Biomedical subjects
Publications and source records attributed to S Saleun.
We describe a novel mutation identified in the antithrombin III (AT-III) propeptide responsible for AT-III deficiency. This mutation, a C to A transversion in exon 2, converts the cysteine at position -4 into a stop codon (TGC-->TGA). In this family with severe thrombophilia, the mutation co-segregates with the clinical phenotype.
Platelet prothrombinase activity and aggregation were studied in parallel in the same platelet suspensions with the aim of determining the relationship between these two responses. Stimulation of platelets with thrombin (0.12 U/ml) plus collagen (20 mu g/ml) led to maximum thrombin generation when the aggregation intensity was still only 30%. Since the rate of thrombin formation then remained constant as aggregation intensity was still only 30%. Since the rate of thrombin formation then remained constant as aggregation increased up to its maximum intensity, the aggregation processes did not appear to result in partial masking of any procoagulant surface. This was confirmed by the fact that thrombin generation was the same on aggregated platelets as on activated platelets prevented from aggregating by addition of RGDS. Finally, maximum 5-hydroxy-tryptamine release could be obtained in the absence of thrombin generation. Thus platelet procoagulant activity did not appear to be related to aggregation or secretion and thrombin formation on the surface of activated platelets was in fact two to three times more important in the absence of stirring than under the conditions of continuous stirring required for aggregation. In conclusion, these results suggest procoagulant activity and aggregation to be two independent platelet responses which occur in different physiological situations.
BACKGROUND: The preparation of platelet concentrates (PCs) from buffy coats (BCs) stored at room temperature is controversial, because of the strong metabolic activity of cells in BCs and the possible detrimental effect of neutrophil enzymes on platelets when the holding time before separation is prolonged. Despite good in vitro and in vivo behavior of BC-PCs stored in synthetic solution, little is known of the quality of BC-PCs stored in plasma. STUDY DESIGN AND METHODS: Comparison was made of PCs prepared from BCs held at 22 degrees C for 3 hours (3-hour BC-PCs) or overnight (12-hour BC-PCs) and stored in plasma. Platelet and white cell counts, pH, response to osmotic shock, and morphologic scores were determined on 20 PCs of each type. The decrease in dense granule and alpha granule content, a marker of platelet activation, were estimated by mepacrine counting and beta-thromboglobulin measurement, respectively (n = 8-10). Platelet function was studied in terms of aggregation and thromboxane production in response to various concentrations of collagen and thrombin (n = 8-17). PCs prepared from unstored BCs (n = 15) and from BCs held for 90 minutes (n = 15) were used as controls. RESULTS: Platelet yield was increased from 53 +/- 10 percent of donated platelets to 73 +/- 4 percent by increasing the BC holding time from 0 to 90 minutes to 3 hours (p < 0.001). Similar yields (7.8 +/- 1.8 vs. 7.9 +/- 2 x 10(10) platelets) and white cell contamination (0.9 +/- 0.8 vs. 1.0 +/- 0.9 x 10(7)) were obtained with 3-hour and 12-hour BC-PCs. At the end of the storage period (Day 5), all variables known to correlate with platelet survival in vivo were well maintained in both 3-hour and 12-hour BC-PCs: pH > or = 6.9, response to osmotic shock > or = 70 percent, and morphology scores always > or = 240. During storage, the dense granule content decreased moderately (30% after 5 days), whatever the conditions. By contrast, the total platelet beta-thromboglobulin content was better preserved in 12-hour BC-PCs than in 3-hour BC-PCs (p < 0.04). No significant differences were observed in collagen-induced aggregation and thromboxane production in the two PC preparations. However, aggregation responses to thrombin were higher in 12-hour BC-PCs on Day 5 of storage (p < 0.01). CONCLUSION: BCs can be held at 22 degrees C for up to 12 hours, with no detrimental effect on the quality of PCs stored for up to 5 days in plasma. Such a holding time might help overcome logistic problems in blood banks.
Explore the source record for details and available documents.
During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.
BACKGROUND: Recently, several reports have described the ability of recombinant baculoviruses to transduce a variety of mammalian cells. Yet, mechanisms involved in baculovirus entry in those cells remain largely unexplored, particularly at the primary binding step of the virions to the cell membrane. METHODS: This report focused on the primary virus-cell interactions that lead to in vitro transduction of human 293 cells using a polyhedrin-deleted baculovirus harboring a CMV-driven beta-galactosidase gene (BacLacZ). RESULTS: Infection rate monitored for 8 h and transduction rate with a multiplicity of infection of up to 800 were, both, non-saturable. Temperatures from 37 degrees C to 4 degrees C dramatically impaired BacLacZ but not adenovirus cell attachment. Competitive infections performed with an excess of a non LacZ-expressing baculovirus hardly competed at a 1/1 ratio. Consistent with an adsorptive binding process onto the cell surface, interactions through electrostatic charges between both viral and cell membranes appeared to be critical for BacLacZ transduction. The addition of polybrene to the cells prior to or during the infection prevented both virus binding and LacZ gene transfer, suggesting the involvement of negatively charged epitopes exposed at the cell surface. The simultaneous presence of the highly charged heparin abrogated BacLacZ binding to the cell surface and subsequent gene transfer. Lastly, direct in vitro binding of BacLacZ to heparin but not BSA columns could be demonstrated after elution of infectious BacLacZ virus in high salt molarity. CONCLUSION: Electrostatic charges play a critical role during the first step in mammalian cell transduction mediated by a recombinant baculovirus.