Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms

Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C- adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ- induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen- induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.

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Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms

Blood ◽

10.1182/blood.v64.6.1200.1200 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1200-1206 ◽

Cited By ~ 90

Author(s):

RC Reimers ◽

SP Sutera ◽

JH Joist

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Red Blood Cells ◽

Blood Cells ◽

Platelet Rich Plasma ◽

Adenine Nucleotides ◽

Laminar Shear Stress ◽

Platelet Surface ◽

Surface Transport ◽

Laminar Shear

Abstract Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C- adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ- induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen- induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.

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The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645696 ◽

1990 ◽

Vol 63 (01) ◽

pp. 112-121 ◽

Cited By ~ 18

Author(s):

David N Bell ◽

Samira Spain ◽

Harry L Goldsmith

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Red Blood Cells ◽

Blood Cells ◽

Platelet Rich Plasma ◽

Mean Transit Time ◽

White Blood Cells ◽

Aggregate Size ◽

Human Platelets ◽

Induced Aggregation

SummaryThe effect of red blood cells, rbc, and shear rate on the ADPinduced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23°C the rate of single platelet aggregtion was upt to 9× greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates Ḡ = 41.9,335, and 1,920 s−1, and at both 0.2 and 1.0 µM ADP. At 0.2 pM ADP, the rate of aggregation was greatest at Ḡ = 41.9 s−1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At Ḡ ≥335 s−1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 µM ADP, the initial rate of single platelet aggregation was still highest at Ḡ = 41.9 s1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at Ḡ ≥335 s−1 but the rate of single platelet aggregation was markedly greater than at 0.2 pM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.

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ULTRASTRUCTURAL INVESTIGATIONS ON THE MECHANISM OF “SHEAR-INDUCED PLATELET ACTIVATION”

10.1055/s-0038-1642845 ◽

1987 ◽

Author(s):

L J Wurzinger ◽

R Opitz ◽

H Schmid-Schönbein

Keyword(s):

Shear Stress ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Adenine Nucleotides ◽

Coagulation System ◽

Laminar Shear Stress ◽

Shear Forces ◽

Platelet Factor ◽

Membrane Defects ◽

Biochemical Activation

High shear forces are suspected to play a triggering role in the initiation of arterial thrombosis, by activating platelets and the coagulation system. In an earlier study a shear stress of 170 N/m2 acting for only 7 milliseconds (ms) on platelet rich plasma (PRP) was found to induce a significant increase in platelet factor 3 availability (Thromb. Haemost. 54: 381-386; 1985). To clarify the question whether platelets can be activated directly by mechanical forces in analogy to smooth muscle cells, electron micrographs of platelets subjected to laminar shear stress were analysed with morphometric methods. The level of activation of platelet suspensions was quantified by assessing 1) the elongation of platelet profiles giving a measure for the “flatness” of the discoid resting platelets, and 2) the centralization of granules.Exposure to a shear stress of 170 N/m2 for 113 ms leaves ca. 15 % of the platelets irreversibly damaged, featuring degenerative ballooning, with break-down of internal structure and cell membrane defects. The remaining 85 % appear typically activated with rounded shape, extension of pseudopods and centralization of granules. Addition of “ADP-scavengers” to the suspension medium totally changes the appearance of sheared platelets: still a comparable proportion of them has undergone irreversible degenerative changes, but the “surviving” population lacks ultrastructural signs of platelet activation. This is reflected in values of the morphometric parameters which are close to the level of unsheared control samples.It is therefore concluded that “shear-induced platelet activation” cannot be ascribed to a direct stimulating effect of shear forces, but rather to secondary biochemical activation by adenine nucleotides leaking from a small percentage of shear destroyed platelets. The latter process, however, requires a well stirred though undiluted environment, as it is provided in vortices and eddies.

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The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin

Thrombosis and Haemostasis ◽

10.1160/th07-01-0010 ◽

2007 ◽

Vol 98 (07) ◽

pp. 192-200 ◽

Cited By ~ 33

Author(s):

Joseph Jakubowski ◽

You FuLi ◽

Marc Barnard ◽

Marsha Fox ◽

Matthew Linden ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Whole Blood ◽

Blood Cells ◽

Inflammatory Markers ◽

Active Metabolite ◽

Platelet Rich Plasma ◽

Shape Change ◽

Flow Cytometric Analysis ◽

Platelet Surface

SummaryThe novel thienopyridine prodrug prasugrel, a platelet P2Y12 ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation,there are few data on the direct effects of the prasugrel’s active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates,platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent in-hibition of these ADP-stimulated thrombo-inflammatory markers.These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC50 values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y12-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y12. In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y12-mediated up-regulation of thromboinflammatory markers of platelet activation.This inhibition is enhanced in the presence other blood cells and calcium,but not aspirin.

