Continuum modeling of thrombus formation and growth under different shear rates

The interaction of platelets with subendothelium requires the transport of platelets to the vicinity of the surface, as well as the basic cell-surface reaction. Exposure of subendothelium to human citrated blood flowing in an annular perfusion chamber at wall shear rates (α) of 50-10,000 sec-1 indicates that a diffusion controlled (DC) transport regime exists below 650 sec- l in which platelet adhesion (C+S) was strongly dependent on α, and thrombus formation (T) was absent . Above 800 sec-1, an apparently reaction controlled (RC) regime predominates in which C+S was independent of α, and T increased in both extent and size. Variation of hematocrit (H) from 0-701. in the RC regime (2600 sec-1) lead to a steady increase of C+S with H, and an exponential increase in T as H increased from 30 to 70%. In the DC regime (200 sec-1) virtually no thrombi were formed for all H, and C+S increased as H increased to 40%; above 40%, C+S became independent of H. Thus, at low α (venous), the platelet-subendothelial reaction is controlled primarily by the arrival rate of platelets at the surface and the red cells increase this transport for H up to 40%. At high α (microcirculatory) , the platelet-vessel wall reactivity becomes more dominant and red cells increase the ability of platelets to attach to the subendothelium.

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Prostacyclin (prostaglandin I2, PGI2) inhibits platelet adhesion and thrombus formation on subendothelium

Blood ◽

10.1182/blood.v53.2.244.244 ◽

1979 ◽

Vol 53 (2) ◽

pp. 244-250 ◽

Cited By ~ 118

Author(s):

HJ Weiss ◽

VT Turitto

Keyword(s):

Blood Vessels ◽

Human Blood ◽

Platelet Adhesion ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Rabbit Aorta ◽

Prostaglandin I2 ◽

Shear Rates ◽

Platelet Spreading ◽

Aggregation Of Platelets

Abstract Prostaglandin I2 (prostacyclin, PGI2), a substance synthesized in the wall of blood vessels, has been previously shown to inhibit the aggregation of platelets in stirred platelet-rich plasma. We used a method in which segments of deendothelialized rabbit aorta are perfused at arterial shear rates with human blood and found that both platelet adhesion and thrombus formation on subendothelium was inhibited in blood containing 10 nM PGI2. PGI2 appears to reduce adhesion by inhibiting platelet spreading. These findings suggest that PGI2 could regulate the deposition of platelets on vascular surfaces.

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Elevated Threshold Shear Rate for the Dependence of Glycoprotein Ibα-Mediated Platelet Thrombus Formation onto Immobilized von Willebrand Factor in Mouse Blood.

Blood ◽

10.1182/blood.v104.11.3662.3662 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3662-3662

Author(s):

Patrizia Marchese ◽

Taisuke Kanaji ◽

Denisa D. Wagner ◽

Jerry Ware ◽

Zaverio M. Ruggeri

Keyword(s):

