scholarly journals The in Vivo Evaluation of the Hemostatic Function of Stored Human Platelets

1977 ◽  
Author(s):  
M. Blajchman ◽  
A. Senyi ◽  
J. Hirsh
Keyword(s):  
Platelet Count ◽  
Platelet Function ◽  
Bleeding Time ◽  
Human Subjects ◽  
Human Platelets ◽  
In Vitro Tests ◽  
In Vivo Evaluation ◽  
Ethyl Palmitate

The assessment of the hemostatic function of stored human platelets is difficult to assess in human subjects. The use of thrombocytopenic rabbits treated with ethyl palmitate to produce reticuloendothelial blockade, has made it possible to study the hemostatic function of human platelets in vivo. The assessment of hemostatic function has been made using both a jugular bleeding time technique and an ear bleeding time technique, and in both, a close correlation between bleeding time and platelet count has been established. Using both methods, both fresh and human platelets stored for 72 hours at 22°C correct the bleeding time of thrombocytopenic animals to levels appropriate to the platelet count achieved. Platelets stored at 4°C using standard methods of preparation and storage were ineffective hemostatically after 24 hours storage. Platelets prepared and stored at 4°C at a pH of 6.4 were hemostatically effective in thrombocytopenic rabbits for as long as 10 days of storage. No correlation, however, was noted between the hemostatic effect of stored platelets and in vitro tests of platelet function. Similarly, the intravenous infusion of ADP and collagen produced similar falls in platelet count for both hemostatically effective and non-effective platelets. These studies provide further evidence for the limitations of in vitro tests of platelet function for the assessment of the potential in vivo function of stored human platelets. Furthermore, these findings raise the possibility for the prolongued liquid storage of human platelets at conditions which minimize bacterial contamination, yet maintain hemostatic efficacy.

Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ahmed Alarabi ◽  
Zubair Karim ◽  
Victoria Hinojos ◽  
Patricia A Lozano ◽  
Keziah Hernandez ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

scholarly journals IκB kinase 2 is not essential for platelet activation

2020 ◽  
Vol 4 (4) ◽  
pp. 638-643
Author(s):  
Manuel Salzmann ◽  
Sonja Bleichert ◽  
Bernhard Moser ◽  
Marion Mussbacher ◽  
Mildred Haase ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Active Site ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Iκb Kinase ◽  
Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

scholarly journals Pharmaceutical Benefits of Fluticasone Propionate Association to Delivery Systems: In Vitro and In Vivo Evaluation

Pharmaceutics ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 521 ◽  
Author(s):  
Marina G. Dogbe ◽  
Ambinintsoa Yattussia Mafilaza ◽  
Carla Vânia Eleutério ◽  
Helena Cabral-Marques ◽  
Sandra Simões ◽  
...  
Keyword(s):  
Extraction Procedure ◽  
A549 Cells ◽  
Free Form ◽  
In Vitro Tests ◽  
In Vivo Evaluation ◽  
Pulmonary Administration ◽  
Cell Compatibility ◽  
Intranasal Instillation

The objective of the present work was to characterize the ability of liposomes and cyclodextrin (CyD) complexes to modulate the in vivo profile of fluticasone (FTZ). In vitro cell compatibility tests were performed, exposing A549 cells to FTZ in the free form and FTZ associated to liposomes and complexed with CyD. The in vivo fate of a selected FTZ liposomal formulation and of several FTZ CyD complexes was achieved following intranasal instillation or pulmonary administration in BALB/c mice, respectively. For pulmonary administration, an inhalation chamber was constructed to enable the simultaneously pulmonary administration to six mice. Thirty minutes and 3 h after administration, mice were sacrificed, their blood, lungs, livers, and spleens were removed, and FTZ level was determined by HPLC using an extraction procedure. The in vitro tests revealed no toxic effects of FTZ formulations, as cellular viability was always superior to 90% for FTZ concentrations ranging from 5 to 60 µM 72 h after incubation. The in vivo biodistribution results showed that FTZ incorporated in liposomes resulted in 20 and 30 times higher accumulation in the lungs in comparison with free FTZ, at 0.5 and 3 h after i.n. administration, respectively. FTZ associated to Hydroxypropyl-γ-cyclodextrin (HP-CyD) was the complex that permitted the higher accumulation of FTZ in the lungs in comparison with the respective free form. The results also suggest that the inhalation chamber apparatus can effectively facilitate the evaluation of in vivo inhalation. The establishment of an animal model of asthma allows us to further study the therapeutic efficacy of the developed FTZ formulations.

