Enhanced Outside-In Signaling Through the Prothrombotic Pro33 Variant of Integrin αIIbβ3 in Adherent Platelets Under Static and Flow Dynamic Conditions

Abstract Abstract 2199 Objectives: Platelet integrin αIIbβ3 is polymorphic at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b). In patients with coronary artery disease, the Pro33 isoform is associated with premature manifestation of myocardial infarction (Zotz et al., JTH 2005). Based on this finding, it has been postulated that the Pro33 variant of αIIbβ3 has a prothrombotic phenotype. Moreover, several studies have shown that elevated shear stress can activate platelets leading to shear-induced platelet aggregation contributing to acute myocardial infarction. Thus, it has been reported that pathological shear stress directly regulates αIIbβ3 (Feng et al., Am J Physiol Cell Physiol 2006). We have shown that adherent Pro33 platelets have a higher resistance than Leu33 platelets upon exposure to high shear rates (Loncar et al, Thromb J 2007). We now studied the impact of the Leu33/Pro33 polymorphism on αIIbβ3-mediated outside-in signaling under static and flow dynamic conditions in the presence or absence of Mn2+, analyzing the Src pY418 and FAK pY397 activities. Methods: Adhesion assays were performed with 10 or 100 μg/ml of immobilized fibrinogen for different incubation times with washed human platelets in the presence or absence of Mn2+ (0.5 mM). Mn2+ induces the active conformation of αIIbβ3. Src pY418 and FAK pY397 activities were determined by Western blotting and quantified densitometrically. Control experiments were performed with 1% BSA. Adhesion experiments under flow conditions were carried out with a cone-plate viscometer. Results: Under static conditions, Pro33 platelets adherent onto fibrinogen exhibited a 2.5-fold higher Src pY418 activity than Leu33 platelets after incubation for 20 min (p<0.01). Presence of Mn2+ (0.5 mM) in suspended Leu33 platelets stimulated their Src pY418 activity in an extent comparable to that of platelets adherent onto fibrinogen, while addition of Mn2+ (0.5 mM) to platelets adherent onto fibrinogen yielded a 3.5-fold increase in Src pY418 activity (p<0.05). Increase of the Mn2+ concentration (2 mM) raised the Src pY418 activity 1.5-fold, as compared to 0.5 mM Mn2+ (p<0.05). In parallel experiments with both HPA-1 isoforms of αIIbβ3, Pro33 platelets adherent onto fibrinogen in the presence of 0.5 mM Mn2+ revealed a 2.5-fold higher Src pY418 activity than Leu33 platelets after 5 min (p<0.05). This difference further increased upon prolonged adhesion for 10 or 20 min with a 5-fold increase after 40 min (p<0.01). The concentration of immobilized fibrinogen (10 or 100 μg/ml) had no influence on this effect. Addition of abciximab completely abolished the Src pY418 activation. Upon exposure to abnormal (5000 s−1) but not physiological (500 s−1) shear rates for 2 or 5 min, Pro33 platelets adherent onto 100 μg/ml fibrinogen exhibited a 2- or 2.5-fold higher Src pY418 activity than under static conditions (p<0.05 or 0.02, respectively). Under the same conditions, the Src pY418 activity of Pro33 platelets was 2-fold higher than that of Leu33 platelets (p<0.05). Again, different concentrations of fibrinogen (10 μg/ml and 100 μg/ml) did not affect these results. In comparison to Src (pY418), phosphorylation of FAK (pY397) increased slower both in Leu33 and Pro33 platelets adherent onto fibrinogen, when exposed to a shear rate of 5000 s−1 for 10min. Both isoforms (Leu33 or Pro33) exhibited a significantly higher FAK pY397 activity than under static conditions (3-fold increase, p<0.05). The Pro33 variant of αIIbβ3 showed a higher FAK pY397 activation than the Leu33 isoform (p<0.05). Again, abciximab completely blocked both the Src and FAK activation not only under static conditions but also upon exposure to shear. Conclusion: Our observations indicate that the HPA-1 polymorphism of αIIbβ3 has a considerable impact on the integrin-mediated outside-in signaling. The significantly higher Src (pY418) and FAK (pY397) activities of Pro33 platelets adherent onto fibrinogen under static and flow conditions are in agreement with the contention that the Pro33 (HPA-1b) isoform of αIIbβ3 is indeed a prothrombotic integrin variant. Disclosures: No relevant conflicts of interest to declare.