Shear Rate ◽

Collagen Type ◽

Thrombus Formation ◽

Collagen Type I ◽

Type I ◽

Shear Rates ◽

Normal Platelet ◽

Platelet Thrombus ◽

Von Willebrand ◽

Willebrand Factor

Abstract The interaction between platelet glycoprotein (GP) Ibα and von Willebrand Factor (VWF) is essential to initiate platelet deposition at sites of vascular injury and sustain platelet thrombus formation when the shear rate exceeds a threshold value. With human blood, the dependence of normal platelet adhesion and aggregation on VWF-GP Ibα function becomes evident at shear rates above 1,000 s−1. In the last several years, mouse models have been increasingly used to study the mechanisms of thrombus formation in circulating blood, and mice deficient in both VWF and GP Ibα have been generated. These animals offer the opportunity to evaluate whether the pathways of platelet adhesion and aggregation mediated by VWF and GP Ibα are equally important in mouse and human blood as well as to define the threshold shear rate at which the function of these pathways may become essential in the mouse circulation. To address this issue, we used an ex vivo perfusion system using fibrillar collagen type I as the thrombogenic surface and a flow chamber in which the shear rate varied according to a predictable function from the inlet to the outlet in relation to the x,y position in the flow path. Thus, wall shear rates between 5,000 at the inlet and 0 s−1 at the outlet could be evaluated in a single experiment, allowing a precise definition of the threshold at which platelet deposition on the surface could initiate. In these studies we used wild type control animals (WT), mice deficient in VWF (VWF-KO) and mice in which most of the extracellular domain of GP Ibα was replaced by a domain of the human interleukin 4 receptor (GPIb-KO/IL-4R). In the latter case, the ligand binding function of GP Ibα was obliterated, but unlike in GP Ib-KO mice platelet morphology and count were essentially normal. Blood was obtained from the retroorbital vein plexus and contained 100 u/ml heparin as an anticoagulant. Experiments were recorded in real time for the visualization of platelet-surface contacts and confocal videomicroscopy was used for the direct measurement of platelet thrombus volume. With normal mouse blood, platelet formed large thrombi throughout the tested range of shear rates. In contrast, with VWF-KO and GPIb-KO/IL-4R blood, thrombus volume was less than 5% of normal at 5,000 s−1, approximately 50% of normal at 3,000 s−1, but entirely normal at 1,500 s−1. Essentially the same results were observed when the extracellular matrix of mouse fibroblasts, which may better represent the complex thrombogenic properties of the vascular wall, was used as a reactive substrate instead of isolated collagen type I. The different threshold shear rate at which VWF and GP Ibα function are essential for thrombus formation with human and mouse platelets may be explained by the smaller size of the latter, which consequently are subjected to a lower drag at equivalent shear rate levels. Moreover, the similar behavior of VWF-KO and GPIb-KO/IL-4R platelets suggests that, under the conditions of these studies, VWF binding is the predominant GP Ibα function required for normal platelet thrombus formation at high shear rates. The present results should allow a more critical evaluation of the findings derived from mouse models of hemostasis and thrombosis.

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Tissue Factor-Driven Enhanced Platelet Aggregation in Flowing Blood in a Rabbit Model of Childhood Hemolytic Uremic Syndrome.

Blood ◽

10.1182/blood.v106.11.2166.2166 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2166-2166

Author(s):

Eric F. Grabowski ◽

Eirini Nestoridi ◽

Alexander Garcia ◽

James G. Fox ◽

Julie R. Ingelfinger

Keyword(s):

Hemolytic Uremic Syndrome ◽

Tissue Factor ◽

Thrombus Formation ◽

Rabbit Model ◽

Renal Tissue ◽

Shear Stresses ◽

Normal Donor ◽

Shear Rates ◽

Uremic Syndrome ◽

And Control

Abstract The Dutch belted rabbit constitutes a promising animal model of the childhood hemolytic uremic syndrome (HUS; A. Garcia, J. Infect. Dis. 2002). When Shiga toxin (Stx)-producing E. coli are administered orally, these rabbits develop HUS. Platelet-fibrin thrombus formation in this model was examined on unfixed renal tissue (5 μm sections) after mounting on 150-μm glass slides in test animal-control animal pairs. Normal donor blood, collected into 4U/ml low MW heparin and 10 μM quinacrine dihydrochoride, was drawn at shear rates of 270–650 sec-1 through a parallel-plate flow chamber for which one surface was one of the above slides. Such shear rates give rise to shear stresses which approach the 20–25 dynes/cm2 estimated to exist in glomerular arterioles. Platelets depositing on the sections were imaged in real time using epifluorescence digital videomicroscopy, with quantitation of adherent platelets via image processing. For 16 pairs of test and control sections, the percent of image pixels occupied by adherent platelets after 6–9 min of blood flow was 17.7 ± 3.06 (mean ± SD) for test sections vs 5.71 ± 5.18 for controls (P<0.001). This 3.1-fold increase for sections from Stx-treated rabbits is all the more striking considering that individual platelet aggregates on these sections were also larger and thicker, as judged from greater local pixel intensities. In 4 additional experiments, preincubation of sections with 300 nM of a monoclonal antibody (courtesy of Y. Nemerson, Mt. Sinai Medical Center, NY, NY) directed against human tissue factor (TF) with known cross-reactivity to rabbit TF markedly reduced platelet adhesion/aggregation on both test and control sections. Residual platelet deposition, which only slightly favored test sections, may be due to either a non-TF-dependent mechanism or incomplete neutralization of TF activity by the amount of antibody used. Studies of parallel fibrin deposition are in progress. Thus, renal tissue from the Dutch belted HUS model is reactive to platelets in large part via expression of TF, an expression augmented on test sections. These observations support the hypothesis that the TF pathway of coagulation plays a major role in childhood HUS.

