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.

scholarly journals Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1414
Author(s):  
Josep M. Cambra ◽  
Emilio A. Martinez ◽  
Heriberto Rodriguez-Martinez ◽  
Maria A. Gil ◽  
Cristina Cuello
Keyword(s):  
Culture Medium ◽  
Embryo Quality ◽  
Transcriptional Profiling ◽  
Defined Medium ◽  
Platelet Factor ◽  
Defined Media ◽  
Defined Culture ◽  
The Impact

The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

scholarly journals RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival

Blood ◽  
2020 ◽  
Vol 136 (15) ◽  
pp. 1773-1782 ◽  
Author(s):  
Daniel DeHelian ◽  
Shuchi Gupta ◽  
Jie Wu ◽  
Chelsea Thorsheim ◽  
Brian Estevez ◽  
...  
Keyword(s):  
Platelet Count ◽  
G Protein ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Rgs Proteins ◽  
Platelet Recovery ◽  
Platelet Production ◽  
Platelet Survival

Abstract G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

Plasma Deposition of Biomolecules for Enhanced Biomedical Applications

2015 ◽  
Vol 1723 ◽  
Author(s):  
Liam O’Neill ◽  
Barry Twomey ◽  
Peter Dobbyn ◽  
John O’Donoghue
Keyword(s):  
Wound Healing ◽  
Chronic Wounds ◽  
Plasma Deposition ◽  
Preclinical Trial ◽  
Minimal Impact ◽  
Plasma Device ◽  
Biological Performance ◽  
The Impact

ABSTRACTBiomolecules have been traditionally immobilised onto surfaces using wet chemical techniques for various medical applications. Recent decades have seen plasma methods being used to prepare these surfaces through various forms of surface modification, but the direct exposure of biomolecules to plasma has been avoided due to fears that the molecules would be denatured by the energetic plasma species. Recent results are now demonstrating that direct plasma deposition of biomolecule coatings can be achieved. This creates the possibility to directly modify the surface of implants without any form of surface pre-treatment and this opens up the possibility to alter the healing processes. Materials such as collagen, chitosan, catalase and heparin can be effectively deposited onto surfaces with minimal impact on biological performance and without any chemical binders, linkers or impurities. The performance of these materials has been characterised using both in vitro and in vivo methodologies. In a further step, the results of a preclinical trial are presented which reveal that direct deposition of biomolecules onto open wounds can also be achieved and the impact of this on wound healing is measured in an immunocompromised animal model. A non-thermal plasma device was used to deliver collagen on to chronic wounds and the treatment was shown to promote wound closure in a rabbit wound healing model.

Abstract 451: Platelet Activation and Thrombosis by Antibodies against VEGF and CD40 Ligand: Extending the mechanism of HIT

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ali Amirkhosravi ◽  
Todd Meyer ◽  
Florian Langer ◽  
Theresa Robson ◽  
Liza Robles ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Cd40 Ligand ◽  
Serotonin Release ◽  
Heparin Binding ◽  
Wild Type ◽  
Platelet Factor ◽  
Cardiovascular Comorbidity ◽  
Platelet Aggregometry

Introduction: The vascular endothelial growth factor (VEGF) monoclonal antibody, bevaci-zumab (Avastin), has been associated with arterial thromboembolic events in some cancer patients. Another therapeutic antibody, hu5c8, which targets CD40L, also produced unexpected thrombosis in lupus clinical trials. Because platelets play a crucial role in arterial thrombosis, we hypothesized that antibodies against VEGF and CD40L may activate platelets via a mechanism similar to that responsible for thrombosis in Heparin-Induced Thrombocytopenia (HIT). Methods: Immune complexes (ICs) were prepared by combining these monoclonal IgG antibodies with their antigens: M90 (or hu5c8) with CD40L (“M90+CD40L”), or bevacizumab with VEGF and heparin (“BVH”). VEGF binds heparin (as does platelet factor 4, the HIT antigen). We measured platelet activation by serotonin release assay, platelet aggregometry, and flow cytometry. We also evaluated IC-induced thrombosis in hFc mice, transgenic for the human IgG receptor, CD32. Results: Similar to HIT antibodies, these ICs potently induced platelet activation dependent on CD32, IC concentration (>10nM) and optimal stoichiometry. Intravenous injection of M90+CD40L or BVH into hFc, but not wild-type mice rapidly produced signs of thrombotic shock, thrombocytopenia and pulmonary thrombosis. However, wild-type control mice (lacking platelet CD32) were unaffected by IC injection. Similarly as with HIT antibodies, bevacizumab IC activity was reduced in the absence or excess of heparin both in vitro and in vivo, whilst M90+CD40L-induced platelet activation was abolished in vitro by blockade of the platelet CD40L receptor, CD40, demonstrating a requirement for Fab-dependent anchoring. Furthermore, VEGF 121 (which lacks the heparin-binding domain of VEGF 165 ) and bevacizumab with or without heparin failed to activate platelets or cause thrombosis in hFc mice. Conclusions: Together, these findings demonstrate that Fab-dependent anchoring of anti-CD40L and anti-VEGF ICs is required for potent platelet activation and thrombosis, as is the case in HIT, suggesting common mechanistic elements. Clinical implications may apply in patients with cardiovascular comorbidity receiving immunotherapy.