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The Impact of Shear Stress On the Interaction of Human Platelet Integrin αIIbβ3 with Fibrinogen.

Blood ◽

10.1182/blood.v120.21.2167.2167 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2167-2167

Author(s):

Marianna Gyenes ◽

Volker R. Stoldt ◽

Khon C. Huynh ◽

Rüdiger E. Scharf

Keyword(s):

Shear Stress ◽

Shear Rate ◽

Fold Increase ◽

Shear Rates ◽

High Shear Rates ◽

Platelet Signaling ◽

Src Activation ◽

Static Conditions ◽

The Impact

Abstract Abstract 2167 Objectives: Shear stress can activate platelets resulting in subsequent platelet aggregation. The so-called “shear-induced platelet aggregation” (SIPA) contributes to various vascular diseases (Speich et al., Am J Physiol Cell Physiol 2008). Several signaling pathways were proposed to be involved in this process, e.g., αIIbβ3-mediated signaling (Feng et al., Am J Physiol Cell Physiol 2006). We investigated the impact of shear stress on the αIIbβ3–ligand interaction in human platelets adherent onto fibrinogen. Platelets on immobilized fibrinogen were exposed to various shear rates and signaling of Src and FAK tyrosine kinases, both essential in the integrin downstream signaling pathways, were examined. Specifically, we analyzed the role of αIIbβ3 in shear-induced platelet signaling (i) by comparing the Src Y418 and FAK Y397 phosphorylation activities between platelets on immobilized fibrinogen and platelets on BSA matrix in response to shear stress, and (ii) by performing experiments in the presence of the αIIbβ3 antagonist abciximab. Methods: Human washed platelets were incubated on immobilized fibrinogen 100 μg/ml or 1% BSA either under static conditions or exposed to shear rates of 500 s−1 or 5000 s−1, respectively. Specific phosphorylation of Src (pY418) and FAK (pY397) was determined by Western blot and quantified densitometrically. Experiments under flow conditions were performed in a cone-plate viscometer. Results: Both Src and FAK exhibited phosphorylation under static conditions on immobilized fibrinogen after 2 min of adhesion. A shear rates of 500 s−1 did not increase the phosphorylation activities. By contrast, high shear rates (5000 s−1) significantly enhanced both Src and FAK phosphorylations in fibrinogen-adherent platelets (3-fold increase each, p<0.05). In the absence of immobilized fibrinogen, platelets incubated with BSA matrix did not show any Src activation under static conditions and only a very low Y418 phosphorylation activity in response to a shear rate of 500 s−1. A shear rate of 5000 s−1 considerably induced Src pY418 activity compared to platelets exposed to physiological shear stress (10-fold increase, p< 0.01). In response to shear rates of 500 s−1 or 5000 s−1, we detected a significantly higher Src activation in platelets adherent onto fibrinogen (500 s−1: 10-fold higher, p<0.01; 5000 s−1: 2-fold higher, p<0.05) than in platelets incubated over a BSA matrix indicating a ligand-dependent signaling. When platelets over BSA were exposed to a shear rate of 5000 s−1, FAK also exhibited a significant elevation of pY397 activity (9-fold increase, p<0.05). By contrast to Src, in platelets exposed to a shear rate of 500 s−1 or 5000 s−1, we observed approximately equal FAK pY397 activation, independent of the presence or absence of immobilized fibrinogen. In platelets incubated for 10 min on a fibrinogen matrix under static conditions, we did not detect any change in the Src activation compared to 2 min incubation. The activity of FAK pY397, however, was time-dependent and showed a 3-fold higher phosphorylation extent after 10 min than after 2 min adhesion (p<0.05). In response to a shear rate of 500 s−1 both Src Y418 and FAK Y397 phosphorylations exhibited a considerable time-dependent enhancement (comparing the phosphorylation activities after incubation for 2 or 10 min). This enhancement could be seen both in platelets adherent onto fibrinogen and in platelets over BSA (3 to 6-fold increase, p<0.05). In platelets exposed to a shear rate of 5000 s−1 for 10 min, the Src and FAK phosphorylation activities were similar to platelets after 2 min. Abciximab inhibited the Src and FAK signaling in platelets exposed to 5000 s−1 on immobilized fibrinogen. The same inhibition was seen in platelets exposed to 5000 s−1 over BSA (p<0.05). Conclusions: Exposure of platelets to high shear rates induces a significant increase of both Src and FAK signaling compared to platelets under static conditions. Whereas Src activation remains predominantly ligand-dependent in fibrinogen-adherent platelets even under shear stress, FAK signaling appears to be shear-induced. The finding that, abciximab inhibits the activation of both Src and FAK in the absence of fibrinogen, emphasizes the role of integrin αIIβ3 in the shear-induced platelet signaling. Disclosures: No relevant conflicts of interest to declare.