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PLATELET ALTERATIONS IN RESPONSE TO REPETITIVE, SHORT-DURATION LAMINAR SHEAR STRESS

10.1055/s-0038-1642843 ◽

1987 ◽

Author(s):

J H Joist ◽

J E Bauman ◽

S P Sutera

Keyword(s):

Shear Stress ◽

Pulse Duration ◽

Platelet Rich Plasma ◽

Pulse Amplitude ◽

Pulse Number ◽

Dense Granule ◽

Laminar Shear Stress ◽

Ramp Function ◽

Laminar Shear ◽

Granule Release

We examined platelet aggregation (PAG = loss of single platelets), platelet dense granule release, and platelet injury (LDH loss) in normal human citrated platelet-rich plasma subjected to biologically more relevant repetitive, laminar shear stress of 25 and 50 dyn/cm2 in a computer-controlled cone-plate viscometer. Shear pulse duration (1-3 sec), shear pulse ramp function (rate of shear stress increase and decrease per pulse, 0.6-4 sec), number of shear pulses (1-20) and pauses between shear pulses (0-5 sec) were varied in different combinations to assess the effects of each variable on platelet alterations. Maximum PAG (92±8%) was observed with three 1 sec shear pulses, 0.6 sec ramp function and 1 sec between shear pause. PAG decreased with increasing ramp function, increasing number of shear pulses (>10), and increasing pause duration. Rapid platelet deaggregation (starting at 5 sec) was observed after a single 1 sec shear exposure. The rate of deaggregation decreased with increasing shear pulse number, increasing shear pulse amplitude, and increasing shear pulse duration. In contrast to PAG, dense granule release increased progressively with increasing shear pulse number, duration, and amplitude. No appreciable platelet injury (LDH loss) was observed under the conditions used. The findings indicate that massive reversible PAG can be induced by a single 1 sec shear pulse and that the extent of PAG with more prolonged, repetitive shear exposure is largely a function of platelet deaggregation rather than PAG. Thus, data previously reported from our laboratory and other investigators using prolonged (>5 sec) exposure of platelets to shear stress may require reevaluation.

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The Ullrastructure of Platelet Participation in Hemostasis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656288 ◽

1965 ◽

Vol 13 (01) ◽

pp. 065-083 ◽

Cited By ~ 7

Author(s):

Shirley A. Johnson ◽

Ronaldo S. Balboa ◽

Harlan J. Pederson ◽

Monica Buckley

Keyword(s):

Platelet Aggregation ◽

Red Blood Cells ◽

Blood Cells ◽

Guinea Pigs ◽

Platelet Factor ◽

Mesenteric Vessels ◽

Platelet Aggregates ◽

Electron Lucent

SummaryThe ultrastructure of platelet aggregation in vivo in response to bleeding brought about by transection of small mesenteric vessels in rats and guinea pigs has been studied. Platelets aggregate, degranulate and separating membranes disappear in parallel with fibrin appearance which is first seen at several loci after 30 seconds of bleeding. About 40 per cent of the electron opaque granules, some of which contain platelet factor 3 have disappeared after one minute of bleeding while the electron lucent granules increase by 70 per cent suggesting that some of them may be empty vesicles. Most of the platelet aggregates of the random type disappear leaving clumped red blood cells entrapped by a network of fibrin fibers which emanate from the remains of platelet aggregates of the rosette type to maintain hemostasis.

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In Vitro Assay for Single-cell Characterization of Impaired Deformability in Red Blood Cells under Recurrent Episodes of Hypoxia

Lab on a Chip ◽

10.1039/d1lc00598g ◽

2021 ◽

Author(s):

YUHAO QIANG ◽

Jia Liu ◽

Ming Dao ◽

E Du

Keyword(s):

Shear Stress ◽

Red Blood Cells ◽

Single Cell ◽

Blood Cells ◽

Blood Circulation ◽

In Vitro Assay ◽

Vitro Assay ◽

Cyclic Shear

Red blood cells (RBCs) are subjected to recurrent changes in shear stress and oxygen tension during blood circulation. The cyclic shear stress has been identified as an important factor that...

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Epigenetic histones modification and cardiovascular lineage programming in mouse embryonic stem cells exposed to laminar shear stress

Vascular Pharmacology ◽

10.1016/j.vph.2006.08.148 ◽

2006 ◽

Vol 45 (3) ◽

pp. e56

Author(s):

Barbara Illi ◽

Alessandro Scopece ◽

Simona Nanni ◽

Antonella Farsetti ◽

Liliana Morgante ◽

...

Keyword(s):

Stem Cells ◽

Shear Stress ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Laminar Shear Stress ◽

Laminar Shear

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Laminar shear stress upregulates endothelial Ca2+-activated K+ channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00642.2012 ◽

2013 ◽

Vol 305 (4) ◽

pp. H484-H493 ◽

Cited By ~ 18

Author(s):

Jun Takai ◽

Alexandra Santu ◽

Haifeng Zheng ◽

Sang Don Koh ◽

Masanori Ohta ◽

...

Keyword(s):

Shear Stress ◽

Transcriptional Activation ◽

Static Condition ◽

Fold Increase ◽

Laminar Shear Stress ◽

Human Coronary Artery ◽

Intracellular Ca ◽

Kinase Cascade ◽

Dependent Protein ◽

Laminar Shear

In endothelial cells (ECs), Ca2+-activated K+ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca2+ levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca2+/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca2+/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm2) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8–30), whereas oscillatory shear (OS; ± 5 dyn/cm2 × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

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