Fatal Dysfunction and Fragmentation of Thrombin-Stimulated Platelets

Abstract Introduction: Platelets play a key role in formation of protective hemostatic blood clots and pathological obstructive thrombi. Under (patho)physiological conditions, chemically activated platelets change their morphology, express adhesive molecules, undergo aggregation, secrete procoagulant substances, and induce mechanical contraction (retraction) of the blood clots. Despite the vital importance of these platelet functions, the subsequent fate of activated platelets is largely unknown. We hypothesize that activated platelets undergo late alterations that determine their fate and may have a pathogenic importance in thrombotic and hemostatic disorders. Methods: We used a combination of confocal microscopy, immunofluorescence, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements to study deferred structural, metabolic, and functional consequences of thrombin-induced activation of viable human platelets, either suspended in platelet-rich plasma or isolated by gel-filtration. Results: Visualized by confocal microscopy, fluorescently labeled platelets in thrombin-induced plasma clots initially underwent shape changes characteristic of platelet activation, but in about 30 min many platelets and platelet aggregates broke up into organelle-containing vesicular fragments. There were two types of platelet-derived vesicles differing in their size and cellular origin: one smaller type was shedding from the tips of filopodia, while the other type resulted from fragmentation of platelet bodies. Concurrently with the fragmentation, thrombin-activated platelets displayed dramatically altered intracellular distribution of F-actin and septins detected as intense fluorescent clusters with a ~2-fold increase in the intensity of septins 2 and 9 and a ~300-fold increase in the F-actin staining. Synchronously with the structural alterations, thrombin induced a time-dependent reduction of the mitochondrial membrane potential (Δψm) in platelets. The overall fluorescence intensity of the Δψm-sensitive MitoTracker dye in freshly formed thrombin-initiated plasma clots dropped 2- and 4-fold after 60 min and 90 min, respectively. A drop of Δψm inversely correlated with an increase of the fraction of disintegrated platelets (r=-0.93, p<0.01). Flow cytometry showed enhanced phosphatidylserine exposure in thrombin-activated platelets, either with or without mitochondrial depolarization. Thrombin caused a significant 59% decrease of the average ATP content in activated platelets relative to untreated platelets after 60 min of incubation. Remarkably, the initial drop of Δψm and ATP content was almost concurred with the termination of contraction of the platelet-rich plasma clot measured as a 90%-decrease of platelet-generated contractile stress. Unexpectedly, no activation of caspase 3/7 was detected in platelets after 90 min of treatment with thrombin. Meanwhile, calpain activity detected in platelets 90 min after thrombin treatment was 6.5-fold higher compared to untreated platelets. Moreover, calpain inhibition caused a ~30-min delay in the commencement of thrombin-induced platelet fragmentation. Conclusions: Our findings indicate that following thrombin-induced platelet activation, a substantial fraction of platelets undergo time-dependent dysfunction and structural disintegration into subcellular particles. The fragmentation of platelets is accompanied by dramatic rearrangements of platelet cytoskeletal components, including polymerization, clustering, and redistribution of actin and septins. Thrombin-induced platelet fragmentation is concurrent with severe impairment of platelet functionality, including mitochondrial depolarization, ATP depletion, and loss of platelet contractility. The lack of caspase activity and increased calpain activity in energetically exhausted thrombin-treated platelets undergoing fragmentation suggests a calpain-dependent platelet death pathway. These studies indicate that such a form of platelet death may be an underappreciated mechanism for enhanced elimination of platelets from the circulation in (pro)thrombotic conditions or under other conditions once they have performed their functions. Work supported by the Program for Competitive Growth at KFU and AHA grant 17SDG33680177. Disclosures No relevant conflicts of interest to declare.

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Evaluation Of Bcma As a Therapeutic Target In Multiple Myeloma Using An Antibody-Drug Conjugate

Blood ◽

10.1182/blood.v122.21.4447.4447 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4447-4447 ◽

Cited By ~ 5

Author(s):

Kwee L Yong ◽

Fiona M Germaschewski ◽

Manuel Rodriguez-Justo ◽

Danton Bounds ◽

Lydia Lee ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Confocal Microscopy ◽