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Characterization of Fibronectin Assembly by Adherent Platelets Under Flow Conditions: Effect of Shear Stress and Role of β3 Integrins

Blood ◽

10.1182/blood.v120.21.5191.5191 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5191-5191

Author(s):

Khon C. Huynh ◽

Volker R. Stoldt ◽

Marianna Gyenes ◽

Rüdiger E. Scharf

Keyword(s):

Shear Stress ◽

Shear Rate ◽

Exposure Time ◽

Total Protein ◽

High Shear ◽

Flow Conditions ◽

Platelet Surface ◽

Shear Rates ◽

Static Conditions

Abstract Abstract 5191 Introduction: To fulfill their role in hemostasis, circulating platelets need to irreversibly adhere to the site of vascular injury and to resist to shear stress generated by the flowing blood. We previously reported that there is a relationship between the conformation of fibronectin (Fn) and its role in platelet adhesion and aggregation (Huynh, K. C. et al., ASH Annual Meeting Abstract, 2011. 118(21): p. 2209). In the present study, we examined the effect of shear stress on the assembly of Fn by adherent platelets. Moreover, we studied the role of β3 integrins (αIIbβ3 and αvβ3) in Fn assembly under flow conditions. Methods: Alexa fluor 488-conjugated fibronetin (Fn488) was added to suspensions of washed platelets (108/ml) in HEPES Tyrode buffer. CaCl2 (2 mM) and ADP (10 μM) were added immediately prior to the experiments. The samples (150 μl) were subsequently applied onto plates precoated with 50 μg/ml Fn. A DiaMed Impact-R device was used to generate shear rates of 500 s−1 or 5000 s−1 for 2 min or 10 min. Nonadherent platelets were removed by washing with PBS buffer followed by addition of 150 μl of 2 % DOC lysis buffer. Lysates were collected and total protein concentrations were determined by Bradford assay. The DOC-insoluble pellets containing Fn fibrils were isolated by centrifugation at 13, 500 rpm for 20 min. Pellets were then solubilized with 100 μl of 1 % SDS buffer. Equal amounts of samples based on total protein concentrations were loaded onto wells of 96-well microplates. Fluorescence signals from Fn488 of samples were recorded by a Fluoroskan microplate reader. In some experiments, abciximab (anti-β3, 10 μg/ml) or LM609 (anti-αvβ3, 5 μg/ml) antibody, were added to platelet mixtures before loading onto Fn precoated plates. All data were collected from at least three different experiments and analyzed using GraphPad Quickcals. To test for statistical differences, student's t-test was used. Results: Fn assembly by adherent platelets was strongly affected by the applied shear rate but not by the exposure time to shear. At a shear rate of 500 s−1, there were no insoluble Fn fibrils detectable in samples with adherent platelets after 2 or 10 min. When shear rates increased from 500 s−1 to 5000 s−1, the amount of insoluble Fn detectable on platelets after 2 and 10 min increased significantly (p < 0. 05) suggesting that adherent platelets exposed to high shear rates assemble more Fn fibrils on their surface. However, prolongation of exposure time to shear from 2 to 10 min did not result in significantly more Fn assembled by adherent platelets. By contrast, there were no insoluble fibrils that could be detected with adherent platelets under static conditions for 2 and 10 min. After 2 min at a shear rate of 5000 s−1, platelets blocked with abciximab showed a significant decrease in the amounts of insoluble Fn fibrils in comparison with control experiments (no antibody) (p = 0. 02). Similar inhibitory effects could be seen with platelets treated with LM609. In parallel experiments in which 10 min at 5000 s−1 were applied, both abciximab and LM609 had an inhibitory effect on Fn fibrillogenesis with a stronger effect by abciximab. Taken together, these data show that αvβ3 even at the low expression on platelets plays a major role in initiating the fibrillogenesis of Fn under high shear rate conditions, whereas αIIbβ3 contributes to the progression of Fn fibrils formation subsequently. Conclusion: Our observations document that the assembly of Fn on the surface of adherent platelets is strongly affected by shear rate conditions. In addition, our data imply that, despite its lower expression on platelet surface, αvβ3 provides a significant contribution in initiating the Fn assembly under high flow conditions, as compared with αIIbβ3. By contrast, αIIbβ3 with its abundant amount on the platelet surface probably exerts its effect in the later phase of Fn fibrillogenesis. The present findings support the contention that not a single integrin or Fn binding domain, but multiple interaction steps including different molecules and Fn domains may be involved in assembling Fn. Disclosures: No relevant conflicts of interest to declare.