Heparin-Induced Thrombocytopenia Antibodies Inhibit PF4-Dependent Enhancement of Activated Protein C Formation by Binding to Antigenic Complexes Formed with the Chondroitin Sulfate Side-Chain of Thrombomodulin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 721-721
Author(s):  
M. Anna Kowalska ◽  
Lubica Rauova ◽  
Vincent Hayes ◽  
Douglas B. Cines ◽  
Daniel W. Bougie ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Protein C ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Activated Protein C ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Heparin Complexes

Abstract Abstract 721 Previous studies have shown that platelet factor 4 (PF4) increases activated protein C (aPC) generation both in vitro and in vivo. PF4 increase of aPC generation by thrombin (IIa) and thrombomodulin (TM) complex followed a bell-shaped curve when tested in solution, on human TM expressing HEK293 and on endothelial cells. PF4 failed to enhance aPC in the presence of chondroitin sulfate (CS)-free TM. These results were consistent with PF4 binding to the CS on the TM glycosaminoglycan (GAG) domain and forming complexes that are similar to PF4/GAG antigenic complexes seen in heparin-induced thrombocytopenia (HIT). We tested the hypothesis that PF4 forms a HIT-like antigenic complex with the TM-CS using the HIT-like monoclonal antibody KKO. KKO abolished the potentiating effects of PF4 on aPC formation measured with TM in solution or with a TM-expressing cell line. To further address the nature of complexes formed between PF4 and TM, we used a mutant of PF4, PF4T38Q, which forms complexes with GAGs that are not recognized by KKO and a subgroup of HIT antibodies. Similar to PF4, PF4T38Q potentiated TM-dependent aPC generation in a bell-shaped manner, but this potentiation was not blocked by KKO. Moreover, KKO did not have any effect when PF4 was replaced with protamine sulfate (PS), which can also form macromolecular complexes with heparin/GAGs and can also enhance aPC generation. We also tested HIT antibodies isolated from patients that developed HIT with thrombocytopenia and thromboembolism developing >4 days after the last exposure to heparin. Patient IgGs specific for PF4/GAG complex were purified using PF4 bound to heparin columns. Specific binding of antibodies to PF4/heparin complexes was checked by ELISA. Complex-specific antibodies were then tested in an aPC generation assay in the presence IIa and TM and near peak concentration of PF4 and compared to a control human IgG. Three of four patient‘s antibodies significantly inhibited the increase in aPC generation in the presence of PF4. These studies provide evidence that HIT-like PF4/GAG complexes develop naturally in vivo. In this case, the ability of HIT or HIT-like antibodies to specifically inhibit the PF4-dependent increase in aPC formation may contribute to the prothrombotic state in HIT. Disclosures: No relevant conflicts of interest to declare.

In Vivo Platelet Activation and Its In Vitro Inhibition by Prasugrel's Active Metabolite In Adolescents with Sickle Cell Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2141-2141
Author(s):  
Andrew L. Frelinger ◽  
Joseph A. Jakubowski ◽  
Julie K. Brooks ◽  
Anu Nigam ◽  
Michelle A. Berny-Lang ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Whole Blood ◽  
Research Funding ◽  
Normal Subjects ◽  
Cell Disease ◽  
Platelet Surface ◽  
Dependent Inhibition

