Role of P-Selectin and GPIbα in the Fast and Delayed Clearance of Transfused Platelets.

Abstract Platelets become activated during preparation and storage of platelet concentrates (PCs) for transfusion. Flow cytometric assays of platelet activation can be employed for quantifying in vitro quality of PCs. It remains, however, unclear whether the level of in vitro platelet activation in stored PCs correlates with in vivo survival of the platelets after transfusion. Platelet surface glycoprotein (GP) Ibα and P-selectin (CD62) can be involved in regulation of posttransfusion PC clearance, mediating adhesive interactions of platelets with counter-receptors on leukocytes and endothelial cells. Recently, we described a rabbit model for analyzing posttransfusion kinetics of human PCs (Leytin et al, Transfusion42:711, 2002, Transfusion43:983, 2003). In the present work, we used this validated model for studying the implication of CD62 and GPIbα expression in posttransfusion PC clearance. Platelet activation in vitro was determined by flow cytometry using anti-CD62 and anti-GPIbα antibodies. PC clearance in vivo was evaluated in rabbits with inhibited reticuloendothelial system, as measured by 0.5 hr (R0.5), 24 hr (R24) and total (R∑) platelet recoveries, and survival time (ST). Correlations were analysed between in vitro assays of platelet activation and in vivo clearance of conventional (Day 2–5) and outdated (Day 7–8) PCs stored at 22°C, and refrigerated PCs. We found that the binding of anti-CD62 antibody was significantly increased in outdated and refrigerated PCs compared to conventional PCs, reflecting an increased exposure of CD62 on the platelet surface. In contrast, binding of anti-GPIbα antibody was significantly decreased during prolonged and refrigerated PC storage. The clearance of conventional (Day 2–5) PCs from the circulation can be described by a biphasic survival curve. The first (early) phase of platelet clearance is characterized by fast (≥ 14 x 109 platelets per hour) platelet removal, whereas the second (delayed) phase has a much slower rate of platelet clearance (approximately 0.4 x 109 platelets per hour). The biphasic survival curves were also obtained for outdated and refrigerated PCs, and were employed for determining fast (R0.5), delayed (ST and R24) and overall (fast + delayed; R∑) platelet clearance in vivo. We found that when stored PCs are cold-damaged, their in vivo viability decreased significantly, in comparison to conventional PCs, as reflected by the fast, delayed and overall platelet clearances. Viability of Day 7–8 PCs is also decreased, compared to Day 2–5 PCs, but only the fast and overall platelet clearance increased significantly. Negative correlation was observed between in vitro anti-CD62-binding to platelets and their fast, but not delayed, clearance. In contrast, anti-GPIbα-binding showed positive correlations with delayed, but not fast, platelet clearance. Overall clearance correlated better with anti-GPIbα- than with anti-CD62-binding. We also demonstrated that CD62 is shed from the platelet surface after transfusion, whereas GPIbα remains unchanged on the surface of circulating platelets. The data suggest that CD62 exposure during PC storage triggers fast CD62-mediated PC clearance. However, after CD62 shedding during platelet circulation, in vitro GPIbα alterations, such as cleavage, clustering or conformation changes, may determine long-term GPIbα-mediated PC clearance.

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

Thrombosis and Haemostasis ◽

10.1160/th15-07-0539 ◽

2016 ◽

Vol 115 (02) ◽

pp. 324-332 ◽

Cited By ~ 4

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.

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Interaction between Lactobacillus reuteri and periodontopathogenic bacteria using in vitro and in vivo (G. mellonella) approaches

Pathogens and Disease ◽

10.1093/femspd/ftaa044 ◽

2020 ◽

Vol 78 (8) ◽

Cited By ~ 1

Author(s):

Thaís Aguiar Santos ◽

Liliana Scorzoni ◽

Raquel Correia ◽

Juliana Campos Junqueira ◽

Ana Lia Anbinder

Keyword(s):