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Glutathione regulates integrin αIIbβ3-mediated cell adhesion under flow conditions

Thrombosis and Haemostasis ◽

10.1160/th08-02-0095 ◽

2008 ◽

Vol 100 (05) ◽

pp. 857-863 ◽

Cited By ~ 11

Author(s):

Chalmette Ball ◽

K. Vinod Vijayan ◽

Trung Nguyen ◽

Kim Anthony ◽

Paul F. Bray ◽

...

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Fluid Shear Stress ◽

Platelet Rich Plasma ◽

Early Observation ◽

Flow Conditions ◽

Fluid Shear ◽

Physical Force ◽

Integrin Αiibβ3 ◽

Static Conditions

SummaryThe platelet integrin αIIbβ3 mediates the final step of platelet aggregation that requires pre-activation through an inside-out signal initiated by agonists. Experiments conducted under static conditions using platelet-rich plasma show that platelet activation and adhesion activity of αIIbβ3 are regulated by glutathione (GSH-GSSG) redox potential.However,it remains unclear as to whether GSH-GSSG exerts its regulatory role in platelets by direct targeting of αIIbβ3 or intracellular signals that activate the integrin. A role of fluid shear stress is also not known. We examined the effects of GSH-GSSG on the adhesion of CHO cells expressing two HPA variants of human αIIbβ3 to the immobilized fibrinogen and von Willebrand factor (VWF) under flow conditions. GSH-GSSG dose-dependently reduced the number of adherent cells to fibrinogen and VWF under 2.5 dyn/cm2 of shear stress, a physical force calculated to be 110 dyne on platelets. GSH treatment also abolished the hyperadhesion activity of cells expressing the Pro33 variant of αIIbβ3.The inhibition was also observed with washed platelets. The data differ from the early observation that GSH enhanced platelet aggregation induced by sub-threshold concentrations of platelet agonists. The results suggest that GSH may have distinct effects on agonist-induced αIIbβ3 activation and on the αIIbβ3-fibrinogen or αIIbβ3-VWF bonds when exposed to fluid shear stress. They further suggest that the HPA phenotype may be redox-regulated.