Abstract Abstract 2141 Introduction: In patients with sickle cell disease (SCD), erythrocytes contribute to microvessel occlusion resulting in tissue damage and platelet activation. Platelet activation, aggregation, local thrombus formation and platelet activation-dependent leukocyte recruitment potentially amplify tissue ischemia. Antiplatelet therapy may therefore be useful in SCD. Here we evaluate levels of platelet activation markers in adolescents with SCD vs. normal controls and the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Methods: Blood was obtained from adolescents (10 – 18 yr) with SCD and healthy adult subjects. Platelet function was evaluated by: light transmission aggregation (LTA) in platelet-rich plasma with 20 μM ADP and in whole blood by VerifyNow P2Y12; Multiple Electrode Aggregometry (MEA) with 6.5 μM ADP; vasodilator stimulated phosphoprotein (VASP) P2Y12 assay; and whole blood flow cytometric analysis of basal and in vitro ADP-stimulated levels of platelet surface activated GPIIb-IIIa (reported by monoclonal antibody PAC1) and P-selectin, platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA). These endpoints were also evaluated after in vitro incubation of whole blood with R-138727 (0.1 – 10 μM). Results: In SCD patients compared with normal subjects, circulating PMA and PNA levels were significantly higher (76.5 ± 20.3% and 55.1 ± 21.8% vs. 20.1 ± 7% and 13.9 ± 4.2% [mean ± SD], respectively, p<0.0001 for both), and in vitro ADP-stimulated platelet surface activated GPIIb-IIIa and P-selectin levels (mean fluorescence, MFI) were significantly lower (128.7 ± 66.2 and 78.1 ± 11.5 vs. 257.3 ± 50.8 and 91.6 ± 5.8, p<0.05 for both). ADP-stimulated platelet aggregation by LTA, VerifyNow and MEA, did not significantly differ between SCD and normal subjects, although whole blood platelet aggregation by MEA and VerifyNow tended to be greater in blood from SCD patients (92.5 vs. 70.4 AU, p=0.064 and 362.9 vs. 314.8 PRU, p=0.488, respectively). Treatment of whole blood in vitro with R-138727 resulted in a concentration-dependent inhibition of platelet function in both SCD patients and normal subjects. However, compared with normal subjects, the IC50 in SCD subjects was significantly lower for LTA but significantly higher for VerifyNow and VASP (Table). R-138727 inhibition of platelet function in SCD patients was similar to normal subjects as judged by MEA, whole blood flow cytometry for ADP-stimulated platelet surface P-selectin and activated GPIIb-IIIa expression, and % PMAs (Table). Sensitivity to R-138727 inhibition in SCD patient blood was greatest as measured by ADP-stimulated platelet surface P-selectin MFI, LTA, and MEA, less with ADP-stimulated platelet surface activated GPIIb-IIIa, and least with VASP, VerifyNow P2Y12 and % P-selectin-positive platelets (Table). Conclusions: 1) The markedly higher circulating PMA and PNA levels in SCD patients relative to normal donors demonstrates increased in vivo platelet activation in SCD patients and suggests that PMA and PNA may be useful markers of the in vivo pharmacodynamic effects of antiplatelet therapy in SCD patients. 2) Blockade of platelet P2Y12 with R-138727 produces dose-dependent inhibition of platelet function in SCD platelets. 3) Assay-dependent differences in IC50 values between SCD patients and normal donors suggest the presence of additional variables that affect these measures of platelet function. Further studies are needed to determine the relationship between platelet inhibition measured by these assays and clinical events in SCD patients. Disclosures: Frelinger: GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Consultancy, Research Funding; Takeda: Research Funding. Jakubowski:Eli Lilly and Company: Employment, Equity Ownership. Heeney:Lilly: Consultancy. Michelson:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

Protective mechanisms of adenosine 5′-monophosphate in platelet activation and thrombus formation

2014 ◽  
Vol 111 (03) ◽  
pp. 491-507 ◽  
Author(s):  
Eduardo Fuentes ◽  
Lina Badimon ◽  
Julio Caballero ◽  
Teresa Padró ◽  
Gemma Vilahur ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Adenosine Receptor ◽  
Thrombus Formation ◽  
Antiplatelet Agent ◽  
Binding Pocket ◽  
In Vivo Model ◽  
Platelet Surface ◽  
Camp Levels

SummaryPlatelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5′-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

scholarly journals Development and assessment of the performance of a shared ventilatory system that uses clinically available components to individualize tidal volumes

2020 ◽  
Author(s):  
David M. Hannon ◽  
Tim Jones ◽  
Jack Conolly ◽  
Conor Judge ◽  
Talha Iqbal ◽  
...  
Keyword(s):  
Invasive Mechanical Ventilation ◽  
Ventilation System ◽  
List Type ◽  
Test Lung ◽  
Minimal Impact ◽  
Wide Range ◽  
The Impact ◽  
Ventilator Settings

