Modulation of Platelet Adhesion by Antithrombin.

Abstract In this study, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating venous and arterial flow. Platelets in anticoagulated whole blood (PPACK, 40μM) were labeled with mepacrine (10μM). Adhesion of platelets onto fibrinogen-coated glass cover slips was assessed in a rectangular flow chamber (shear rates of 13 s−1 to 1500 s−1). Platelets were visualized at 15 sec, 1 and 5 min following perfusion using a fluorescence laser-scan microscope. In parallel, the effects of supraphysiological supplementation of blood with antithrombin (2.8 IU/ml of blood) on platelet adhesion rates was evaluated. During perfusion, platelet adhesion onto fibrinogen linearly increased with exposure time and shear rates. Within the first min of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin activity was observed at shear rates of 13 s−1 and 50 s−1 (r=−0.48 and r=−0.7, p each <0.05). Significant differences in platelet adhesion (1786±516 U vs. 823±331 U, p<0.05) related to low (92±3.3%) and high (117±4.1%) antithrombin activity was observed at a flow rate of 13s−1 within first minute. Further supplementation of anticoagulated whole blood with antithrombin (activity up to 280 %) decreased the rate of platelets adhesion (ratio of adhesion at 1 and 5 min) about 35% when compared to nonsupplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4, p=0.008). Application of heparine as anticoagulant did not enhance the antiadhesion properties of antithrombin. Our findings are in accordance with the “low shear phenomenon” of arterial thrombus progression, i.e. thrombus enlargement at distal areas with reduced flow or even stasis. Moreover, the observation that antithrombin significantly suppressed platelet adhesion onto immobilized fibrinogen under low flow conditions is of therapeutic interest and needs further evaluation.

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Investigating Platelet Interactions in Sickle Cell Disease Using a Novel Multi-Shear "Endothelialized" Microfluidic System

Blood ◽

10.1182/blood.v124.21.4155.4155 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4155-4155

Author(s):

Margo Renee Rollins ◽

Byungwook Ahn ◽

Yumiko Sakurai ◽

Jordan C Ciciliano ◽

Wilbur A Lam

Keyword(s):

Endothelial Cells ◽

Sickle Cell ◽

Whole Blood ◽

Blood Cells ◽

Platelet Adhesion ◽

Blood Samples ◽

Shear Rates ◽

Healthy Control ◽

Platelet Aggregates

Abstract Sickle Cell Disease (SCD) is an inherited disorder of the β-globin chain of hemoglobin, in which a single point mutation leads to decreased deformability of red blood cells (RBCs) and increased cellular adhesion to endothelium. The effect of this mutation on RBCs has been well characterized, and the interplay of endothelial cells, RBCs, and white blood cells (WBCs) have also been well characterized. However, few studies have specifically investigated how platelets interact with endothelial cells and other blood cells in the context of SCD and the role these cell fragments may have in vaso-occlusion. To that end, we utilized microfluidic technology previously developed in our lab to perform a real time in vitro analyses of platelet-endothelial cell interactions in SCD patient samples. This microvasculature-on-a-chip enables the visualization of blood cell-endothelial cell interactions under a controlled hemodynamic environment (Tsai et al, JCI, 2012). As shear stress can trigger platelet activation, we further modified and optimized our standard microfluidic devices to encompass 3 different physiologic shear rates. Our device features microchannels 50µm in diameter with human umbilical vein endothelial cells (HUVEC) confluently lining the channels; there are 12 channels in each device, grouped in 3 sets of 4 channels with graduating shear rates spanning 3 orders of magnitude (Figure 1). Our initial experiments were performed under normoxic conditions allowing characterization of platelet-endothelial interactions in an arterial in vitro environment. Whole blood samples were obtained from 3 patient populations: patients with HgbSS SCD on hydroxyurea (HgbSS+HU), patients with HgbSS SCD not on hydroxyurea (HgbSS-no HU), and normal healthy controls. Over 30 minutes, whole blood stained with fluorescently labeled CD41 to identify platelets and Hoeschst to identify HUVEC nuclei was perfused at a rate of 1.5µl/minute under videomicroscopy. Accumulation of platelets on the endothelialized channels and platelet aggregates were quantified based on anti-CD41 fluorescence. Within 1 minute of perfusion, HgbSS-no HU whole blood samples exhibited extensive platelet aggregates at 1 and 10 dyne/cm2 (Figure 2); this phenomenon did not occur under any of the shear conditions in blood samples from Hgb SS+HU or healthy control samples. In HgbSS-no HU blood samples, some of these thrombi-like aggregates were stable under flow, increased in size, and persisted for the remainder of the 30 minute experiments. In contrast, mild, uniform, platelet adhesion slowly developed at high shear conditions in Hgb SS+HU with fewer platelet aggregates forming as compared to patients with HgbSS- no HU. Healthy control samples did not exhibit this platelet aggregation. There appears to be an attenuating effect of hydroxyurea on platelets that prevents platelet clumping from occuring as frequently under various shear conditions that is not present in the Hgb SS-no HU samples (Figure 3). In conclusion, using our novel in vitro system, we have demonstrated the platelets from Hgb SS-no HU patients have a significantly increased propensity to adhere, aggregate, and accumulate in endothelialized microvasculature-sized microchannels. Interestingly, this effect appears to be attenuated in blood samples from Hgb SS+HU patients and not present in healthy controls, demonstrating that hydroxyurea appears to be an important modifier of this phenomenon. Experiments investigating the underlying mechanisms of this phenomenon, the effects of deoxygenation and the potential role of platelets in vaso-occlusion, the effects of sickle cell platelet adhesion/aggregation on endothelial function, and how hydroxyurea may or may not affect any or all of these parameters, are all currently ongoing. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Sphingosine kinase 1 (Sphk1) negatively regulates platelet activation and thrombus formation

