Amidolytic assay for procoagulant activity of lymphoid and tumor cells

Among the effector molecules induced in monocytes by the cellular immune response is tissue factor (TF), the initiating receptor/cofactor of the extrinsic coagulation protease cascade that is also frequently observed on human tumor cells. Other cellular activators have also been described on monocytes and tumor cells. Analyses of the cellular immune procoagulant response would be aided by a simple and efficient form of quantitation. An assay for cellular procoagulant activity (PCA) induction and expression was developed utilizing the chromogenic thrombin substrate tosyl-Gly-Pro-Arg-p-nitroanilide acetate. The constitutive or induced PCA of a variety of cells was analyzed. Peripheral blood mononuclear cells, peritoneal exudate cells, 13762 Mat B (III) mammary carcinoma cells, 1591-RE fibrosarcoma cells, the macrophage cell line WEHI-265, a detector of PCA inducing lymphokines, or mixtures of these cells were incubated with or without stimuli, e.g., endotoxin, in 96-well microplates. After incubation the cells were assayed for PCA by addition of the chromogenic substrate for thrombin using fibrinogen depleted plasma as a source of the coagulation proteins factors VII, X, V and prothrombin. The absorbance at 405 nm was determined. Spontaneous cleavage of the chromogenic substrate restricted the assay to total analysis times of less than 14 min. The 13762 Mat B (III) rat tumor which constitutively expressed tissue factor-like procoagulant activity induced measurable substrate hydrolysis with as few as 100 cells/well. It was observed that the chromogenic substrate assay was approximately twice as sensitive as conventional clotting assays for procoagulant activity. Endotoxin stimulated human peripheral blood mononuclear cells and mouse peritoneal exudate cells were readily analyzed. The procoagulant activity of approximately 280 LPS-stimulated human monocytes generated sufficient thrombin to provide a significant measurable signal within 10 min. Also supernatants from mixed lymphocyte cultures as well as from immune lymphocyte responses to syngeneic tumor cell cultures induced procoagulant activity in the macrophage like cell line WEHI-265 as determined with the assay for thrombin generation. The hydrolysis of the substrate was attributed to thrombin formation since the induced cleavage was abolished by hirudin, the highly specific active site inhibitor of thrombin. This chromogenic thrombin assay can be used for measuring induction of viable cell expression or total cellular procoagulant activity rapidly and efficiently in large replicate numbers suitable for a variety of analyses of cellular immune responses including clonal analyses of gene induction.     

Role of sialylation in determining the pharmacokinetics of neutrophil inhibitory factor (NIF) in the Fischer 344 rat

1. Recombinant neutrophil inhibitory factor (NIF) is a glycoprotein. Its amino acid sequence remains constant and has a molecular weight of 28.9 kD. However, approximately 40% of the total molecular weight consists of glycans with variable structure. 2. The pharmacokinetics of 11 different NIF batches with varying extents and patterns of sialylation have been investigated in the Fischer 344 rat following intravenous administration. These data indicate that reducing the extent of NIF sialylation reduces the half-life of the molecule due to an increase in the systemic clearance. Also, an increase in the number of unsialylated or neutral glycans may increase the volume of distribution of NIF, although this effect is marginal. 3. Isolated perfused rat liver (IPRL) investigations have shown that sialylated NIF has a low hepatic extraction (< 1%), while asialo NIF has an extraction that is > 20-fold higher. Co-administration of asialo NIF with asialo fetuin (a protein cleared by hepatic asialoglycoprotein receptor (possibly galactose)-mediated uptake reduced the hepatic extraction of asialo NIF. 4. These data suggest that NIF molecules that have free sugar moieties (possibly galactose) interact with an asialoglycoprotein receptor (possibly galactose-mediated) in the liver (parenchymal cells/hepatocytes). Interaction with this receptor leads to cellular internalization and degradation.     

Two sites in the tissue factor extracellular domain mediate the recognition of the ligand factor VIIa.

Tissue factor (TF) binds the serine protease coagulation factor VIIa and initiates the coagulation protease cascade by forming a catalytic cofactor-enzyme complex. Using a photoactivatable crosslinking reagent coupled to factor VIIa, we have identified interactive sites in the amino-terminal (residues 44-84) and the carboxyl-terminal (residues 129-169) aspect of the extracellular domain of TF. Epitopes of inhibitory antibodies have previously indicated participation of these regions in TF function. The presence of the gamma-carboxyglutamic acid domain in factor VIIa appears to facilitate the interaction with the negatively charged, amino-proximate site, whereas crosslinking of TF with VIIa or des-(1-38)-VIIa at the positively charged carboxyl-proximate site was similar. Lack of alpha-helical secondary structure in the TF extracellular domain is consistent with the proposed structural similarity of TF with the cytokine receptor family. The interactive sites identified for TF are located in sequence spans that demonstrate a low degree of sequence conservation among the members of this receptor family. Regions with highly conserved residues, such as sequences encoded by exon 2 and 5 in TF, were not implicated in ligand recognition, suggesting that conserved residues in the receptor family may maintain the common beta-strand architecture, and variable regions provide a pair of nonidentical motifs for oriented ligand recognition.     

Perioperative risks associated with the operative treatment of clavicle fractures

Background

Clavicle fractures are a common injury among young adults who were historically treated non-operatively with satisfactory outcomes. However, more recent studies have shown a higher nonunion rate for displaced clavicle fractures treated conservatively. The purpose of this study is to investigate the midterm complications, clinical outcomes and overall patient satisfaction after osteosynthesis of midshaft clavicular fractures.