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Impact of the Leu33Pro Polymorphism of αIIbβ3 on Integrin – Ligand Interaction Under Abnormally High Shear Conditions

Blood ◽

10.1182/blood.v124.21.4156.4156 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4156-4156

Author(s):

Marianna Gyenes ◽

Volker R. Stoldt ◽

Khon C. Huynh ◽

Rudiger E. Scharf

Keyword(s):

Shear Stress ◽

Shear Rate ◽

Human Platelets ◽

Dependent Manner ◽

Ligand Interaction ◽

Shear Rates ◽

Src Signaling ◽

Src Activation ◽

Static Conditions ◽

The Impact

Abstract Objectives: Several studies have shown that shear stress can activate platelets leading to shear-induced platelet aggregation. Moreover, it has been reported that pathological shear stress can directly regulate platelet αIIbβ3 (Feng et al. 2006). This integrin is polymorphic at residue 33 (Leu or Pro) of the β subunit. The Pro33 isoform of αIIbβ3 may be a prothrombotic integrin variant. For example, patients with coronary artery disease, who are carriers of Pro33 platelets, experience their myocardial infarction 5.2 years (median) earlier than those with Leu33 platelets (Zotz et al. 2005). In this work, we studied the impact of the Leu33Pro polymorphism on Src pY418 and FAK pY397 signaling in human platelets adherent onto fibrinogen and subsequently exposed to physiological (500 s-1) or abnormally high (5000 s-1) shear rates. Methods: Washed platelets were placed onto immobilized fibrinogen (100 µg/ml) or BSA (2 %) either under static conditions or upon exposure to shear rates of 500 s-1 or 5000 s-1. Incubation times were 2, 5 or 10 min. Specific phosphorylations of Src (pY418) and FAK (pY397) were determined by Western blot and quantified densitometrically. Experiments under flow conditions were performed in a cone-plate viscometer. Results: Adherent Pro33 positive platelets exhibited a significantly higher Src activity than Leu33 platelets throughout under static conditions (2-fold higher after 2 and 5 min, 3-fold after 10 min, p<0.01, each). A physiological shear rate of 500 s-1 did not have a further effect on the Src signaling in fibrinogen-adherent Pro33 or Leu33 platelets over an incubation time of 10 min. By contrast, in response to a shear rate of 5000 s-1, both genotypes exhibited a rapid, significant increase in Src (pY418) activation compared to platelets under static conditions (3-fold higher Src phosphorylation after 2 min, p <0.01). While Src activation in Pro33 platelets remained constantly elevated during 10 min of adhesion, a decrease in pY418 activity of Leu33 platelets was found, resulting in a 4-fold higher Src signaling of Pro33 platelets, as compared to Leu33 platelets (p<0.001). For FAK, we observed low Y397 phosphorylation both in adherent Leu33 and Pro33 platelets after 2 min under static conditions. Upon prolonged adhesion (> 5 min), Pro33 platelets exhibited a significantly higher FAK (pY397) activity than Leu33 platelets (5-fold higher after 5 min, 3-fold higher after 10 min, p<0.01, each). In contrast to Src activation, both shear rates (500 s-1 and 5000 s-1) affected specific FAK phosphorylation considerably in Pro33 platelets. This resulted in significantly higher FAK signaling of Pro33 than of Leu33 platelets (5-fold higher, p<0.01). In Leu33 platelets, we observed an enhanced pY397 activity only in response to 5000 s-1. Next, we examined the impact of the Leu33Pro polymorphism of αIIbβ3 on Src and FAK signaling without fibrinogen, exclusively in response to shear. Under this condition, we detected only a slight increase in Src activity in Pro33 but not in Leu33 platelets. By contrast, FAK signaling in response to shear rates of 500 s-1 and 5000 s-1 exhibited a significantly higher pY397 activity in Pro33 than in Leu33 platelets (p<0.01). In control experiments, abciximab completely inhibited Src and FAK signaling in fibrinogen-adherent platelets exposed to a shear rate of 5000 s-1 but also in non-adherent platelets exposed to 5000 s-1over BSA (p<0.01, each). Conclusion: Exposure of human platelets to shear enhances specific phosphorylation of Src and FAK in an integrin-dependent manner. This effect is modulated by the Leu33Pro polymorphism of αIIbβ3. Pro33 platelets exhibit higher phosphorylation activities than Leu33 platelets both under static and flow dynamic conditions. The observation of higher phosphorylation activities of both tyrosine kinases, Src and FAK, in Pro33 than in Leu33 platelets in suspension upon exposure to shear (without concomitant adhesion) suggests that the Leu33Pro polymorphism of αIIbβ3 plays a role in shear-induced signaling. This contention is also supported by the strong inhibition of abciximab, abrogating shear-induced activation of both kinases not only in fibrinogen-adherent platelets but also in platelets in suspension. Overall, these observations suggest that platelet αIIbβ3 plays a relevant role in shear-induced integrin signaling. Disclosures No relevant conflicts of interest to declare.