scholarly journals Assessment of the hemostatic effectiveness of human platelets treated with aminomethyltrimethyl psoralen and UV A light using a rabbit ear bleeding time technique

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3489-3492
Author(s):  
SJ Wagner ◽  
L Bardossy ◽  
G Moroff ◽  
RY Dodd ◽  
MA Blajchman
Keyword(s):  
Bleeding Time ◽  
Human Platelet ◽  
Human Platelets ◽  
Ethyl Palmitate ◽  
Rabbit Ear ◽  
Immunodeficiency Virus ◽  
New Zealand White Rabbits ◽  
The Mean ◽  
Human Immunodeficiency Virus 1

The photochemical aminomethyltrimethyl psoralen (AMT), in conjunction with UV A light (UVA), has been shown to inactivate human immunodeficiency virus-1 and model viruses in platelet suspensions under conditions that have only a minimal effect on in vitro platelet properties. A rabbit ear bleeding time technique was used to assess the hemostatic effectiveness of human platelet suspensions treated with AMT/UVA. New Zealand White rabbits were made thrombocytopenic by a combination of irradiation and heterologous antirabbit platelet antiserum. Reticuloendothelial function in these rabbits was suppressed by the intravenous administration of ethyl palmitate. The hemostatic function of 1- and 5-day-old human platelet suspensions (14.5% plasma) that had been treated on day 1 with 40 micrograms/mL AMT and 24 kJ/m2 UVA (1 x UVA) was evaluated by measuring microvascular bleeding times after a standard incision. Comparable bleeding times were observed after infusion with both control and AMT/UVA-treated platelets stored for either 1 or 5 days. With the transfusion of AMT/1 x UVA-treated platelets stored for 5 days, the mean (+/- SD) bleeding time was 156.3 +/- 39.2 seconds (n = 10). With untreated platelets (no AMT/no UVA), stored for 5 days, the mean bleeding time was 189.2 +/- 36.4 seconds (n = 10). Neither AMT nor 1 x UVA treatment alone influenced the observed bleeding times. In contrast, the hemostatic effectiveness of human platelet suspensions was diminished if they were exposed to three times the standard UVA dose (72 kJ/m2) on day 1 and stored for 4 more days, regardless of whether AMT was present, with the mean bleeding time increasing to 442.2 +/- 122.6 seconds (n = 15, AMT present) or 396.0 +/- 45.9 seconds (n = 10, AMT absent). These results are consistent with data obtained from in vitro studies and indicate that virucidal AMT/1 x UVA treatment does not influence platelet hemostatic function. However, the final conditions to achieve these results must be carefully controlled.

scholarly journals The platelet function defect of cardiopulmonary bypass [see comments]

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 107-117 ◽  
Author(s):  
AS Kestin ◽  
CR Valeri ◽  
SF Khuri ◽  
J Loscalzo ◽  
PA Ellis ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Function ◽  
Peripheral Blood ◽  
Bleeding Time ◽  
Platelet Reactivity ◽  
Platelet Surface ◽  
Shed Blood ◽  
Function Defect