Patients and methods

A total of 37 patients treated for a clavicle fracture from January 2007 to December 2008 with at least 12 months’ follow-up were identified from a billing code search. At the latest follow-up appointment, the patients completed the Constant Shoulder, the Disabilities of the Arm, Shoulder and Hand scale (DASH) and the Medical Outcomes Study 36-Item Short-Form Health Survey version 2.0 (SF36v2) functional outcome surveys as well as a custom questionnaire to assess hand dominance, employment status, the amount of time taken before returning to work, the presence of numbness around the incision site (a surrogate marker of a supraclavicular nerve palsy), whether the patient desired the plate removed and/or if it was worth another surgery.

Results

With regard to the functional outcome surveys, the average DASH score was 11.8 ± 16.4, the Constant score was 93.3 ± 7.2, the SF36v2 physical component summary (PCS) was 50.7 ± 10.1 and the SF36v2 mental component summary (MCS) 50.6 ± 11.2. From the custom questionnaire, 27 patients (73%) found their cosmetic appearance acceptable while the remaining 10 patients (27%) were bothered by the appearance of the plate. The average time to return to work was 82.1 ± 77.4 days. There were no infections, refractures or nonunions of the clavicle.

Conclusion

As the relative indications for open reduction and internal fixation of clavicle fractures become more popular, such as cosmetic concerns or faster recovery, we wish to demonstrate that the procedure is not without risks, including implant discomfort requiring a subsequent operation for removal, numbness around the incision site and infection. Despite these risks, patients tend to be satisfied with the procedure and are able to function at levels equal to that of the general population. The purpose of this study is not to recommend for or against operative treatment of clavicle fractures but merely to demonstrate risks associated with the procedure.     

Platelet‐induced expression of tissue factor procoagulant activity in freshly isolated human mononuclear cells: implications for experimental use

Summary Monocytes express tissue factor (TF) as a result of cytokine stimulation or endothelial adherence. We evaluated monocyte–platelet interaction in vitro as another trigger for monocyte TF enhancement in human mononuclear cells isolated by density gradient centrifugation from peripheral blood. Cell TF procoagulant activity (TF‐PCA) was quantitated by a one‐stage recalcification clotting time assay. Platelets were counted and identified by whole blood flow cytometry as CD61 positive particles, activated platelets were characterized by P‐Selectin (CD62) expression, and monocytes by surface CD14 expression. A significant correlation between normalized TF‐PCA of isolated mononuclear cells and platelet count was shown (r = 0.43, P < 0.001). Percentage of activated platelets in baseline samples was 4.2 ± 3.5 while adenosine diphosphate (ADP) increased platelet positivity to 34 ± 17% (P < 0.001). After isolation, 52 ± 12% of platelets within suspensions were activated (P < 0.0001). Percentage of CD62‐positive monocytes (CD14⁺ particles) increased from baseline 5% to 13 ± 6% in ADP‐stimulated samples to 53 ± 17% after isolation (P < 0.001). These findings suggest that density gradient centrifugation activates platelets and that an adhesive interaction between monocytes and platelets may promote TF‐PCA expression in isolated mononuclear suspensions. Enhanced monocyte TF expression as a result of an activated platelet–monocyte interaction seems to be an important laboratory effect requiring consideration when utilizing this technique in an experimental setup.     

Platelet-induced expression of tissue factor procoagulant activity in freshly isolated human mononuclear cells: implications for experimental use

Monocytes express tissue factor (TF) as a result of cytokine stimulation or endothelial adherence. We evaluated monocyte-platelet interaction in vitro as another trigger for monocyte TF enhancement in human mononuclear cells isolated by density gradient centrifugation from peripheral blood. Cell TF procoagulant activity (TF-PCA) was quantitated by a one-stage recalcification clotting time assay. Platelets were counted and identified by whole blood flow cytometry as CD61 positive particles, activated platelets were characterized by P-Selectin (CD62) expression, and monocytes by surface CD14 expression. A significant correlation between normalized TF-PCA of isolated mononuclear cells and platelet count was shown (r = 0.43, P < 0.001). Percentage of activated platelets in baseline samples was 4.2 +/- 3.5 while adenosine diphosphate (ADP) increased platelet positivity to 34 +/- 17% (P < 0.001). After isolation, 52 +/- 12% of platelets within suspensions were activated (P < 0.0001). Percentage of CD62-positive monocytes (CD14+ particles) increased from baseline 5% to 13 +/- 6% in ADP-stimulated samples to 53 +/- 17% after isolation (P < 0.001). These findings suggest that density gradient centrifugation activates platelets and that an adhesive interaction between monocytes and platelets may promote TF-PCA expression in isolated mononuclear suspensions. Enhanced monocyte TF expression as a result of an activated platelet-monocyte interaction seems to be an important laboratory effect requiring consideration when utilizing this technique in an experimental setup.     

The failure of antimotion sickness medication to improve reading in developmental dyslexia: results of a randomized trial

Although there have been no randomized clinical trials of the efficacy of antimotion sickness medication treatment of developmental dyslexia, some children are treated in this way. We have performed two evaluations of such treatments. In Experiment 1, 12 children participated in a double-blind within-subject crossover design to test the acute (2-day) administration of four preparations: 10 mg methylphenidate, 12.5 mg meclizine, both methylphenidate and meclizine, or placebo. Improvements obtained with each drug were scattered across measures of reading fluency, balance and coordination, and eye movements, suggesting that a chronic trial would be justified. In Experiment 2, six children from Experiment 1 received 12.5 mg meclizine b.i.d. for 3 months and placebo for 3 months in a double-blind within-subject crossover design. Meclizine had no effect on reading, but it significantly improved ocular motor stability during steady fixation. This study thus failed to support the hypothesis that meclizine is of benefit in developmental dyslexia.