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Evaluation of Platelet Thrombus Formation under Flow.

Blood ◽

10.1182/blood.v106.11.3964.3964 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3964-3964

Author(s):

Volker R. Stoldt ◽

Jan Peveling ◽

Robert Loncar ◽

Andreas Beck ◽

Volker Aurich ◽

...

Keyword(s):

Shear Rate ◽

Laser Scanning ◽

Type I Collagen ◽

Thrombus Formation ◽

Confocal Laser ◽

Lag Phase ◽

Type I ◽

Shear Rates ◽

Platelet Aggregate

Abstract We have used confocal laser microscopy and a novel “voxel”-based imaging software to study the dynamics of platelet aggregation and thrombus formation when anticoagulated blood was perfused over collagen-coated surfaces at shear rates simulating arterial flow. The objective was to evaluate the three-dimensional growth of platelet thrombi over time (“4-D” imaging). Blood from healthy donors, anticoagulated with either PPACK (80 μM) or, depending on the type of experiment, with trisodium citrate (11 mM), was incubated with mepacrine (10 μM) to render platelets fluorescent. Blood was aspirated with a syringe pump through a rectangular perfusion chamber (flow path height of 80 μm) at a flow rate of 160 or 480 μl per min to provide initial shear rates of 500 or 1,500 sec−1, respectively. Prior to perfusion, glass coverslips were coated with fibrillar type I collagen (Roche Diagnostics, Mannheim, Germany) prepared in 0.5 M acedic acid, pH 2.8, and blocked with 2 % BSA. The chamber was mounted on a Zeiss Axiovert 100M/LSM 510 invert laser scanning confocal microscope (Carl Zeiss, Oberkochem, Germany). Upon perfusion, a series of stacks, i.e. 30 confocal optical sections, from the bottom to the apex of the forming platelet aggregate or thrombus, were obtained every 25 sec with a 488-nm laser and a scanning time of < 500 msec on an area of 26,450 μm2. Images corresponding to an area of 0.202 μm2 were analyzed by a “voxel”-based procedure, whereby a voxel is defined by a volume of 0.202 μm3 (0.45 μm x 0.45 μm x 1 μm). For calibration, fluorescent beads (Invitrogen, Carlsbad, CA, USA) were used, and the volume coresponding to a 1.0 μm thick stack was calculated pursuant to the voxel technique. A threshold was applied to distinguish adherent platelets from the background. Using these procedures, a uniform profile of thrombus formation and volume was observed (n=7). With citrate anticoagulated blood at an initial shear rate of 500 sec−1, thrombus growth begun after a lag phase of 220 sec, and, after 420 sec, thrombus volume reached a maximum (mean ± SD, 5x104 ± 4.9x103 μm3). Thrombus progression occurred in a two-step way with an apical growth (height extension) at the interval of 220 and 300 sec, and a further growth in the plane section at the interval of 300 and 420 sec after perfusion. Prolonged perfusion resulted in markedly abnormal flow pattern due to thrombus growth and increased shear rates. Again at an initial shear rate of 500 sec−1, platelet aggregate formation and thrombus progression were completely suppressed in the presence of anti-αIIbβ3 antibody (abciximab, 4 μg/ml). Interestingly, the polymorphism (HPA-1, PlA) of the β subunit of αIIbβ3 had a dramatic effect on thrombus growth. Thus, when comparing blood from homozygous carriers of HPA-1b (n=8) and HPA-1a (n=8), thrombus formation and progression occurred more rapidly with HPA-1b than with HPA-1a platelets, resulting in significantly larger thrombi from HPA-1b than from HPA-1a individuals (p=0.001). In conclusion, the voxel-based determination of thrombus formation and progression in vitro provides an appropriate technique to assess volumina of thrombi. Moreover, this technique can detect phenotypic differences related to an αIIbβ3 polymorphism which is postulated to modulate platelet thrombogenicity.

