scholarly journals Vesiculation of platelets during in vitro aging

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 887-895
Author(s):  
AP Bode ◽  
SM Orton ◽  
MJ Frye ◽  
BJ Udis
Keyword(s):  
Surface Area ◽  
Platelet Activation ◽  
Lactic Dehydrogenase ◽  
Prostaglandin E ◽  
Platelet Factor ◽  
Storage Container ◽  
Fluorescent Beads ◽  
In Vitro Aging ◽  
Two Populations

Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

scholarly journals Vesiculation of platelets during in vitro aging

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 887-895 ◽  
Author(s):  
AP Bode ◽  
SM Orton ◽  
MJ Frye ◽  
BJ Udis
Keyword(s):  
Surface Area ◽  
Platelet Activation ◽  
Lactic Dehydrogenase ◽  
Prostaglandin E ◽  
Platelet Factor ◽  
Storage Container ◽  
Fluorescent Beads ◽  
In Vitro Aging ◽  
Two Populations

Abstract Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

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 Platelet Activation and Biofilm Formation by Aerococcus urinae, an Endocarditis-Causing Pathogen

2010 ◽  
Vol 78 (10) ◽  
pp. 4268-4275 ◽  
Author(s):  
Oonagh Shannon ◽  
Matthias Mörgelin ◽  
Magnus Rasmussen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Biofilm Formation ◽  
Platelet Rich Plasma ◽  
Protein Structures ◽  
Prostaglandin E ◽  
Bacterial Surface ◽  
Fibrinogen Receptor ◽  
Aerococcus Urinae

ABSTRACT The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets, as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and prostaglandin E1. Plasma proteins at the bacterial surface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immunoglobulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeled fibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae, and blockade of the platelet FcRγIIa hindered platelet activation induced by Au3. Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE.

DN.9693 COMPARED WITH PROSTACYCLIN AND PROSTAGLANDIN E1 IN SEVEN PLATELET TESTS

1987 ◽  
Author(s):  
J R O'Brien ◽  
M D Etherington ◽  
G P Salmon
Keyword(s):  
Bleeding Time ◽  
Prostaglandin E ◽  
Minor Effect ◽  
Membrane Glycoproteins ◽  
Clot Retraction ◽  
Platelet Factor ◽  
Active Concentration ◽  
A Minor ◽  
Induced Aggregation

A new drug, DN.9693, has low Km phosphodiesterase inhibitory properties. Its effect on seven broad spectrum platelet "function" tests has been compared with the effects of prostacyclin E1 (PGI1) and prostaglandin E (PGE ). The tests were (1) platelet aggregation induced by ADP, collagen, adrenaline, thrombin, arachidonic acid and ristocetin; (2) a new test in which platelets aggregate after adding distilled water to cause osmotic stress; (3) the loss of platelets washed in buffered saline; (4) clot retraction; (5) the glass bead column platelet retention test; (6) the in vitro filter "bleeding time" (see two other submitted abstracts); (7) the amount of platelet factor 4 (PF4) which "leaks" from platelets at room temperature.PGI2 inhibited all seven tests, 50% inhibition of the various tests required from 0.1 to 44ng/ml of PGI2. PGE1 also inhibited in all tests but on average required 18 times higher concentrations. Thus an increase in cAMP may be relevant to all these tests, but an understanding of the mechanisms involved is incomplete. DN.9693 inhibited only the first four tests; the equi-active concentration was about 600 times that of PGI2. DN.9693, 2.5μg/ml, caused 50% inhibition of ristocetin induced aggregation and at 4μg/ml had a minor effect on the filter bleeding time. Thus DN.9693 may affect the platelet membrane glycoproteins. In conclusion it is confirmed that PGE1 is less active than PGI^ but has similar activities. DN.9693 whenstudied in these tests has many, but notall, of the prostaglandin-like properties.

Single Center Experience with the Nanoparticle-Based Flow Immunoassay for Diagnosis of Heparin-Induced Thrombocytopenia (HIT).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2189-2189
Author(s):  
Susanne Macher ◽  
Nazanin Sareban ◽  
Camilla Drexler ◽  
Gerhard Lanzer ◽  
Katharina Schallmoser
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Laboratory Diagnosis ◽  
Heparin Therapy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Score System ◽  
Single Center Experience