Lactobacillus Reuteri ◽

Galleria Mellonella ◽

Survival Curve ◽

Fusobacterium Nucleatum ◽

In Vitro Assays ◽

Antimicrobial Effects ◽

Bacteria Growth ◽

Periodontopathogenic Bacteria

ABSTRACT Periodontitis is a multifactorial inflammatory disease, and the major cause of tooth loss in adults. New therapies have been proposed for its treatment, including the use of probiotics such as Lactobacillus reuteri. The objective of this study was to evaluate the antimicrobial effects of L. reuteri: live, heat-killed and culture filtrate (cell-free supernatant), on periodontopathogenic bacteria (Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans) in vitro, as well as the in vivo survival curve, hemocyte density and microbial recovery using Galleria mellonella. For in vitro assays, all preparations reduced colony forming units of F. nucleatum, while only live L. reuteri reduced the growth of A. actinomycetemcomitans. All treatments reduced periodontopathogenic bacteria growth in vivo. The treatment with the supernatant increased the survival of larvae infected with F. nucleatum more than the treatment with live L. reuteri, and none of the treatments altered the survival of A. actinomycetemcomitans-infected larvae. In addition, the treatment with L. reuteri preparations did not alter the hemocyte count of F. nucleatum- and A. actinomycetemcomitans-infected larvae. This study demonstrated that L. reuteri preparations exerted antimicrobial effects and increased the survival of G. mellonella infected by F. nucleatum, although only live L. reuteri was able to reduce the growth of A. actinomycetemcomitans in vitro.

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

Blood ◽

10.1182/blood.v118.21.2141.2141 ◽

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.

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

Thrombosis and Haemostasis ◽

10.1160/th13-05-0386 ◽

2014 ◽

Vol 111 (03) ◽

pp. 491-507 ◽

Cited By ~ 34

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.

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Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound [see comments]

Blood ◽

10.1182/blood.v77.4.770.770 ◽

1991 ◽

Vol 77 (4) ◽

pp. 770-779 ◽

Cited By ~ 124

Author(s):

AD Michelson ◽

PA Ellis ◽

MR Barnard ◽

GB Matic ◽

AF Viles ◽

...

Keyword(s):

Whole Blood ◽

Actin Polymerization ◽

Flow Cytometric Analysis ◽

Adenosine Diphosphate ◽

Surface Expression ◽

Surface Glycoprotein ◽

Platelet Surface ◽

Granule Secretion

Abstract In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642495 ◽

1994 ◽

Vol 71 (05) ◽

pp. 633-640 ◽

Cited By ~ 269

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.

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Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

Blood ◽

10.1182/blood.v88.9.3288.bloodjournal8893288 ◽

1996 ◽

Vol 88 (9) ◽

pp. 3288-3298 ◽

Cited By ~ 105

Author(s):

CJ O'Malley ◽

JE Rasko ◽

RL Basser ◽

KM McGrath ◽

J Cebon ◽

...

Keyword(s):

Platelet Count ◽

Growth And Development ◽

Atp Release ◽

Adenosine Diphosphate ◽

In Vitro Assays ◽

Initial Increase ◽

Platelet Surface ◽

Development Factor

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.

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Redistribution of Activation-Dependent Antigens on the Platelet Surface In Vivo and In Vitro.

Blood ◽

10.1182/blood.v106.11.3946.3946 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3946-3946

Author(s):

Yue Han ◽

De Pei Wu ◽

Lan Dai ◽

Wenrong Sheng ◽

Changgeng Ruan

Keyword(s):

Cerebral Infarction ◽

Platelet Activation ◽

Normal Subjects ◽

Platelet Membrane ◽

Dependent Manner ◽

Membrane Glycoproteins ◽

Platelet Surface ◽

Maximum Decrease

Abstract Platelets play a crucial role in thrombosis, platelet activation induces an alteration of platelet membrane glycoproteins between the platelet surface and surface connected canalicular system (SCCS). In an attempt to better understand this process and assess its significance in the study of thrombosis disease, we used flowcytometry for detecting the redistribution of antigens on the platelet surface following activation. The expression of platelet membrane glycoproteins (GP) Ib, IIIa and P-selectin as well as the platelet agglutination induced by ristocetin were measured in 20 normal subjects, whose platelets were stimulated by the peptide SFLLRNP(TRAP, thrombin receptor activated peptide) at different time points (0~30minute). The same antigens were also analyzed in whole blood in 30 patients with cerebral infarction. As expected, a stable increase of P-selectin was obtained from 2 minute upon TRAP activation, while a reversible reduction of GPIbα and agglutinability by ristocetin were showed at the same time, with a maximum decrease at 2 minute, then followed by a return of GP Ibα to platelet surface. In addition, the platelet membrane GPIIIa was not significantly changed in the initial stage after platelet stimulation and increased by degrees 10 minutes later. Compared with those in normal group, there was an apparent increase of P-selectin and a lower expression of GPIb in the patients with cerebral infarction, while GPIIIa was not significantly changed in all the samples. In summary, TRAP can mediate platelet activation, which leads to the redistribution of GPIb and GPIIIa in a time-dependent manner, together with the release of P-selectin. Meanwhile, the change of activation-dependent antigens on the platelet surface could be an important index reflecting the platelet activation in vivo.

