Mass cytometry of platelet-rich plasma: a new approach to analyze platelet surface expression and reactivity

Platelets ◽  
2021 ◽  
pp. 1-8
Author(s):  
Melissa Klug ◽  
Kilian Kirmes ◽  
Jiaying Han ◽  
Olga Lazareva ◽  
Marc Rosenbaum ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Mass Cytometry ◽  
Platelet Surface ◽  
New Approach

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021 ◽  
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

Platelet Activation by Antiphospholipid Antibodies Depends on Epitope Specificity and is Prevented by mTOR Inhibitors

2019 ◽  
Vol 119 (07) ◽  
pp. 1147-1153 ◽  
Author(s):  
Anne Hollerbach ◽  
Nadine Müller-Calleja ◽  
Svenja Ritter ◽  
Friederike Häuser ◽  
Antje Canisius ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Antiphospholipid Antibodies ◽  
Platelet Rich Plasma ◽  
Mtor Inhibitors ◽  
Surface Expression ◽  
Human Platelets ◽  
Antigen Specificity ◽  
Platelet Surface ◽  
Glycoprotein I

AbstractAntiphospholipid antibodies (aPL) have been reported to activate platelets. This is considered to be one of the pathogenic properties of aPL. Even though aPL heterogeneity is quite well established, little is known, if the ability to activate platelets is common to all aPL or depends on antigen specificity. To further study this issue, we analyzed the ability of three human monoclonal aPL with distinctly different antigenic specificities to activate platelets in vitro. The results obtained with human monoclonal aPL were validated with immunoglobulin G (IgG) fractions obtained from patients with antiphospholipid syndrome (APS). A co-factor-independent human monoclonal anticardiolipin aPL had no discernible effect on human platelets. Two monoclonal aPL reactive against β2 glycoprotein I (β2GPI) induced platelet aggregation, integrin αIIbβ3 activation and P-selectin surface expression. These data could be confirmed with patient IgG fractions which could only induce aggregation, if they had anti-β2GPI activity. Anti-β2GPI aPL-induced platelet activation depended on interaction of aPL with the low affinity Fcγ-receptor IIa on the platelet surface. It was completely abolished by pretreatment of platelet-rich plasma with the mechanistic target of rapamycin (mTOR) inhibitors rapamycin or everolimus. This extends previous data showing that mTOR is involved in signaling of anti-β2GPI in monocytes and endothelial cells. In conclusion, anti-β2GPI aPL activate platelets while co-factor-independent anticardiolipin aPL have no effect. mTOR is involved in this signaling process which has implications beyond APS, because so far the role of mTOR signaling in platelets is incompletely explored and requires further study.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

Effects of nitric oxide/EDRF on platelet surface glycoproteins

1996 ◽  
Vol 270 (5) ◽  
pp. H1640-H1648 ◽  
Author(s):  
A. D. Michelson ◽  
S. E. Benoit ◽  
M. I. Furman ◽  
W. L. Breckwoldt ◽  
M. J. Rohrer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Thromboxane A2 Analogue ◽  
Surface Glycoproteins

We examined the effects of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) on platelet surface glycoproteins (GP). As determined by flow cytometry, in both a washed platelet system and platelet-rich plasma, the EDRF congener (S-nitroso-N-acetylcysteine) markedly inhibited both the thrombin-induced and the (stable thromboxane A2 analogue) U-46619-induced upregulation of P-selectin (alpha-granule protein), CD63 (lysosomal protein), and the GPIIb-IIIa complex (fibrinogen receptor) but minimally inhibited downregulation of the GPIb-IX complex (von Willebrand factor receptor). The inhibitory effects of EDRF were markedly reduced in whole blood or by the addition of washed erythrocytes. Platelets in whole blood were still responsive to guanosine 3',5'-cyclic monophosphate (cGMP), as shown by complete inhibition of P-selectin upregulation by the stable analogue N6,2'-O dibutyryl cGMP. These data suggests that 1) cGMP negatively regulates the platelet surface expression of P-selectin, CD63, and the GPIIb-IIIa complex but not the platelet surface expression of the GPIb-IX complex and 2) hemoglobin within erythrocytes inhibits the effects of EDRF/NO on platelet surface glycoproteins.

scholarly journals Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2016-2021 ◽  
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Von Willebrand Factor ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Platelet Surface ◽  
Granule Secretion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Platelet Shape ◽  
Stimulation Of

Abstract Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.

Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of α2β1 but not αIIbβ3 integrin

2005 ◽  
Vol 94 (07) ◽  
pp. 107-114 ◽  
Author(s):  
Christelle Lecut ◽  
Martine Jandrot-Perrus ◽  
Marion A. H. Feijge ◽  
Judith M. E. M. Cosemans ◽  
Johan W. M. Heemskerk
Keyword(s):  
Thrombin Generation ◽  
Type I Collagen ◽  
Platelet Rich Plasma ◽  
Procoagulant Activity ◽  
Type I ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Collagen Receptors ◽  
Fibrillar Collagens

SummaryThe role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in enhancing thrombin generation, in a way dependent on exposure of phosphatidylserine (PS) at the platelet surface. Soluble, non-fibrillar type-I collagen required pre-activation of integrin α2β1 with Mn2+ for enhancement of thrombin generation. With all preparations, blocking of glycoprotein VI (GPVI) with 9O12 antibody abrogated the collagen-enhanced thrombin generation, regardless of the α2β1 activation state. Blockade of α2β1 alone or antagonism of autocrine thromboxane A2 and ADP were less effective. Blockade of αIIbβ3 with abciximab suppressed thrombin generation in platelet-rich plasma, but this did not abolish the enhancing effect of collagens. The high activity of type-I fibrillar collagens in stimulating GPVI-dependent procoagulant activity was confirmed in whole-blood flow studies, showing that these collagens induced relatively high expression of PS. Together, these results indicate that: i) fibrillar type-I collagen greatly enhances thrombin generation, ii) GPVI-induced platelet activation is principally responsible for the procoagulant activity of fibrillar and non-fibrillar collagens, iii) α2β1 and signaling via autocrine mediators facilitate and amplify this GPVI activity, and iv) αIIbβ3 is not directly involved in the collagen effect.

Upregulation of CD40 ligand and enhanced monocyte-platelet aggregate formation are associated with worse clinical outcome after ischaemic stroke

2012 ◽  
Vol 107 (02) ◽  
pp. 346-355 ◽  
Author(s):  
Grzegorz Dworacki ◽  
Joanna Kufel-Grabowska ◽  
Cezary Watala ◽  
Wojciech Kozubski ◽  
Maria Lukasik
Keyword(s):  
Clinical Outcome ◽  
Predictive Marker ◽  
Cd40 Ligand ◽  
Surface Expression ◽  
Vascular Events ◽  
Platelet Surface ◽  
Increased Risk ◽  
Platelet Aggregate ◽  
Platelet Aggregates ◽  
Long Term Prognosis

SummaryThe white blood cell count and mean platelet volume determined shortly after the symptom onset are known as independent predictors for clinical outcome after stroke. In the present study we sought to evaluate the prognostic value of platelet-derived inflammatory biomarkers measured prospectively after an ischaemic event. Using five-colour flow cytometry, the platelet surface expression of CD40L, CD62P and subpopulations of leukocyte-platelet aggregates were assessed in 93 stroke patients on the first (V0), 10th (V1) and 90th (V2) day after stroke, and once in 65 disease controls. The clinical outcome was evaluated using the Scandinavian Stroke Scale (SSS) and modified Rankin Scale (mRS) at the same time points as blood sampling and 24 months after the event. Patients with either CD40L surface expression or the percentage of monocyte-platelet aggregates (M-plt) in the third tertile (T3) at V0 had a significantly lower score on the SSS at V1. Patients with the percentage M-plt at V0 higher than the median value of M-plt in controls were at increased risk of SSS < 40 at V1 (odds ratio: 2.6; 95% confidence interval [CI]: 1.4 – 8.7; p=0.006). Patients with the percentage of M-plt in T3 at V0 showed progressive decline in survival (hazard ratio [HR]: 1.6; 95% CI: 1.1–1.9; p=0.02) and a significantly higher number of recurrent vascular events (HR: 2.64; 95% CI: 1.3–3.2; p=0.02) when compared to the first tertile. In conclusion, increased levels of M-plt could be a predictive marker for both early outcome and long-term prognosis while increased CD40L was correlated with worse clinical outcome.The preliminary results of this study were presented in part during a poster session at the 62nd Annual Meeting of the American Academy of Neurology in Toronto, 7–17 April 2010.