AJP Cell Physiology ◽

10.1152/ajpcell.00029.2014 ◽

2014 ◽

Vol 307 (10) ◽

pp. C920-C927 ◽

Cited By ~ 6

Author(s):

Patrick Münzer ◽

Evi Schmid ◽

Britta Walker ◽

Anna Fotinos ◽

Madhumita Chatterjee ◽

...

Keyword(s):

Platelet Adhesion ◽

Thrombus Formation ◽

Sphingosine Kinase ◽

Flow Chamber ◽

Negative Regulator ◽

Sphingosine Kinase 1 ◽

Shear Rates ◽

Dense Granules ◽

Platelet Formation

Sphingosine 1-phosphate (S1P) is a powerful regulator of platelet formation. Enzymes generating S1P include sphingosine kinase 1. The present study thus explored the role of sphingosine kinase 1 in platelet formation and function. Activation-dependent platelet integrin αIIbβ3activation and secretion of platelets lacking functional sphingosine kinase 1 ( sphk1−/−) and of wild-type platelets ( sphk1+/+) were determined utilizing flow cytometry and chronolume luciferin assay. Cytosolic Ca2+activity ([Ca2+]i) and aggregation were measured using fura-2 fluorescence and aggregometry, respectively. In vitro platelet adhesion and thrombus formation were evaluated using a flow chamber with shear rates of 1,700 s−1. Activation-dependent increase of [Ca2+]i, degranulation (release of alpha and dense granules), integrin αIIbβ3activation, and aggregation were all significantly increased in sphk1−/−platelets compared with sphk1+/+platelets. Moreover, while platelet adhesion and thrombus formation under arterial shear rates were significantly augmented in Sphk1-deficient platelets, bleeding time and blood count were unaffected in sphk1−/−mice. In conclusion, sphingosine kinase 1 is a powerful negative regulator of platelet function counteracting degranulation, aggregation, and thrombus formation.

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Inhibition of Platelet Adhesion to Fibrin(ogen) in Flowing Whole Blood by Arg-Gly-Asp and Fibrinogen γ-Chain Carboxy Terminal Peptides

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646346 ◽

1992 ◽

Vol 68 (06) ◽

pp. 694-700 ◽

Cited By ~ 25

Author(s):

Roy R Hantgan ◽

Silvia C Endenburg ◽

I Cavero ◽

Gérard Marguerie ◽

André Uzan ◽

...

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Platelet Adhesion ◽

Integrin Receptor ◽

Shear Rates ◽

Perfusion Chamber ◽

Receptor Recognition ◽

Adhesive Interactions ◽

Coated Surfaces ◽

Carboxy Terminal

SummaryWe have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen-and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s–1 and 1,300 s–1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s–1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAK-QAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb: III a, as the primary receptor responsible for platelet: fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.