Surface Expression ◽

Tumour Cells ◽

Time Dependent ◽

Antibody Drug Conjugate ◽

Drug Conjugate ◽

Antibody Drug

B-cell maturation antigen (BCMA) is upregulated at the terminal stages of plasma cell (PC) differentiation, and is expressed on normal and malignant PC. Apart from low levels of mRNA detected on dendritic cells, expression appears absent on other tissues, indicating the potential as a target for novel antibody therapeutics for multiple myeloma (MM). We generated a humanised anti-BCMA antibody, modified for Fc-enhanced function, and conjugated to mcMMAF (anti-BCMA antibody drug conjugate, ADC). Flow cytometric studies on human myeloma cell lines (HMCL) showed rapid internalisation of anti-BCMA antibody by flow cytometry and confocal microscopy. The internalised antibody was transported to the lysosome and was nearly undetectable by confocal microscopy after 6 hours, indicating the suitability of BCMA as a target for an antibody drug conjugate (ADC). BCMA expression reached original surface levels by 6 hours post antibody treatment, thus maintaining the cell as a target for effector mediated killing. Evaluation of BCMA expression on HMCL revealed variable surface expression (1/11 high, 5/11 moderate, 5/11 low). Tumour cell killing by the ADC was expression level, dose and time dependent. The highest expressing HMCL, H929, showed significant killing (60% at 100ng/mL) at 24 hours, and up to 90% after 2 days. Cells expressing moderate levels of BCMA required incubation for up to 4 days to show maximal levels of cell death, suggesting the importance of continued internalisation of the antibody/antigen complexes over this period. ARH77 cells were transduced to express varying levels of BCMA, and killing at 3 days (200ng/ml) was directly proportional to level of surface expression (NT 0% killing, Low BCMA 75% killing, High BCMA 90%). We studied surface antigen levels in a cohort of patients to ascertain the need for patient selection. Like the HMCL, patient CD138+ plasma cells (PC) displayed a range of expression. Of 67 patients tested, CD138+ PC from 12 expressed high levels, 52 expressed intermediate, and 3 had low/negative surface BCMA as determined by MFI ratio of specific antibody to isotype control. Non-CD138+ cells from the bone marrow (BM) were negative for BCMA. Immuno-histochemistry (IHC) on paraffin-embedded BM sections, using a murine antibody and dual staining with anti-Blimp1 to identify tumour cells, revealed both membrane and diffuse, or punctate, cytoplasmic staining. Expression levels varied, from high uniform, to heterogeneous and patchy, to uniform low level. In no patient were the tumour cells entirely negative for BCMA. There was broad correlation between FACS analysis and IHC, thus patients were divided into high, moderate and low expressing groups. Examination of patient and disease characteristics revealed no correlation between BCMA expression and disease stage, response to last treatment, time from diagnosis, isotype, CD56 expression, or cyclin D-type, but there was a trend towards higher BCMA levels in tumours with adverse genetics (90% of patients with adverse genetics had high/moderate levels cf 64% of patients with standard CGN (p=0.06, Fisher’s exact test, 2-tailed). CD138+ cells in LPL (n=3) were positive for BCMA, but CD20+ lymphocytes were negative. Serum BCMA levels in MM patients (175.6±242.6ng/mL; mean±SD, n=34) were higher than in normal subjects (9.28±1.9ng/mL; n=38) but no correlation with bone marrow plasmacytosis or surface BCMA was noted. Levels appeared similar between new diagnosis (147.6±190.8ng/mL; mean±SD, n=8) and relapsed disease (184.3±259.1ng/mL; n=26). We tested ADC activity on primary tumour cells in whole BM cultures, enumerating viable CD138+ cells by flow cytometry. As with the HMCL, ADC mediated cytotoxicity was expression level, dose and time dependent, with a slower time course than with HMCL, perhaps reflecting the slower turn-over of these cells. In samples expressing moderate levels of BCMA, ADC-mediated cytotoxicity increased from 23.1±2.9% (mean±SEM, n=6) at 1-2 days to 48.3±5.1% at 4 days, and 61.2±6.2% by 6-7 days. Optimal doses of ADC ranged from 500ng-1ug/ml. In summary, these preclinical data in MM support the potential utility of an anti-BCMA ADC across the whole MM population, perhaps with particular efficacy in patients with adverse genetics, for whom an unmet need remains. Disclosures: Yong: GSK: Research Funding. Germaschewski:GSK: Employment. Mayes:GlaxoSmithKline: Employment. Sully:GlaxoSmithKline: Employment. Seestaller-Wehr:GlaxoSmithKline: Employment. Fieles:GlaxoSmithKline: Employment. Tunstead:GlaxoSmithKline: Employment. McCahon:GlaxoSmithKline: Employment. Craigen:GlaxoSmithKline: Employment.

