membrane fluidity, agonist-mediated release of arachidonic acid, thromboxane formation, calcium mobilization, as well as irreversible aggregation and secretion of granule contents. Platelets of patients with AD responded poorly when stirred with weak or potent agonists on a platelet aggregometer. Although capable of agonist-mediated calcium mobilization and synthesis of thromboxanes, the aggregation response of platelets of patients with AD to thrombin and archidonate was considerably compromised. In view of the normal biochemistry and signal transduction capabilities, the compromised response of these cells to potent agonists like thrombin suggested an extrinsic defect. The present study has shown that a plasmatic factor is at least in part responsible for the functional abnormalities of AD platelets.

The kinetics of chondroitin 4-sulfate release from stimulated platelets and its relation to thromboxane A2 formation and granule secretion

1. In platelet rich plasma (PRP), chondroitin 4-sulfate release from platelets occurred after stimulation with ADP (5µM), collagen (5-10µM). Release started within 60 s and maximum release (0.7-2.0 mg/l) was reached within 180 s. TXA2 formation and dense granule release reached a maximum within 90 s after stimulation. 2. Using washed platelets (1.5 x 10**8 cells/ml), the platelet responses were faster. Release of chondroitin 4-sulfate and TXA2 started within 20-30 s after thrombin addition (100 mU/ml). Maximum release was reached within 60 s in both cases. Dense granule release started in the first 5 s of stimulation (34.6 ñ 12.4 por cento) reaching maximum secretion (74.4 ñ 8.7 por cento) within 60 s. 3. Our results demonstrate that maximal chondroitin 4-sulfate release occurs after the dense granule release reaction in both PRP and washed platelets. This observation suggests that chondroitin 4-sulfates is unlikely to be stored in the dense granules but may be stored in the alfagranules

Effects of procoagulants on wound healing.

As blood coagulation is a prelude for wound healing, a systemic haemocoagulant (Botropase) and local procoagulants (thrombin and fibrin) were evaluated on physical (wound breaking strength, wound half-closure time and period of epithelization), biochemical (granuloma-hydroxyproline and hexosamine) and histological attributes of healing wounds in albino rats. Botropase prompted all phases of tissue repair. Thrombin delayed wound contraction whereas fibrin had no discernable action. The findings that procoagulants modify healing process has bearing on their surgical use.

Effects of cadmium treatment in vitro on the uptake and release of [3H]serotonin by human blood platelets.

Effects of cadmium treatment on human platelets were studied with respect to uptake and release of 5-[3H]hydroxytryptamine (5-HT). The uptake of 5-[3H]HT in the presence of varying concentrations of CdCl2 (0.001-10 mM) was inhibited significantly with respect to control platelets and the inhibition was maximum at 1 mM CdCl2 concentration. From studies on the kinetics of 5-[3H]HT uptake a higher Km and significantly lower Vmax for CdCl2-treated platelets were observed. CdCl2 stimulated spontaneous release but inhibited thrombin-induced release of 5-[3H]HT. Spontaneous release of 5-[3H]HT induced by CdCl2 was not significantly altered in the presence of externally available CaCl2 (1 mM).

A simple gravimetric method for estimation of plasma fibrinogen.

The study was undertaken to evaluate a new method of estimating plasma fibrinogen levels, gravimetrically using calcium chloride in albino rats. The results were compared with the standard method in which thrombin was used as clotting agent and were found to be statistically not significant. Advantages of using calcium chloride instead of thrombin have been discussed.