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Desensitization of Adrenaline-Induced Red Blood Cell H+ Extrusion in Vitro After Chronic Exposure of Rainbow Trout to Moderate Environmental Hypoxia

Journal of Experimental Biology ◽

10.1242/jeb.156.1.233 ◽

1991 ◽

Vol 156 (1) ◽

pp. 233-248 ◽

Cited By ~ 1

Author(s):

S. THOMAS ◽

R. KINKEAD ◽

P. J. WALSH ◽

C. M. WOOD ◽

S. F. PERRY

Keyword(s):

Rainbow Trout ◽

Blood Cell ◽

Red Blood Cell ◽

Whole Blood ◽

Inverse Correlation ◽

Adrenergic Stimulation ◽

Plasma Adrenaline ◽

Arterial Blood Gas ◽

Arterial Blood

The sensitivity of red blood cell Na+/H+ exchange to exogenous adrenaline was assessed in vitro using blood withdrawn from catheterized rainbow trout (Oncorhynchus mykiss) maintained under normoxic conditions [water PO2, (PwO2)=20.66 kPa] or after exposure to moderate hypoxia (PwO2=6.67-9.33 kPa) for 48 h, which chronically elevated plasma adrenaline, but not noradrenaline, levels. Peak changes in whole-blood extracellular pH over a 30 min period after adding 50–1000 nmoll−1 adrenaline were employed as an index of sensitivity; the blood was pre-equilibrated to simulate arterial blood gas tensions in severely hypoxic fish (PaO2=2.0 kPa, PaCO2=0.31 kPa). Blood pooled from normoxic fish displayed a dose-dependent reduction in whole-blood pH after addition of adrenaline. Blood pooled from three separate groups of hypoxic fish, however, displayed diminished sensitivity to adrenaline, ranging from complete desensitization to a 60%reduction of the response. Subsequent experiments performed on blood from individual (i.e. not pooled) normoxic or hypoxic fish demonstrated an inverse correlation between the intensity of H+ extrusion (induced by exogenous adrenaline addition) and endogenous plasma adrenaline levels at the time of blood withdrawal. However, acute increases in plasma adrenaline levels in vitro did not affect the responsiveness of the red blood cell to subsequent adrenergic stimulation. The intensity of H+ extrusion was inversely related to the PaO2in vivo between 2.67 and 10.66 kPa, and directly related to the logarithm of the endogenous plasma adrenaline level. The results suggest that desensitization of Na+/H+ exchange in chronically hypoxic fish is related to persistent elevation of levels of this catecholamine. This desensitization can be reversed in vitro as a function of time, but only when blood is maintained under sufficiently aerobic conditions.

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Results of in vitro whole blood coagulation assays using ROTEM and the flow-chamber T-TAS system are affected by hematocrit

Thrombosis Research ◽

10.1016/j.thromres.2020.06.030 ◽

2020 ◽

Vol 194 ◽

pp. 98-100

Author(s):

Anna Ågren ◽

Gustaf Edgren ◽

Paul Hjemdahl ◽

Gunilla Gryfelt ◽

Anders Östlund ◽

...

Keyword(s):

Blood Coagulation ◽

Whole Blood ◽

Flow Chamber ◽

Coagulation Assays

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Microfibrils (MF) Platelet Interaction: Requirement Of Von Willebrand Factor In Platelet Adhesion To A Placenta Microfibrillar Component (PMC)

10.1055/s-0038-1652578 ◽

1981 ◽

Author(s):

F Fauvel ◽

Y J Legrand ◽

N Gutman ◽

J P Muh ◽

G Tobelem ◽

...

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Human Platelets ◽

Human Artery ◽

Von Willebrand ◽

Willebrand Factor ◽

Adhesion Of Platelets ◽

Aggregation Of Platelets

It has been shown that collagenase resistant arterial microfibrils (MF) are able to interact with platelets and therefore represents, besides collagen, a second thrombogenic structure in the vessel wall. In vitro observation using a PMC purified from the villosities of human placenta by a mechanical non denaturing procedure confirm this interaction between platelets and MF. PMC was homogenous under electron microscope (feltwork of MF with a mean diameter of 120 – 130 A) and was glycoproteic in nature. PMC were able to induce an aggregation of human platelets only if the platelets were in plasma. The role of Von Willebrand factor (F VIII/WF) as a cofactor of the aggregation of platelets by MF has been postulated from the fact that twice washed platelets from normal subject resuspended in PPP obtained from a severe Von Willebrand deficient patient were not aggregated by the PMC. Furthermore, aggregation was restored after resuspension of the same platelets in the PPP of the same patient 30 and 120 minutes after perfusion of cryoprecipitate (40 units F VIII/RA per kg).F VIII/WF mediates platelet adhesion after binding to subendothelium of human artery. Our observation strongly supports the idea that MF are the subendothelial components to which F VIII/WF binds, thus promoting an adhesion of platelets.