Abstract Abstract 2189 Heparin-induced thrombocytopenia (HIT), caused by antibodies against heparin/platelet factor 4 (HPF4) complex, is a rare but potentially serious side effect of heparin therapy where due to high mortality, rapid diagnosis is crucial. For the detection of HPF4 antibodies we compared the new nanoparticle-based lateral-flow immunoassay (LFI-HIT, Milenia Biotec, Germany) and a particle gel immunoassay (PaGIA, BioRad, Germany) with an IgG-specific-PF4/polyanion enzyme-linked immunosorbent assay (IgG-ELISA, GTI Diagnostics, USA). Sera from 121 patients (54/67 f/m, median 73 years, range 14–94) with suspected HIT were prospectively tested. The LFI-HIT and the PaGIA were evaluated visually, the IgG-ELISA was positive at an optical density (OD) cutoff > 0.4. For most of the positive samples, the functional heparin-induced platelet activation (HIPA) assay was additionally performed to detect false positive serological results and to confirm a clinically relevant HIT by in vitro platelet-activation. Regarding HIT as a clinico-pathological syndrome, characteristics for HIT were evaluated for each patient by the 4Ts scoring system and divided into high, intermediate or low risk. Results of serological analyses and OD values are summarized in the table. Ten of 121 samples were positive in the LFI-HIT, 10/10 positive in the PaGIA and 8/10 positive in the IgG-ELISA. The HIPA was tested in 9/10 samples and was positive in 8/9 samples. Of the 2 samples positive for LFI-HIT and PaGIA but negative in the ELISA, 1 was HIPA positive, 1 HIPA negative, resulting in a specificity of 88.9% for the LFI-HIT assay correlated to the HIPA. From 111/121 LFI-HIT-negative samples, 2 were positive in the PaGIA, the IgG-ELISA (OD 1.318 and 2,019) and in the HIPA. Seven of the 111 LFI-HIT negative samples were positive only in the IgG-ELISA. Due to marginal positive reactions of 5/7 samples in the ELISA with OD values between 0.4 to 0.5, only 2 LIF-HIT negative IgG-ELISA positive samples were tested by HIPA and 1/2 was positive. Based on the ELISA, the sensitivity of the LFI-HIT was 91.9% (102/111 negative samples also negative in the ELISA) in contrast to 93.1% of the PaGIA. The specificity of the LFI-HIT was 80% (LFI-HIT and IgG-ELISA positive), compared to 57.9% of the PaGIA. Notably, the clinical risk estimated by the 4Ts score system (received from 92/121 patients) did not correlate with laboratory diagnosis of HIT, probably due to inadequate evaluation. Concluding our data, a reliable exclusion of HIT by rapid testing with the LFI-HIT only seems possible with additional analysis of HPF4 antibodies by IgG-ELISA and/or HIPA assay. LFI-HIT PaGIA IgG-ELISA OD IgG-ELISA HIPA assay Median (range) Samples n=121 Pos 10 Pos 10 Pos 8 2.366 (0.902-3.000) 7/7 pos Neg 2 0.199 and 0.170 1/2 pos, 1/2 neg Neg 0 - - - - Neg 111 Pos 9 Pos 2 1.318 and 2.019 2/2 pos Neg 7 0.110 (0.054-0.139) 6/6 neg Neg 102 Pos 7 0.436 (0.404-1.463) 1/2 pos, 1/2 neg Neg 95 0.082 (0.013-0.376) Disclosures: No relevant conflicts of interest to declare.

scholarly journals Crotalocytin: characterization of the timber rattlesnake platelet activating protein

Blood ◽  
1980 ◽  
Vol 56 (6) ◽  
pp. 1020-1028
Author(s):  
AH Schmaier ◽  
RW Colman
Keyword(s):  
Serine Protease ◽  
Lactic Dehydrogenase ◽  
Metabolic Energy ◽  
Prostaglandin E ◽  
Amidolytic Activity ◽  
Platelet Factor ◽  
Timber Rattlesnake ◽  
Dense Granules ◽  
Rattlesnake Venom ◽  
Platelet Secretion

Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.

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.

A Small Molecule Inhibitor of the Gas6/Mer Pathway Inhibits Platelet Activation and Thrombosis with Equal Efficacy to, but Greater Potency Than, iMer, the Novel MerTK Splice Variant

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3303-3303
Author(s):  
Brian R. Branchford ◽  
Luke Law ◽  
Susan Sather ◽  
Gary Brodsky ◽  
H. Shelton Earp ◽  
...  
Keyword(s):  
Surface Area ◽  
Platelet Activation ◽  
Artery Occlusion ◽  
Area Coverage ◽  
Average Percentage ◽  
Microfluidic Flow ◽  
Molecule Inhibitor ◽  
Vehicle Treated Control