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Dose-Dependent Effects of Platelet-Rich Plasma Powder on Chondrocytes In Vitro

The American Journal of Sports Medicine ◽

10.1177/0363546520911035 ◽

2020 ◽

Vol 48 (7) ◽

pp. 1727-1734 ◽

Cited By ~ 5

Author(s):

Olga Hahn ◽

Matthias Kieb ◽

Anika Jonitz-Heincke ◽

Rainer Bader ◽

Kirsten Peters ◽

...

Keyword(s):

Cell Culture ◽

Growth Factor ◽

Platelet Rich Plasma ◽

Cell Activity ◽

Fold Increase ◽

Cell Number ◽

Time Dependent ◽

Human Chondrocytes ◽

Enzyme Linked Immunosorbent Assay ◽

Unstimulated Control

Background: Platelet-rich plasma (PRP) is widely used in sports medicine. However, neither preparation nor parameters for clinical application, such as concentration, timing, and number of applications, are standardized, making research and clinical utilization challenging. Purpose: To investigate the effect of varying doses of PRP powder in terms of different concentrations, timing, and number of applications on human chondrocytes in a reproducible cell culture model. Study Design: Controlled laboratory study. Methods: A standardized lyophilized platelet growth factor preparation (PRP powder) was used to stimulate human chondrocytes. Chondrocytes were cultivated for 2 weeks with different stimulation frequencies (2×, 3×, 6×) and different concentrations of PRP powders (0.5%, 1%, 5%). Cell proliferation and metabolic cell activity were analyzed on days 7 and 14. Phenotypic changes were visualized through live-dead staining. Chondrogenic differentiation was quantified with enzyme-linked immunosorbent assay to assess the synthesis of procollagen types 1 and 2. Furthermore, sulfated proteoglycans and glycosaminoglycans were analyzed. Results: Human chondrocytes exhibited a significant dose- and time-dependent increase after 14 days in cell number (1% and 5% PRP powder vs unstimulated control: 7.95- and 15.45-fold increase, respectively; 2× vs 6× stimulation with 5% PRP powder: 4.00-fold increase) and metabolic cell activity (1% and 5% PRP powder vs unstimulated control: 3.27-fold and 3.58-fold change, respectively). Furthermore, cells revealed a significant increase in the amount of bone-specific procollagen type 1 (14 days, 1.94-fold) and sulfated glycosaminoglycans (14 days, 2.69-fold); however, no significant change was observed in the amount of cartilage-specific collagen type 2. Conclusion: We showed that chondrocytes exhibit a significant dose- and time-dependent increase in cell number and metabolic cell activity. The standardized use of growth factor concentrates in cell culture models can contribute to clinical knowledge in terms of dosage and timing of PRP applications. Clinical Relevance: Problems with PRP, such as the absence of standardization, lack of consistency among studies, and unknown dosage, could be solved by using characterized PRP powder made by pooling and lyophilizing multiple platelet concentrates. The innovative PRP powder generates new possibilities for PRP research, as well as for the treatment of patients.

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Analysis of Human Platelet Glycoprotein llb-llla by Fluorescein Isothiocyanate-Conjugated Disintegrins with Flow Cytometry

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648984 ◽

1994 ◽

Vol 72 (06) ◽

pp. 919-925 ◽

Cited By ~ 22

Author(s):

Chao-Zong Liu ◽

Yi-Wen R Wang ◽

Ming-Ching Shen ◽

Tur-Fu Huang

Keyword(s):

Flow Cytometry ◽

Fluorescence Intensity ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Fluorescein Isothiocyanate ◽

Platelet Membrane ◽

Platelet Glycoprotein ◽

Type I ◽

Activated Platelets ◽

Very High

SummaryDisintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein Ilb-IIIa (GPIIb-Ilia) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-Ilia complex on platelet membrane with a very high affinity (Kd, 10−7 ∼ 10−8 M). In this study, we analyzed GPIIb-Ilia complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-Ilia. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann’s thrombasthenia were probed with FITC-disintegrins. As a result these three patients could be classified as type I thrombasthenia based on the extremely low level of GPIIb-Ilia detected by this method (<5% of normal value).

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Binding of Coagulation Factor XIII Zymogen to Activated Platelet Subpopulations: Roles of Integrin αIIbβ3 and Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0039-1683912 ◽

2019 ◽

Vol 119 (06) ◽

pp. 906-915 ◽

Cited By ~ 1

Author(s):

Yana N. Kotova ◽

Nadezhda A. Podoplelova ◽

Sergey I. Obydennyy ◽

Elizaveta A. Kostanova ◽

Alexander A. Ryabykh ◽

...