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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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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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Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

Blood ◽

10.1182/blood.v76.2.345.bloodjournal762345 ◽

1990 ◽

Vol 76 (2) ◽

pp. 345-353 ◽

Cited By ~ 1

Author(s):

RR Hantgan ◽

G Hindriks ◽

RG Taylor ◽

JJ Sixma ◽

PG de Groot

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Von Willebrand Factor ◽

Whole Blood ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Shear Rates ◽

Wall Shear ◽

Von Willebrand ◽

Willebrand Factor

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.

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The Effect of Rigid Red Blood Cells on Platelet Adhesion in Blood Flow and Implications in Sickle Cell Disease

Blood ◽

10.1182/blood-2019-128733 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 986-986

Author(s):

Alison Leigh Banka ◽

Mark Shamoun ◽

Mario Gutierrez ◽

Tyler Tanski ◽

Lola Eniola-Adefeso

Keyword(s):

Blood Flow ◽

Sickle Cell Disease ◽

Red Blood Cells ◽

Whole Blood ◽

Blood Cells ◽

Platelet Adhesion ◽

Model System ◽

Cell Disease ◽

The Impact

Introduction: Sickle cell disease (SCD) occurs due to a mutation in the β-subunit of hemoglobin, causing stiffening of red blood cells (RBCs) and leading to RBC sickling and vaso-occlusive crises (VOC) in SCD patients. While sickled RBCs remain a hallmark of SCD, they are prone to lysis and represent a small fraction of the total RBCs present in patients at a given time. The remaining RBCs maintain a normal, discoid shape and are either healthy or stiff due to polymerization of the hemoglobin β-globin subunit. In healthy blood flow, RBCs form a core in the center of the vessel and the remaining cells, platelets and white blood cells (WBCs), marginate towards the endothelium. However, the increased stiffness of RBCs in SCD disrupts this neat segregation of blood cells to different areas of the blood vessel and can contribute to VOC, the root cause of many acute and chronic complications for SCD patients. Despite the presence of normally shaped, stiffened RBCs in SCD patients, the impact of these RBCs on other cell types in blood flow is currently not well understood. Our laboratory previously demonstrated that the presence of artificially rigidified RBCs leads to an expansion of the RBC core and significantly decreases WBC adhesion to an inflamed endothelium in vitro. Here, we examine the impact of stiffened RBCs on platelet adhesion to a damaged endothelium in vitro by first using a model system with artificially rigidified RBCs and second, utilizing SCD patient blood to further support our model and understand platelet-RBC interactions in SCD patients. Methods: In our model system, we artificially rigidified RBCs taken from healthy donors and reconstituted them into whole blood before perfusing the mixture over an activated, damaged endothelium using a parallel plate flow chamber. We quantified platelet adhesion to the endothelium in comparison to healthy, non-rigidified controls using fluorescent microscopy. To determine if our model findings translated to SCD, we recruited a cohort of hemoglobin SS and SC patients during routine visits and similarly perfused their whole blood over the same damaged endothelium and quantified platelet adhesion. Results and conclusions: The inclusion of artificially rigidified RBCs in otherwise healthy subject blood flow significantly increased platelet adhesion to a damaged endothelium with a maximum increase in platelet adhesion of six-fold over a healthy, non-rigid control in our model system. Both RBC rigidity and the percentage of RBCs that were artificially rigidified had a large impact on the increase in platelet adhesion. SCD platelet adhesion to the damaged endothelium model varied from donor to donor based on variables such as treatment method and disease severity. Overall, this work experimentally elucidates the biophysical impact of stiffened RBCs on platelet adhesion using both an artificial model utilizing healthy blood as well as SCD blood, which can help determine the mechanism of action causing VOC in SCD. Disclosures No relevant conflicts of interest to declare.

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