Abstract Abstract 3303 Background: Growth Arrest Specific gene 6 (Gas6) signals through platelet-surface Mer receptors, leading to platelet activation and thrombus stabilization via activation of PI3K and Akt, and β3 integrin phosphorylation. This amplifies outside-in signaling via αIIb β3, a necessary step for stable platelet aggregation. iMer is a truncated form of the Mer receptor tyrosine kinase's extracellular domain, produced by alternative splicing, that inhibits Gas6 signaling. A selective UNC Mer small molecule inhibitor (UNC Mer TKI) inhibits signaling by inhibiting Mer tyrosine phosphorylation. Objectives: We hypothesized that inhibiting the Gas6/Mer pathway with UNC Mer TKI would decrease platelet activation responses and thrombus formation to a greater degree than iMer. So, we comparatively evaluated iMer's and UNC Mer TKI's inhibition of Gas6/Mer signaling in vitro and in vivo. Methods: We measured the effect on platelet inhibiton of iMer and UNC Mer TKI using lab assays of human platelet function and murine thrombosis models. In vitro studies included standard aggregometry and aggregate formation on collagen surfaces in a microfluidic flow chamber. In vivo studies included a FeCl3-induced model of carotid artery injury and a collagen/epinephrine-induced pulmonary embolism (PE) model to compare thrombosis protection between littermate C57BL/6 mice treated with inhibitors or vehicle control. A paired t-test was used to compare samples in aggregation, microfluidic flow surface area coverage, as well as elapsed time to initial and stable occlusions in the FeCl3model and survival time in the PE model. Results/Discussion: Inhibitor-treated platelets exhibited significantly decreased aggregation. Collagen-stimulated samples treated with 1.2 μM iMer had mean aggregation of 45 +/− 13% (Stand Dev), compared to 72 +/− 8% in controls (n=6, p=0.02). 1.2 μM UNC Mer TKI-treated samples had mean aggregation of 56 +/−15%, compared to 74 +/−10% in controls (n=5, p<0.01). Adhesion of platelets to collagen under physiologic flow conditions resulted in 1.9% (+/− 1%) coverage in 1.2 μM iMer-treated samples compared to 8.7 +/− 2% mean surface area coverage in controls (n=7, p<0.001). 1.2 μM UNC Mer TKI-treated samples exhibited 8.5 +/−8.8% surface area coverage, compared to 19.2 +/− 15.7% for control-treated samples (n=5 in quadruplicate, p<0.01), and 2.4 +/−1.7% for abciximab-treated positive controls. Both iMer- and UNC Mer TKI-treated samples have a higher average percentage of small platelet aggregates (1–10 platelets/aggregate) compared to controls, which had a higher average percentage of large aggregates (>10 platelets/aggregate). Following 6% FeCl3-induced carotid artery injury for analysis of iMer's thrombosis protection, vehicle-treated control mice (n=6) had a hazard ratio for stable artery occlusion of 7.9 (95%CI 1.7–36.3) compared to 30mg/kg iMer-treated mice (n=8, p<0.01). The 1.2 μM iMer treated mice also demonstrated significantly prolonged time to both initial (p=0.03) and stable occlusion (p=0.02), suggesting decreased thrombus stability. In analysis of UNC Mer TKI's thrombosis inhibition, vehicle-treated control mice (n=10) had a hazard ratio for stable artery occlusion of 4.8 (95%CI 1.4–15.8) compared 3 mg/kg UNC Mer TKI-treated mice (n=9, p=0.02). Times to initial (p=0.06) and stable (p<0.01) occlusions were also prolonged in the UNC Mer TKI-treated mice compared to controls. In the PE model, 1.2 μM iMer-treated mice (n=5) had longer mean survival (14.1 +/− 14.5 min) following collagen/epinephrine injection than controls (n=5, 1.78 +/12.4 min, p=NS), with 2 iMer–treated mice and no controls surviving for 30 min. A much greater effect was seen with the 3 mg/kg UNC Mer TKI-treated mice (n=5) who had a mean survival time of 37.24 +/−31.2 min, with 3 surviving for a full 60 min, compared to 1.63 +/− 0.6 min (p=0.03) for controls (n=5), of which only 1 survived for 60 min. Conclusions: iMer and UNC Mer TKI are Gas6/Mer inhibitors that decrease platelet activation and protect mice from thrombosis, and may have translational applications as novel anti-platelet agents. UNC Mer TKI has similar efficacy, and higher potency, especially in the animal models where similar (FeCl3 model) or superior (PE model) results were seen with a 10-fold lower concentration. UNC Mer TKI also has the advantage of possible oral preparation, which may increase its potential therapeutic appeal. Disclosures: Branchford: University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Sather:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Brodsky:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.