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Revised Model for Platelet Adhesion to Collagen.

Blood ◽

10.1182/blood.v114.22.2999.2999 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2999-2999

Author(s):

Lucia Stefanini ◽

Moritz Stolla ◽

Sean F Maloney ◽

Timothy Daniel Ouellette ◽

Claire Roden ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Mapk Signaling ◽

Low Flow ◽

Flow Conditions ◽

Integrin Activation ◽

Shear Rates ◽

Slow Kinetics ◽

Rap1 Activation

Abstract Abstract 2999 Poster Board II-968 The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions. Disclosures: No relevant conflicts of interest to declare.

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Anthocyanins Inhibit Platelet Activation and Attenuate Thrombus Growth In Both Human and Murine Thrombosis Models

Blood ◽

10.1182/blood.v116.21.3197.3197 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3197-3197 ◽

Cited By ~ 2

Author(s):

Yan Yang ◽

Zhenyin Shi ◽

Adili Reheman ◽

Wuxun Jin ◽

Conglei Li ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Intravital Microscopy ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Vessel Occlusion ◽

Shear Rates ◽

Perfusion Chamber ◽

Platelet Adhesion And Aggregation

Abstract Abstract 3197 Background: Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods: Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results: Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions: our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures: No relevant conflicts of interest to declare.

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CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction

Blood ◽

10.1182/blood.v122.21.3501.3501 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3501-3501

Author(s):

Jiansong Huang ◽

Xiaofeng Shi ◽

Wenda Xi ◽

Ping Liu ◽

Xiaodong Xi

Keyword(s):

Molecular Mechanisms ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Human Platelets ◽

Flow Conditions ◽

Cho Cell ◽

Integrin Αiibβ3 ◽

Shear Rates ◽

Platelet Adhesion And Aggregation

Abstract The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Thrombus formation and occlusion at high shear rates of blood flow in micro-channels

The Proceedings of Conference of Hokuriku-Shinetsu Branch ◽

10.1299/jsmehs.2017.54.d024 ◽

2017 ◽

Vol 2017.54 (0) ◽

pp. D024

Author(s):

Mizuki KOMATSU ◽

Shunichi KOBAYASHI

Keyword(s):

Blood Flow ◽

Thrombus Formation ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Micro Channels

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CD44 sensitivity of platelet activation, membrane scrambling and adhesion under high arterial shear rates

Thrombosis and Haemostasis ◽

10.1160/th14-10-0847 ◽

2016 ◽

Vol 115 (01) ◽

pp. 99-108 ◽

Cited By ~ 5

Author(s):

Kousi Alzoubi ◽

Madhumita Chatterjee ◽

Britta Walker ◽

Patrick Münzer ◽

Dong Luo ◽

...

Keyword(s):

Caspase 3 ◽

Thrombus Formation ◽

Blood Platelets ◽

Protein Abundance ◽

Integrin Activation ◽

Platelet Surface ◽

Shear Rates ◽

Occlusive Vascular Disease ◽

Phosphatidylserine Exposure

SummaryCD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44-/- ) were compared to platelets from corresponding wild-type mice (cd44+/+ ). In resting platelets, P-selectin abundance, αllbβ3 inte-grin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44-/- and cd44+/+ platelets. Platelet degranulation and αllbβ3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca2+-store depletion with thapsigargin (1 µM), effects more pronounced in cd44-/- than in cd44+/+ platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44-/- than in cd44+/+ platelets. Thrombin further decreased forward scatter in cd44-/- and cd44+/+ platelets, an effect which tended to be again more pronounced in cd44-/- than in cd44+/+ platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s-1) were significantly augmented in cd44-/- mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and prothrombotic potential of platelets.

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