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Impaired Platelet Function in Sept8-Deficient Mice In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0040-1718733 ◽

2020 ◽

Author(s):

Kerstin Jurk ◽

Katharina Neubauer ◽

Victoria Petermann ◽

Elena Kumm ◽

Barbara Zieger

Keyword(s):

Platelet Function ◽

Thrombin Generation ◽

Human Platelets ◽

Platelet Surface ◽

Integrin Αiibβ3 ◽

Glycoprotein Vi ◽

Fibrinogen Receptor ◽

Static Conditions ◽

The Impact

AbstractSeptins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

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Antibiofilm Properties of Temporin-L on Pseudomonas fluorescens in Static and In-Flow Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms21228526 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8526

Author(s):

Angela Di Somma ◽

Federica Recupido ◽

Arianna Cirillo ◽

Alessia Romano ◽

Alessandra Romanelli ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Biofilm Formation ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Antibiofilm Activity ◽

Dead Cell ◽

Flow Conditions ◽

Dynamic Conditions ◽

Ability To Act ◽

Static Conditions

Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.

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Flow Analysis of Von Willebrand Factor Collagen Binding Mutants

Blood ◽

10.1182/blood.v118.21.2213.2213 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2213-2213

Author(s):

Thomas A J McKinnon ◽

Agata Anna Nowak ◽

Alina Hua ◽

Carolyn Millar ◽

Michael Laffan

Keyword(s):

Shear Stress ◽

Collagen Type ◽

Collagen Type I ◽

Type I ◽

Flow Conditions ◽

Collagen Binding ◽

Binding Function ◽

Static Conditions ◽

A1 Domain ◽

Von Willebrand

Abstract Abstract 2213 Von Willebrand Factor (VWF) binds to exposed sub-endothelial collagen at sites of vessel injury principally via its A3 domain, although some evidence suggests that the A1 domain can compensate for the A3 domain under flow conditions if the A3 domain is absent or non-functional. Recently, several naturally occurring Von Willebrand disease-causing mutations have been indentified in the A3 domain; S1731T, W1745C, S1783, H1786D and most recently M1761K, as well as one mutation in the A1 domain (I1343V) all of which have defective collagen binding. While the collagen binding function of these mutations has been assessed under static conditions it remains to be established if these affect collagen binding under shear stress. In the present study the collagen binding mutants were expressed in HEK293T cells and collagen binding function determined using an in vitro flow assay. All of the mutations were expressed at similar levels to wild type (wt) VWF and demonstrated normal multimeric patterns and binding to GPIbα under static conditions. As expected, collagen binding analysis under static conditions confirmed the collagen binding defect of all the mutants, with reduced or abolished binding to both collagens type I and III for all the mutants except S1731T which demonstrated normal binding to collagen type III and slightly reduced binding to collagen type I. Analysis of platelet capture under flow conditions confirmed that all the mutants were able to capture platelets similarly to wtVWF. Analysis of VWF mediated platelet capture to a collagen surface under flow conditions confirmed the phenotype of the collagen binding mutants. With the exception of S1731T, which demonstrated normal platelet capture on both collagens, none of the mutants were able to bind to collagen type I or III under flow conditions, or mediate platelet capture at high shear stress. The collagen binding function of these mutants under flow was partially restored when co-expressed with wtVWF. Interestingly, in contrast to a previous study, a VWF variant lacking the A3 domain (VWF-ΔA3) failed to bind to collagen under shear stress and was not able to mediate platelet capture to collagen. Together these data confirm that the major collagen binding site in VWF is located in the A3 domain and demonstrate that collagen binding mutations affect VWF mediated platelet capture under shear stress. 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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Transition to Turbulence Downstream of a Stenosis for Whole Blood and a Newtonian Analog Under Steady Flow Conditions

Journal of Biomechanical Engineering ◽

10.1115/1.4052370 ◽

2021 ◽

Author(s):

Rayanne Pinto Costa ◽

Blaise Simplice Talla Nwotchouang ◽

Junyao Yao ◽

Dipankar Biswas ◽

David Casey ◽

...