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Factor Xiii ◽

Coagulation Factor ◽

Fibrin Clot ◽

Factor Xiiia ◽

Integrin Αiibβ3 ◽

Activated Platelets ◽

Preferential Binding ◽

Platelet Thrombi

AbstractFactor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbβ3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbβ3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi.

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A Flow Cytometry Method for Characterizing Platelet Activation

2020 Design of Medical Devices Conference ◽

10.1115/dmd2020-9070 ◽

2020 ◽

Author(s):

Brian Alzua ◽

Mark Smith ◽

Yan Chen

Keyword(s):

Flow Cytometry ◽

Medical Device ◽

Platelet Activation ◽

Medical Devices ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Standard Regimen ◽

Activated Platelets ◽

Wide Range

Abstract Hemocompatibility testing is critical for assessing the safety of blood-contacting medical devices. Comprehensive hemocompatibility testing requires examining a wide range of possible adverse effects cause by direct or indirect blood contact, such as hemolysis, complement activation, and thrombus formation [1]. Moreover, these domains each encompass complex intercellular processes with many potential targets for analysis. For example, the current testing paradigm of platelet function may involve exposing the device to human whole blood and performing simple blood counts and/or macroscopic evaluation to determine the extent of platelet activation and clot formation as described in ASTM F2888-19. However, this approach does not capture any observations for device-mediated initiation of any steps in the platelet activation pathway prior to aggregation. We have validated a method to evaluate platelet activation by quantifying surface p-selectin expression after exposure to various materials. This method will provide an additional level of detail about potential platelet activating properties of a medical device. Flow cytometry has been used previously to measure platelet activation for clinical and research purposes. We sought to adapt this method to test for platelet activation induced by exposure of blood to medical devices or materials. We determined that processing fresh whole blood to platelet-rich plasma (PRP) by gentle centrifugation enhanced the signal compared to fresh blood itself. In each experiment, devices were exposed to PRP according to an extraction ratio of 6 cm2/mL for 1 hour. A blank control consisting of untreated PRP, and a positive control consisting of ADP, a potent agonist, were also used. After the exposure, excess plasma was removed from the articles and combined with anti-CD61 (to stain for platelets) and anti-CD62P (to stain for activated platelets) antibodies. Flow cytometry was then performed to quantify the percentage of CD62P+ over the total CD61+ cells to measure the percentage of activated platelets. In order to optimize the method, we investigated the effect of several experimental factors, including anticoagulant usage, donor variability, and selection of reference materials to serve as controls. Our results indicate that the flow cytometry-based method is consistent and reproducible, quick and easy to perform, and is well-correlated with results from the standard platelet and leukocyte count assay. The flow cytometry-based platelet activation method is a powerful supplement to the standard regimen of medical device hemocompatibility testing.

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Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614247 ◽

1998 ◽

Vol 79 (01) ◽

pp. 177-185 ◽

Cited By ~ 10

Author(s):

Ashia Siddiqua ◽

Michael Wilkinson ◽

Vijay Kakkar ◽

Yatin Patel ◽

Salman Rahman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Functional Characterization ◽

Human Platelets ◽

Western Blots ◽

Activated Platelets ◽

Reducing Conditions ◽

Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

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A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648952 ◽

1994 ◽

Vol 72 (05) ◽

pp. 745-749 ◽

Cited By ~ 7

Author(s):

Elza Chignier ◽

Maud Parise ◽

Lilian McGregor ◽

Caroline Delabre ◽

Sylvie Faucompret ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Activation ◽

Peripheral Blood ◽

Vascular Trauma ◽

Activated Platelets ◽

Group I ◽

Group Ii ◽

Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

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The Effect of Collagen Mediated Platelet Release on Plasma Prekallikrein Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661014 ◽

1984 ◽

Vol 51 (01) ◽

pp. 037-041 ◽

Cited By ~ 2

Author(s):

K M Weerasinghe ◽

M F Scully ◽

V V Kakkar

Keyword(s):