Glucose Transporter 3 in Platelets Facilitates Alpha-Granule Mediated Glucose Uptake, Driving Intragranular Glycolysis That Is Required for Platelet Degranulation and Activation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 417-417
Author(s):  
Fidler P.L Trevor ◽  
Elizabeth Middleton ◽  
Jesse W Rowley ◽  
Luc Boudreau ◽  
Robert A. Campbell ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Plasma Membrane ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Platelet Activation ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Conflicts Of Interest ◽  
Platelet Factor

Abstract Patients with diabetes display increased thrombosis and platelet activation. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. In vitro studies of platelets under hyperglycemic conditions suggest that glucose metabolism may lead to increased platelet activation. Platelets import glucose via two glucose transporters GLUT1, which is expressed on the plasma membrane, and GLUT3, which is expressed on the plasma membrane (15%) and the remaining 85% on α-granule membranes. Following stimulation, platelet α-granules translocate to the plasma membrane and release their cargo. To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. GLUT3 KO platelets displayed a 23% reduction in basal glucose uptake compared to littermate controls. Control platelets stimulated with thrombin displayed a significant increase in glucose uptake whereas KO platelets failed to show any change. Additionally, platelet glycogen content and glycolysis intermediates were significantly reduced in KO platelets, which exhibited reduced glycolytic rates following metabolic stress (mitochondrial uncoupling). Because GLUT3 KO platelets had only a minor decrease in glucose uptake under basal conditions, but platelet glucose metabolism was dramatically altered when stimulated, we hypothesized that under basal conditions, GLUT3 facilitates glucose uptake into α-granules, to generate glycogen and fuel intragranular glycolysis that generates the energy required for α-granule degranulation. To test this hypothesis we permeabilized platelet plasma membranes, but not α-granule membranes using saponin and incubated the platelets with C13-glucose. Under these conditions, control platelets produced 2.5-fold more C13 -lactic acid than KO platelets. In vitro, GLUT3 knockout platelets display a 90% reduction in spreading on fibrinogen and collagen matrices and significant reductions in α-granule degranulation as marked by CD62p surface translocation, platelet factor 4 release, and the persistence of α-granules observed in electron micrographs of stimulated platelets. In vivo in a KBx/N model of rheumatoid arthritis, which is dependent in part on platelet activation, GLUT3 KO mice exhibited significantly reduced severity of disease. Analysis of GLUT3 mice in models of arterial thrombosis, deep vein thrombosis and tail-bleeding indicated no alteration in thrombosis between littermate controls and KO mice. Together these data indicate that GLUT3 mediated α-granule glucose uptake is essential for platelet activation and degranulation. Moreover reducing platelet GLUT3 may ameliorate the course of rheumatoid arthritis. Disclosures No relevant conflicts of interest to declare.

Platelet Factor 4 (PF4) Release In Vitro From Unstimulated Human Platelets

1981 ◽  
Author(s):  
D S Cohen ◽  
J M Strohschein ◽  
R N Saunders ◽  
D I Cargill
Keyword(s):  
Platelet Rich Plasma ◽  
Venous Blood ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Time Period ◽  
Time Periods ◽  
Endogenous Release ◽  
Two Populations

Human venous blood was collected (9.5:0.5) in an anticoagulant (provided in the PF4 RIA kit, Abbott Labs.) consisting of 2.5% EDTA, 0.025% adenosine and 7.0% procaine HCl. Platelet rich plasma (PRP) was stirred (900 RPM) at 37°C in a Payton aggregometer for intervals of 3, 6, 9 and 12 minutes. Unincubated aliquots of PRP were taken as the 0 time controls.A significant (p < 0.02) elevation in PF4 was observed with increasing intervals. Furthermore, from the data obtained, two populations of PF4 releasers were delineated: A high releasing (HR) group and a low releasing (LR) group. HR (n=10) displayed a baseline release of 10.3 ± 0.8 ng/ml and values of 36.1 ± 9.0 (p < 0.05 compared to baseline), 46.8 ± 8.2 (p < 0.01), 54.6 ± 7.9 (p < 0.01) and 87 ± 13.1 (p < 0.01) for the corresponding time periods. LR (n=7) exhibited significant but diminished release at each time period with a basal level of 10.1 ± 1.0 and 14.7 ± 1.4 (p < 0.05 compared to baseline), 15.3 ± 1.5 (p < 0.05), 19.7 ± 2.4 (p < 0.05) and 23.6 ± 2.0 (p < 0.01) respectively. Comparison of both populations revealed significant (p < 0.01) differences at the 6, 9 and 12 minute intervals. Total PF4 content from Triton lysed PRP displayed similar levels between groups: 360.0 ±22.6 (HR) and 380.2 ± 15.2 (LR).We conclude that an endogenous release of PF4 occurs in vitro in unstimulated human PRP even in a milieu designed to deter release. This should be a concern when PF4 values are to be assessed.

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