Activation of human platelets by 2-arachidonoylglycerol is enhanced by serotonin

2003 ◽  
Vol 89 (02) ◽  
pp. 340-347 ◽  
Author(s):  
Monica Bari ◽  
Domenico Del Principe ◽  
Alessandro Finazzi-Agrò ◽  
Mauro Maccarrone
Keyword(s):  
Cyclic Amp ◽  
Cannabinoid Receptors ◽  
Cannabinoid Receptor ◽  
Platelet Rich Plasma ◽  
Maximum Velocity ◽  
Human Platelets ◽  
Platelet Surface ◽  
Therapeutic Implications ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Mechanism

SummaryThe endocannabinoid 2-arachidonoylglycerol (2-AG) has been shown to activate human platelets in platelet-rich plasma, by binding to a “platelet-type” cannabinoid receptor (CBPT). Here, washed human platelets were used to characterize the binding of [3H]2-AG to CBPT, showing a dissociation constant (Kd) of 140 ± 31 nM and a maximum binding (Bmax) of 122 ± 10 pmol.mg protein-1. Selective antagonists of both CB1 and CB2 cannabinoid receptors inhibited this binding, which was enhanced up to ~230% over the controls by 1 µM serotonin (5-hydroxytryptamine, 5-HT). Human platelets were also able to bind [3H]5-HT (Kd = 79 ± 17 nM, Bmax = 14.6 ± 1.3 pmol.mg protein-1), and 1 µM 2-AG enhanced this binding up to ~150%. Moreover, they were able to take up [3H]5-HT through a high affinity transporter (Michaelis-Menten constant = 22 ± 2 nM, maximum velocity = 344 ± 15 pmol.min-1.mg protein-1), which was not affected by 2-AG. Interestingly, 5-HT did not affect the activity of the 2-AG transporter of human platelets. Treatment of washed platelets with 1 µM 2-AG led to increased intracellular inositol-1,4,5-trisphosphate (up to ~300%) and decreased cyclic AMP (down to ~50%). Furthermore, treatment of pre-loaded platelets with 1 µM 2-AG induced a ~300% increase in [3H]2-AG release, according to a CBPT-dependent mechanism. Also, 1 µM 5-HT enhanced the effect of 2-AG on inositol-1,4,5-trisphosphate (~500% of the controls), cyclic AMP (~20%) and [3H]2-AG release (~570%), and the latter process was shown to be partly (~50%) involved in the 5-HT-dependent platelet activation. Taken together, reported findings represent the first demonstration that 2-AG and 5-HT can mutually reinforce their receptor binding on platelet surface, which might have therapeutic implications.

Role of the α-subunit326GRV sequence in the surface expression of fibrinogen and vitronectin receptors

1998 ◽  
Vol 275 (5) ◽  
pp. C1239-C1246 ◽  
Author(s):  
Milagros Ferrer ◽  
Matilde S. Ayuso ◽  
Nora Butta ◽  
Roberto Parrilla ◽  
Consuelo González-Manchón
Keyword(s):  
Functional Role ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Platelet Surface ◽  
Α Subunit ◽  
Vitronectin Receptor ◽  
Ovary Cells ◽  
High Affinity

The platelet GPIIb-GPIIIa heterodimer (integrin αIIbβ3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The326GRV motif in GPIIb is highly conserved in the α-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327→His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327in the α-subunit of the vitronectin receptor, to His did not prevent the surface expression of αv-GPIIIa complexes. Thus the conformational constraint imposed by His327seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

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