Human platelets labeled at two discrete biotin densities are functional in vitro and are detected in vivo in the murine circulation: A promising approach to monitor platelet survival in vivo in clinical research

Transfusion ◽  
2021 ◽  
Author(s):  
Catherine Ravanat ◽  
Anaïs Pongérard ◽  
Monique Freund ◽  
Véronique Heim ◽  
Floriane Rudwill ◽  
...  
Keyword(s):  
Clinical Research ◽  
Human Platelets ◽  
Platelet Survival

N-Glycosylation Of Human ITP Autoantibodies Affects Complement Activation, Platelet Phagocytosis, and Platelet Survival In a Murine Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 569-569
Author(s):  
Ulrich J. Sachs ◽  
Kathrin Walek ◽  
Annika Krautwurst ◽  
Mathias J. Rummel ◽  
Gregor Bein ◽  
...  
Keyword(s):  
Complement Activation ◽  
Laser Scanning ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Laser Scanning Microscopy ◽  
Platelet Survival ◽  
Scid Mouse Model ◽  
Rapid Clearance

Abstract Introduction Immune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets. The IgG effector functions of autoantibodies depend on their Fc-constant region which undergoes posttranslational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. Material and Methods AAbs were purified from ITP patients (n=15) and controls (n=10) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs (deAAbs) were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. The capability of AAbs and deAAbs to eliminate human platelets in vivo was studied in a NOD/SCID mouse model in presence and absence of a complement source. Results AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs. 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and induced complement consumption. N-glycan cleavage significantly reduced C1q binding (MFI 16.4 vs. 4.9, p=0.017) and complement consumption. In vivo survival of human PLTs was assessed after cotransfusion with native or deAAbs in NOD/SCID mice. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL5h) 75% vs. 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL5h 82% vs. 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL5h42%, p<0.001). Conclusion Removal of N-glycan from AAbs interferes with Fc-mediated phagocytosis and complement activation and thereby prolongs platelet survival in vivo. Our study provides tools for better characterizing ITP AAbs and sheds light on the heterogeneity of AAbs in ITP. Clinical studies should aim to assess such additional characteristics, since this could lead to the identification of ITP patient subgroups with increased responses to specific or new interventions such as, targetting complement factors. Disclosures: No relevant conflicts of interest to declare.

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.

The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

1972 ◽  
Vol 28 (01) ◽  
pp. 031-048 ◽  
Author(s):  
W. H. E Roschlau ◽  
R Gage
Keyword(s):  
Platelet Aggregation ◽  
Aspergillus Oryzae ◽  
Degradation Products ◽  
Blood Platelet ◽  
Human Platelets ◽  
Fibrinolytic Enzyme ◽  
Inhibitory Effects ◽  
Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

scholarly journals A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro.

1985 ◽  
Vol 26 (4) ◽  
pp. 457-464
Author(s):  
C von Schacky ◽  
W Siess ◽  
S Fischer ◽  
P C Weber
Keyword(s):  
Comparative Study ◽  
Eicosapentaenoic Acid ◽  
Acid Metabolism ◽  
Human Platelets

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.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets

2016 ◽  
Vol 115 (02) ◽  
pp. 324-332 ◽  
Author(s):  
Rabie Jouni ◽  
Heike Zöllner ◽  
Ahmad Khadour ◽  
Jan Wesche ◽  
Anne Grotevendt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Factor ◽  
Standard Drug ◽  
Platelet Surface ◽  
Minimal Impact ◽  
Platelet Survival ◽  
Heparin Complexes ◽  
The Impact

SummaryProtamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/ SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 μg/ml ODSH: 75 %, range 70–81 % vs without ODSH: 49%, range 44–59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83 %, range 77–93 % vs without ODSH: 59 %, range 29–61 %, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations > 16 μg/mL (p< 0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 μg/ml ODSH: 53 ± 9, p< 0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 μg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

Mobilization of Fibrinogen Receptor Sites on Human Platelets Stimulated with ADP is Prevented by Prostacyclin

1979 ◽  
Author(s):  
J. Hawiger ◽  
S. Parkinson ◽  
S. Timmons
Keyword(s):  
Inhibitory Effect ◽  
Human Platelets ◽  
Affinity Constant ◽  
Human Fibrinogen ◽  
Fibrinogen Receptor ◽  
Receptor Sites ◽  
Plasma Factor ◽  
Potent Inhibitory Effect

Fibrinogen is a plasma factor required for aggregation of human platelets by ADP. The mechanism of platelet-ADP-fibrinogen interaction was studied by measuring the equilibrium binding of 125I-fibrinogen to human platelets separated from plasma proteins. Binding of 125I-fibrinogen to platelets not stimulated with ADP was low and unaffected by an excess of unlabel led fibrinogen. However, when platelets were stimulated with 4μM of ADP, there was an eightfold increase In the number of available binding sites for human fibrinogen, with affinity constant of 1.9 x 109M-1. This striking increase in fibrinogen receptor sites on human platelets was specific for ADP as contrasted to ATP, AMP, and adenosine. Prostacyclin (Prostaglandin I2, PGI2), a novel prostaglandin produced by the blood vessel wall, completely blocked this ADP-induced increase in fibrinogen receptor sites on human platelets. The effect of PGI2 was prompt and concentration dependent, reaching maximum at 10-9M. 6-keto PGF2 a stable derivative ot PGI2, did not have such an effect. Thus movement of fibrinogen receptor sites on human platelet membrane stimulated with ADP is prevented by PGI2. This represents a new biologic property of this vascular hormone and contributes to better understanding of its potent inhibitory effect in vitro and in vivo on ADP-induced platelet aggregation requiring mobilization of fibrinogen receptor.

Arginine vasopressin release from human platelets after irreversible aggregation

1990 ◽  
Vol 78 (1) ◽  
pp. 113-116 ◽  
Author(s):  
Giovanni Anfossi ◽  
Elena Mularoni ◽  
Mariella Trovati ◽  
Paola Massucco ◽  
Luigi Mattiello ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Arginine Vasopressin ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Vasopressin Release ◽  
Irreversible Aggregation ◽  
Local Release

1. The release of arginine vasopressin from human platelets was investigated in platelet-rich plasma after irreversible aggregation induced by adenosine 5′-pyrophosphate, collagen, sodium arachidonate, thrombin and adrenaline in vitro. 2. Arginine vasopressin levels were significantly higher in the supernatant from stimulated platelet-rich plasma than from unstimulated samples, reaching 3.5 × 10−12 (range 1.6–12.5 × 10−12) mol/l in the absence of an aggregating agent, 8.8 × 10−12 (range 4.2–17.5 × 10−12) mol/l after adenosine 5′-pyrophosphate, 13.7 × 10−12 (2.2–63.2 × 10−12) mol/l after collagen, 7.8 × 10−12 (2.2–14.6 × 10−12) mol/l after sodium arachidonate, 7.8 × 10−12 (2.2–16.3 × 10−12) mol/l after thrombin and 12.2 × 10−12 (4.8–32.1 × 10−12) mol/l after adrenaline. 3. An arginine vasopressin level of 18 × 10−12 mol/l, which can be achieved physiologically, increased the sensitivity of platelets to adenosine 5′-pyrophosphate and collagen in vitro; the same concentration of arginine vasopressin caused a potentiation of the effect of catecholamines on the response of platelets to sodium arachidonate. 4. These results indicate that intraplatelet arginine vasopressin is released during aggregation and suggest that a local release of arginine vasopressin could occur after complete platelet aggregation in vivo.

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