Platelet Aggregation ◽

Healthy Volunteers ◽

Platelet Rich Plasma ◽

Age Groups ◽

Inhibitory Effect ◽

Age Group ◽

Platelet Factor ◽

Activated Platelets ◽

Platelet Release ◽

Collagen Treatment

SummaryCollagen mediated platelet aggregation caused -5.6 ± 6.7% inhibition and +39.1 ± 15.2% potentiation of prekallikrein activation in plasma from normal healthy volunteers between 20–40 and 50–65 years of age, respectively (n = 15, p <0.01). The amouns of platelet factor-four (PF4) released in the two groups were not significantly different. Collagen treatment in the presence of indomethacin caused +11.5 ± 3.6% and +59.6 ± 19.5% potentiation in the 20–40 and 50–65 age groups respectively (p <0.02). Adrenaline mediated platelet aggregation caused -55.2 ± 7.1% and -35.2 ± 8.3% inhibition in the 20–40 and 50–65 age groups, respectively. Collagen treatment of platelet-deficient-plasma and platelet-rich-plasma in EDTA also caused potentiation of prekallikrein activation.The results indicate that the observed degree of prekallikrein activation after platelet aggregation is a net result of the inhibitory effect of PF4 and the potentiatory effect of activated platelets. The potentiatory effect was greater after collagen treatment as compared to adrenaline treatment, and in the 50–65 age group as compared to the 20–40 age group.

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The Role of P-Selectin in COVID-19 Coagulopathy: An Updated Review

International Journal of Molecular Sciences ◽

10.3390/ijms22157942 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7942

Author(s):

Chiara Agrati ◽

Alessandra Sacchi ◽

Eleonora Tartaglia ◽

Alessandra Vergori ◽

Roberta Gagliardini ◽

...

Keyword(s):

Endothelial Activation ◽

Cardiovascular Complications ◽

Current State ◽

Activated Platelets ◽

Blood Clots ◽

Severe Infections ◽

Platelet Aggregates ◽

Definition Of ◽

Leucocyte Recruitment

In severe COVID-19, which is characterized by blood clots and neutrophil-platelet aggregates in the circulating blood and different tissues, an increased incidence of cardiovascular complications and venous thrombotic events has been reported. The inflammatory storm that characterizes severe infections may act as a driver capable of profoundly disrupting the complex interplay between platelets, endothelium, and leukocytes, thus contributing to the definition of COVID-19-associated coagulopathy. In this frame, P-selectin represents a key molecule expressed on endothelial cells and on activated platelets, and contributes to endothelial activation, leucocyte recruitment, rolling, and tissue migration. Briefly, we describe the current state of knowledge about P-selectin involvement in COVID-19 pathogenesis, its possible use as a severity marker and as a target for host-directed therapeutic intervention.

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Endothelial cell-related autophagic pathways in Sugen/hypoxia-exposed pulmonary arterial hypertensive rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00527.2016 ◽

2017 ◽

Vol 313 (5) ◽

pp. L899-L915 ◽

Cited By ~ 8

Author(s):

Fumiaki Kato ◽

Seiichiro Sakao ◽

Takao Takeuchi ◽

Toshio Suzuki ◽

Rintaro Nishimura ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cell ◽

Vascular Remodeling ◽

Time Dependent ◽

Causative Role ◽

Dependent Manner ◽

Vascular Endothelial ◽

Pulmonary Vascular Remodeling ◽

Pulmonary Arterial

Pulmonary arterial hypertension (PAH) is characterized by progressive obstructive remodeling of pulmonary arteries. However, no reports have described the causative role of the autophagic pathway in pulmonary vascular endothelial cell (EC) alterations associated with PAH. This study investigated the time-dependent role of the autophagic pathway in pulmonary vascular ECs and pulmonary vascular EC kinesis in a severe PAH rat model (Sugen/hypoxia rat) and evaluated whether timely induction of the autophagic pathway by rapamycin improves PAH. Hemodynamic and histological examinations as well as flow cytometry of pulmonary vascular EC-related autophagic pathways and pulmonary vascular EC kinetics in lung cell suspensions were performed. The time-dependent and therapeutic effects of rapamycin on the autophagic pathway were also assessed. Sugen/hypoxia rats treated with the vascular endothelial growth factor receptor blocker SU5416 showed increased right ventricular systolic pressure (RVSP) and numbers of obstructive vessels due to increased pulmonary vascular remodeling. The expression of the autophagic marker LC3 in ECs also changed in a time-dependent manner, in parallel with proliferation and apoptotic markers as assessed by flow cytometry. These results suggest the presence of cross talk between pulmonary vascular remodeling and the autophagic pathway, especially in small vascular lesions. Moreover, treatment of Sugen/hypoxia rats with rapamycin after SU5416 injection activated the autophagic pathway and improved the balance between cell proliferation and apoptosis in pulmonary vascular ECs to reduce RVSP and pulmonary vascular remodeling. These results suggested that the autophagic pathway can suppress PAH progression and that rapamycin-dependent activation of the autophagic pathway could ameliorate PAH.

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