Keyword(s):

Reynolds Number ◽

Steady Flow ◽

Whole Blood ◽

Critical Reynolds Number ◽

Blood Rheology ◽

Transition To Turbulence ◽

Flow Conditions ◽

Shear Rates ◽

The Impact

Abstract Blood, a multiphase fluid comprised of plasma, blood cells, and platelets, is known to exhibit a shear-thinning behavior at low shear rates and near-Newtonian behavior at higher shear rates. However, less is known about the impact of its multiphase nature on the transition to turbulence. In this study, we experimentally determined the critical Reynolds number at which the flow began to transition to turbulence downstream of an eccentric stenosis for whole porcine blood and a Newtonian blood analog (water-glycerin mixture). Velocity profiles for both fluids were measured under steady-state flow conditions using an ultrasound Doppler probe placed 12 diameters downstream of an eccentric stenosis. Velocity was recorded at 21 locations along the diameter at 11 different flow rates. Normalized turbulent kinetic energy was used to determine the critical Reynolds number for each fluid. Blood rheology was measured before and after each experiment. Tests were conducted on five samples of each fluid inside a temperature-controlled in-vitro flow system. The viscosity at shear rate 1000 s 1 was used to define the Reynolds number for each fluid. The mean critical Reynolds numbers for blood and water-glycerin were 470 ± 27.5 and 395 ± 10, respectively, indicating a ~19% delay in transition to turbulence for whole blood compared to the Newtonian fluid. This finding is consistent with a previous report for steady flow in a straight pipe, suggesting some aspect of blood rheology may serve to suppress, or at least delay, the onset of turbulence in vivo.

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Enhanced Integrin Activation of PLD2-Deficient Platelets Accelerates Inflammation after Myocardial Infarction

International Journal of Molecular Sciences ◽

10.3390/ijms21093210 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3210 ◽

Cited By ~ 1

Author(s):

Aglaia Maria Klose ◽

Meike Klier ◽

Simone Gorressen ◽

Margitta Elvers

Keyword(s):

Myocardial Infarction ◽

Plasma Levels ◽

Ischemia Reperfusion ◽

Scar Formation ◽

Border Zone ◽

Myocardial Ischemia And Reperfusion ◽

Integrin Αiibβ3 ◽

Tnf Α ◽

The Impact ◽

Deficient Mice

Background: Phospholipase (PL)D1 is crucial for integrin αIIbβ3 activation of platelets in arterial thrombosis and TNF-α-mediated inflammation and TGF-β-mediated collagen scar formation after myocardial infarction (MI) in mice. Enzymatic activity of PLD is not responsible for PLD-mediated TNF-α signaling and myocardial healing. The impact of PLD2 in ischemia reperfusion injury is unknown. Methods: PLD2-deficient mice underwent myocardial ischemia and reperfusion (I/R). Results: Enhanced integrin αIIbβ3 activation of platelets resulted in elevated interleukin (IL)-6 release from endothelial cells in vitro and enhanced IL-6 plasma levels after MI in PLD2-deficient mice. This was accompanied by enhanced migration of inflammatory cells into the infarct border zone and reduced TGF-β plasma levels after 72 h that might account for enhanced inflammation in PLD2-deficient mice. In contrast to PLD1, TNF-α signaling, infarct size and cardiac function 24 h after I/R were not altered when PLD2 was deleted. Furthermore, TGF-β plasma levels, scar formation and heart function were comparable between PLD2-deficient and control mice 21 days post MI. Conclusions: The present study contributes to our understanding about the role of PLD isoforms and altered platelet signaling in the process of myocardial I/R injury.

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