The use of cardiopulmonary bypass (CPB) during cardiac surgery is associated with a hemostatic defect, the hallmark of which is a markedly prolonged bleeding time. However, the nature of the putative platelet function defect is controversial. In this study, blood was analyzed at 10 time points before, during, and after CPB. We used a whole-blood flow cytometric assay to study platelet surface glycoproteins in (1) peripheral blood, (2) peripheral blood activated in vitro by either phorbol myristate acetate, the thromboxane (TX)A2 analog U46619, or a combination of adenosine diphosphate and epinephrine, and (3) the blood emerging from a bleeding-time wound (shed blood). Activation-dependent changes were detected by monoclonal antibodies directed against the glycoprotein (GP)Ib-IX and GPIIb-IIIa complexes and P-selectin. In addition, we measured plasma glycocalicin (a proteolytic fragment of GPIb) and shed-blood TXB2 (a stable breakdown product of TXA2). In shed blood emerging from a bleeding-time wound, the usual time-dependent increase in platelet surface P-selectin was absent during CPB, but returned to normal within 2 hours. This abnormality paralleled both the CPB-induced prolongation of the bleeding time and a CPB-induced marked reduction in shed-blood TXB2 generation. In contrast, there was no loss of platelet reactivity to in vitro agonists during or after CPB. In peripheral blood, platelet surface P-selectin was negligible at every time point, demonstrating that CPB resulted in a minimal number of circulating degranulated platelets. CPB did not change the platelet surface expression of GPIb in peripheral blood, as determined by the platelet binding of a panel of monoclonal antibodies, ristocetin-induced binding of von Willebrand factor, and a lack of increase in plasma glycocalicin. CPB did not change the platelet surface expression of the GPIIb-IIIa complex in peripheral blood, as determined by the platelet binding of fibrinogen and a panel of monoclonal antibodies. In summary, CPB resulted in (1) markedly deficient platelet reactivity in response to an in vivo wound, (2) normal platelet reactivity in vitro, (3) no loss of the platelet surface GPIb-IX and GPIIb-IIIa complexes, and (4) a minimal number of circulating degranulated platelets. These data suggest that the “platelet function defect” of CPB is not a defect intrinsic to the platelet, but is an extrinsic defect such as an in vivo lack of availability of platelet agonists. The near universal use of heparin during CPB is likely to contribute substantially to this defect via its inhibition of thrombin, the preeminent platelet activator.

A Miniaturized Nafion-Based Glucose Sensor: in vitro and in vivo evaluation in dogs

1994 ◽  
Vol 17 (2) ◽  
pp. 88-94 ◽  
Author(s):  
F. Moussy ◽  
D.J. Harrison ◽  
R.V. Rajotte
Keyword(s):  
Glucose Sensor ◽  
Reference Electrode ◽  
The Body ◽  
In Vitro Tests ◽  
In Vivo Evaluation ◽  
Heat Curing ◽  
Layer Membrane ◽  
Outer Coating

We have developed an implantable glucose sensor based on a new tri-layer membrane configuration. The needle-type sensor integrates a Pt working electrode and a Ag/AgCI reference electrode. Its size is equivalent to a 25 gauge needle (0.5 mm in diamater). Poly (o-phenylenediamine) was used as an inner coating to reduce interference by small compounds present in the body fluids, and the perfluorinated ionomer, Nation as a biocompatible, protective, outer coating. Glucose oxidase trapped in an albumin/glutaraldehyde matrix was sandwiched between these coatings. In vitro tests in buffer showed the sensors had a good selectively, a sensitivity of about 25 nA/mM, and a 90% response time of 33 s. Stabilization of the current following polarization required 10 to 30 min in vitro and 30 to 40 in vivo. Although these sensors remained stable for many weeks in saline solution, their implantation in animals resulted in the degradation of the protective Nation outer coating, which in turn, led to the failure of the incorporated reference electrode. We demonstrated that if unprotected, the AgCI layer of the reference electrode rapidly dissolves in the biological environment. However, we later showed that in vivo degradation of Nation can be prevented by heat curing. When heat cured sensors were subcutaneously implanted in dogs, the sensors' signal closely followed the plasma glucose level during glucose tolerance tests. The response of the sensors implanted in dogs was retained for 10 days.

scholarly journals A Comparative Study of the Effects of Heparin and a Low Molecular Weight Semi-Synthetic Heparin Analogue Upon Platelet Function

1977 ◽  
Author(s):  
R. Michalski ◽  
D. A. Lane ◽  
V. V. Kakkar
Keyword(s):  
Molecular Weight ◽  
Intravenous Injection ◽  
Platelet Function ◽  
Factor Xa ◽  
In Vitro Tests ◽  
Low Molecular Weight ◽  
Clotting Time ◽  
Induced Aggregation

We have already reported O) some in vitro and in vivo properties of a low molecular weight glycosaminoglycan polysulphate. It was found that while this semi-synthetic heparin analogue (SSHA) was virtually inactive in a number of in vitro clotting assays, following intravenous or subcutaneous injection it has a more specific anti-Xa potentiating effect than heparin. In the present communication a comparison has been made of some effects of SSHA and heparin upon platelet function. In several of the in vitro tests performed, such as their potentiating effect on ADP and adrenaline induced aggregation and their effects on the aggregation of washed platelets by Factor Xa, heparin proved to be far more potent than SSHA. It was found that after intravenous injection of both drugs, PRP samples containing comparable anti-Factor Xa activities responded differently to the addition of thrombin as SSHA barely inhibited thrombin induced aggregation. Similarly, SSHA had little effect on the dilute thrombin clotting time of plasma, following intravenous injection. Heparin and analogue were neutralised to approximately the same degree by a crude PF4 preparation, and similar transient thrombocytopenia effects were observed with both drugs.