AbstractObjectivesTo develop and assess the performance of a system for shared ventilation that uses clinically available components to individualize tidal volumes under a variety of clinically relevant conditions.DesignEvaluation and in vitro validation study.SettingVentilator shortage during the SARS-CoV-2 global pandemic.ParticipantsThe design and validation team consisted of intensive care physicians, bioengineers, computer programmers, and representatives from the medtech sector.MethodsUsing standard clinical components, a system of shared ventilation consisting of two ventilatory limbs was assembled and connected to a single ventilator. Individual monitors for each circuit were developed using widely available equipment and open source software. System performance was determined under 2 sets of conditions. First, the effect of altering ventilator settings (Inspiratory Pressure, Respiratory rate, I:E ratio) on the tidal volumes delivered to each lung circuit was determined. Second, the impact of altering the compliance and resistance in one simulated lung circuit on the tidal volumes delivered to that lung and the second lung circuit was determined. All measurements at each setting were repeated three times to determine the variability in the system.ResultsThe system permitted accurate and reproducible titration of tidal volumes to each ‘lung circuit’ over a wide range of ventilator settings and simulated lung conditions. Alteration of ventilator inspiratory pressures stepwise from 4-20cm H2O, of respiratory rates from 6-20 breaths/minute and I:E ratio from 1:1 to 1:4 resulted in near identical tidal volumes delivered under each set of conditions to each simulated ‘lung’. Stepwise alteration of compliance and resistance in one ‘test’ lung circuit resulted in reproducible alterations in tidal volume to the ‘test’ lung, with little change to tidal volumes in the ‘control’ lung (a change of only 6% is noted). All tidal volumes delivered were highly reproducible upon repetition.ConclusionsWe demonstrate the reliability of a simple shared ventilation system assembled using commonly available clinical components that allows individual titration of tidal volumes. This system may be useful as a temporary strategy of last resort where the numbers of patients requiring invasive mechanical ventilation exceeds supply of ventilators.Article SummaryStrengths and limitations of this studyThis solution provides the ability to safely and robustly ventilate two patients simultaneously while allowing differing tidal volumes in each limb.The designed solution uses equipment readily available in most hospitals.Accurate and reproducible titration of tidal volumes to each ‘lung’ was possible over a wide range of ventilator settings.Alteration of one simulated ‘lung’ conditions had minimal impact on the tidal volumes delivered to the unaffected lungThe system relies on patients being sedated and paralysed.We have not yet tested this solution in vivo, on COVID-19 patients.

Surface Sialic Acid Prevents Loss of GPIbα during Platelet Storage and Rescues In Vivo Survival of Murine Platelets.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 138-138 ◽  
Author(s):  
Gerard Jansen ◽  
Emma C. Josefsson ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Shape Change ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
Surface Receptors ◽  
Platelet Survival ◽  
Platelet Receptor

Abstract Platelet processing and storage are associated with platelet lesion, e.g. shape change, activation, release reaction and apoptosis, which is partially due to loss of surface receptors. Surface sialic acid is considered to be a key determinant for the survival of circulating blood cells and glycoproteins. However, its role in platelet receptor loss and platelet survival is unclear. In this study, the relationship between surface sialic acid and platelet receptor loss was investigated in vitro and in vivo. Murine platelets stored at room temperature for 6 hours lost surface sialic acid, as evidenced by flow cytometry using FITC conjugated RCA I lectin, which recognizes exposed galactose residues. This loss correlated with a 30–60% loss of surface receptors GPIbα and GPV, but not GPIX and integrin αIIbβ3, as measured by flow cytometry. Treatment of murine platelets with the neuraminidase (NA) substrate fetuin partially decreases the loss of GPIbα and GPV to 10–20%. In vitro, sialic acid was cleaved from the platelet surface by adding NA (α2-3,6,8-NA (V. cholerae) or α2-3,6,-NA (C. perfringens)) to murine platelets. Removal of sialic acid correlated with the removal of 50–60% of surface GPIbα and GPV, but not GPIX and integrin αIIbβ3. Addition of fetuin, or the more specific NA inhibitor 2,3-dehydro-2-deoxy-, sodium salt (DANA), completely prevented this loss, as determined by both flow cytometry and Western blot analysis. Murine platelets treated with α2-3,6,8-NA (V. cholerae) ± the addition of DANA were labeled with the green dye CMFDA and transfused into age-, strain- and sex-matched C57BL/6 mice to measure platelet survival. NA-treated platelets were cleared within minutes after transfusion, whereas the addition of DANA rescued platelet survival to control-count increments. Our study shows that inhibiting the loss of surface sialic acid prevents platelet surface GPIbα and GPV loss during storage in vitro and rescues platelet survival in vivo.

Reversible Inhibition of Human Platelet Activation by Hypothermia In Vivo and In Vitro

1994 ◽  
Vol 71 (05) ◽  
pp. 633-640 ◽  
Author(s):  
Alan D Michelson ◽  
Hollace MacGregor ◽  
Marc R Barnard ◽  
Anita S Kestin ◽  
Michael J Rohrer ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Bleeding Time ◽  
Human Platelet ◽  
Platelet Surface ◽  
Shed Blood

SummaryA hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22° C and 37° C. the forearm skin temperature was maintained at temperatures between 22° C and 37° C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB 2 (the stable metabolite of TXA 2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37° C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB 2 ggeneration, and the bleeding time. In summary, by a combination of immunologic, biochemical, and functional assays, we demonstrate that hypothermia inhibits human platelet activation in whole blood in vitro and in vivo. Rewarming hypothermic blood completely reverses the activation defect. These results suggest that maintaining normothermia or rewarming a hypothermic bleeding patient may reduce the need for platelet transfusions.

Sign in / Sign up

Export Citation Format

Share Document