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The Bcl-2 Family Protein Inhibitor, ABT-263, Promotes Apoptotic-Like Response in Isolated Platelets.

Blood ◽

10.1182/blood.v108.11.1122.1122 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1122-1122

Author(s):

Chris Tse ◽

Haichao Zhang ◽

Jun Chen ◽

Ryan M. Fryer ◽

Richard Nelson ◽

...

Keyword(s):

Platelet Activation ◽

Reticuloendothelial System ◽

Human Platelets ◽

Dense Granule ◽

Protein Inhibitor ◽

Cytochrome C Release ◽

Family Protein ◽

Granule Release

Abstract Platelets are relatively short-lived, anucleated cells derived from megakaryocytes that are essential for proper hemostasis. Although they lack a nucleus, platelets possess a highly organized structure with multiple cellular organelles including mitochondria and a number of specific granules. Upon recruitment to sites of endothelial injury, platelet activation stimulates their adhesion, aggregation and release of granule components leading to coagulation. Many of the cellular changes observed during platelet activation and platelet senescence resemble those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, cell shrinkage and microparticle formation (analogous to membrane blebbing). ABT-263 is an orally bioavailable Bcl-2 family protein inhibitor currently in clinical development. This compound is a potent antagonist of the anti-apoptotic proteins Bcl-xL, Bcl-2 and Bcl-w and induces apoptosis in cancer cells dependent on these proteins for survival. ABT-263 induces a unique thrombocytopenia in multiple species that is characterized by a rapid clearance of circulating platelets with recovery to normal platelet counts within several days after treatment cessation. To elucidate the mechanisms underlying this thrombocytopenia, a series of in vitro experiments were performed using freshly isolated platelets comparing ABT-263, its enantiomer (which has significantly lower activity against Bcl-2 family proteins) and known platelet activators. ABT-263, but not the enantiomer control, reduced the viability of dog and human platelets and activated key apoptotic processes, including cytochrome c release, caspase 3 activation, and PS externalization. ABT-263 did not induce aggregation of isolated platelets and did not induce surface expression of GPIIb/IIIa that is essential for platelet adhesion to fibrinogen during clot formation. While ABT-263 had no effect on markers of lysosomal- or alpha-granule release, increases in a marker of dense granule release were apparent at high concentrations. Taken together these data suggest that ABT-263 induces an apoptotic-like response in platelets that is distinct from platelet activation. Furthermore, the lack of effect of the enantiomer control compound suggests this effect is mediated through inhibition of antiapoptotic Bcl-2 family members. Loss of membrane asymmetry and PS externalization has been suggested to be the primary signal for the recognition and clearance of senescent platelets by phagocytes of the reticuloendothelial system. Induction of apoptosis in platelets by Bcl-2 family protein inhibitors such as ABT-263 may coopt this process inducing a premature platelet senescence resulting in a novel type of thrombocytopenia, in vivo.

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Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound [see comments]

Blood ◽

10.1182/blood.v77.4.770.bloodjournal774770 ◽

1991 ◽

Vol 77 (4) ◽

pp. 770-779 ◽

Cited By ~ 1

Author(s):

AD Michelson ◽

PA Ellis ◽

MR Barnard ◽

GB Matic ◽

AF Viles ◽

...

Keyword(s):

Whole Blood ◽

Actin Polymerization ◽

Flow Cytometric Analysis ◽

Adenosine Diphosphate ◽

Surface Expression ◽

Surface Glycoprotein ◽

Platelet Surface ◽

Granule Secretion

In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

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