scholarly journals Non-genomic effects of the Pregnane X Receptor negatively regulate platelet functions, thrombosis and haemostasis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gagan D. Flora ◽  
Khaled A. Sahli ◽  
Parvathy Sasikumar ◽  
Lisa-Marie Holbrook ◽  
Alexander R. Stainer ◽  
...  
Keyword(s):  
Platelet Function ◽  
Thrombus Formation ◽  
Pregnane X Receptor ◽  
Human Platelets ◽  
Regulatory Effects ◽  
Granule Secretion ◽  
Species Specific ◽  
Platelet Functions

AbstractThe pregnane X receptor (PXR) is a nuclear receptor (NR), involved in the detoxification of xenobiotic compounds. Recently, its presence was reported in the human vasculature and its ligands were proposed to exhibit anti-atherosclerotic effects. Since platelets contribute towards the development of atherosclerosis and possess numerous NRs, we investigated the expression of PXR in platelets along with the ability of its ligands to modulate platelet activation. The expression of PXR in human platelets was confirmed using immunoprecipitation analysis. Treatment with PXR ligands was found to inhibit platelet functions stimulated by a range of agonists, with platelet aggregation, granule secretion, adhesion and spreading on fibrinogen all attenuated along with a reduction in thrombus formation (both in vitro and in vivo). The effects of PXR ligands were observed in a species-specific manner, and the human-specific ligand, SR12813, was observed to attenuate thrombus formation in vivo in humanised PXR transgenic mice. PXR ligand-mediated inhibition of platelet function was found to be associated with the inhibition of Src-family kinases (SFKs). This study identifies acute, non-genomic regulatory effects of PXR ligands on platelet function and thrombus formation. In combination with the emerging anti-atherosclerotic properties of PXR ligands, these anti-thrombotic effects may provide additional cardio-protective benefits.

scholarly journals Radioactive Diisopropyl Fluorophosphate as a Platelet Label: An Evaluation of in Vitro and in Vivo Technics

Blood ◽  
1967 ◽  
Vol 29 (3) ◽  
pp. 354-372 ◽  
Author(s):  
T. C. BITHELL ◽  
J. W. ATHENS ◽  
G. E. CARTWRIGHT ◽  
M. M. WINTROBE
Keyword(s):  
Specific Activity ◽  
Human Subjects ◽  
Survival Curve ◽  
Human Platelets ◽  
Detectable Effect ◽  
Survival Curves ◽  
Diisopropyl Fluorophosphate ◽  
Platelet Survival

Abstract A technic for the in vitro labeling of human platelets with DFP32 is presented, critically evaluated, and compared to in vivo methods employing DFP32 and to in vitro methods using Cr51. The initial recovery of platelets labeled in vitro with DFP32 averaged 79 per cent, but the survival curve was characterized by an irreversible initial loss of platelet radioactivity. Experiments in which platelets were simultaneously labeled in vitro with both DFP32 and Cr51 suggest that this is not due to elution of DFP32. The survival curve of platelets labeled in vivo with DFP32 shows an initial transient reduction in platelet radioactivity. It is suggested that both of these aberrations in initial survival are the result of platelet injury by DFP32. Significant "tailing" was observed in the survival curves obtained with DFP32, and possible explanations of this phenomenon are discussed. DFP32-labeled platelets circulating after 5 hours apparently survive normally and disappear from the circulation as a rectilinear function over the next 6-8 days. Although both in vitro and in vivo labeling methods employing DFP32 provide a meaningful approximation of platelet lifespan, the initial and terminal aberrations of the survival curves greatly complicate further interpretation. Dextran had no detectable effect on platelet survival, and epinephrine, Mecholyl, and cutaneous vasodilatation did not alter the platelet count or the specific activity of circulating labeling platelets in human subjects. The problem of initial platelet survival and the question of an extravascular or marginal platelet pool is discussed in the light of these data.

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