Decision letter: Activated αIIbβ3 on platelets mediates flow-dependent NETosis via SLC44A2 (2019)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Platelet-neutrophil interactions are important for innate immunity, but also contribute to the pathogenesis of deep vein thrombosis, myocardial infarction and stroke. Here we report that, under flow, von Willebrand factor/glycoprotein Ibα-dependent platelet 'priming' induces integrin αIIbβ3 activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet αIIbβ3 to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in SLC44A2 (rs2288904-A) present in 22% of the population causes an R154Q substitution in an extracellular loop of SLC44A2 that is protective against venous thrombosis results in severely impaired binding to both activated αIIbβ3 and VWF-primed platelets. This was confirmed using neutrophils homozygous for the SLC44A2 R154Q polymorphism. Taken together, these data reveal a previously unreported mode of platelet-neutrophil crosstalk, mechanosensitive NET production, and provide mechanistic insight into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis. eLife digest Platelets in our blood form clots over sites of injury to stop us from bleeding. Blood clots can also occur in places where they are not needed, such as deep veins in our legs or other regions of the body. Developing such clots – also known as deep vein thrombosis (or DVT for short) – is one of the most common cardiovascular diseases and a major cause of death. Although certain inherited factors have been linked to DVT, the underlying mechanisms of the disease remain poorly understood. In addition to platelets, the pathological (or dangerous) clots that cause DVT also contain immune cells called neutrophils which fight off bacterial infections. Platelets are recruited to the wall of the vein by a protein called "von Willebrand Factor" (or VWF for short). However, it remained unclear how these recruited platelets interact with neutrophils and whether this promotes the onset of DVT. To answer this question, Constantinescu-Bercu et al. used a device that mimics the flow of blood to study how human platelets change when they are exposed to VWF. This revealed that VWF 'primes' the platelets to interact with neutrophils via a protein called integrin αIIbβ3. Further experiments showed that integrin αIIbβ3 binds to a protein on the surface of neutrophils called SLC44A2. Once the neutrophils interacted with the 'primed' platelets, they started making traps which increased the size of the blood clot by capturing other blood cells and proteins. Finally, Constantinescu-Bercu et al. studied a genetic variant of the SLC44A2 protein which is found in 22% of people and is associated with a lower risk of developing DVT. This genetic mutation caused SLC44A2 to interact with 'primed' platelets more weakly, which may explain why people with this genetic variant are protected from getting DVT. These findings suggest that blocking the interaction between 'primed' platelets and neutrophils could reduce the risk of DVT. Although current treatments for DVT can prevent patients from forming dangerous blood clots, they can also cause severe bleeding. Since neutrophils are not crucial for normal blood clots to form at the site of injury, drugs targeting SLC44A2 could inhibit inappropriate clotting without causing excess bleeding. Introduction To fulfil their hemostatic function, platelets must be recruited to sites of vessel damage. This process is highly dependent upon von Willebrand factor (VWF). Upon vessel injury, exposed subendothelial collagen binds plasma VWF via its A3 domain (Cruz et al., 1995). Elevated shear, or turbulent/disturbed flow, then unravels tethered VWF and exposes its A1 domain, facilitating specific capture of platelets via glycoprotein (GP)Ibα. As well as capturing platelets under flow, the A1-GPIbα interaction also induces shear-dependent signaling events (Bryckaert et al., 2015). For this, GPIbα first binds the A1 domain of immobilized VWF (Zhang et al., 2015). Rheological forces then cause unfolding of the GPIbα mechanosensitive domain that translates the mechanical stimulus into a signal within the platelet (Ju et al., 2016; Zhang et al., 2015). This leads to release of intracellular Ca2+ stores and activation of the platelet integrin, αIIbβ3 (Gardiner et al., 2010). VWF-mediated signaling transduces a mild signal. Consequently, these signaling events are often considered redundant within hemostasis as platelets respond more dramatically to other agonists present at sites of vessel injury (e.g. collagen, thrombin, ADP, thromboxane A2) (Jackson et al., 2003; Senis et al., 2014). Full platelet activation involves release of α- and δ-granules, presentation of new cell surface proteins, activation of cell surface integrins and alterations in the membrane phospholipid composition. The extent of platelet activation is dependent upon both the concentration, and identity, of the agonist(s) to which the platelets are exposed, which is dictated by the location of the platelets relative to the damaged vessel. For example, platelets in the core of a hemostatic plug/thrombus are exposed to higher concentrations of agonists and are more highly activated (i.e. P-selectin-positive procoagulant platelets) than those in the surrounding shell (P-selectin-negative) (de Witt et al., 2014; Shen et al., 2017; Stalker et al., 2013; Welsh et al., 2014). Thus, platelets exhibit a 'tunable' activation response determined by agonist availability. Aside from hemostasis, platelets also have important roles as immune cells by aiding in targeting of bacteria by leukocytes (Gaertner et al., 2017; Kolaczkowska et al., 2015; Sreeramkumar et al., 2014; Wong et al., 2013). Platelet-leukocyte interactions also influence the development of inflammatory cardiovascular conditions. In deep vein thrombosis (DVT), VWF-dependent platelet recruitment, platelet-neutrophil interactions and the production of highly thrombotic neutrophil extracellular traps (NETs) all contribute to the development of a pathological thrombus (Brill et al., 2011; Brill et al., 2012; Fuchs et al., 2012a; Schulz et al., 2013; von Brühl et al., 2012). Although the precise sequence of events still remains unclear, it appears that during the early stages of DVT, VWF-bound platelets acquire the ability to interact with leukocytes (von Brühl et al., 2012). Exactly how this is mediated given the lack of vessel damage is unclear. It also remains to be determined precisely how platelet-tethered neutrophils undergo NETosis in DVT in the absence of an infectious agent. Known direct platelet-leukocyte interactions involve either P-selectin or CD40L on the surface of platelets binding to P-selectin glycoligand-1 (PSGL-1) and CD40, respectively, on leukocytes (Lievens et al., 2010; Mayadas et al., 1993; Palabrica et al., 1992). As platelets must be potently activated to facilitate P-selectin/CD40L exposure, such interactions unlikely mediate the early platelet-leukocyte interactions that occur in the murine DVT model. Consistent with this, lack of platelet P-selectin has no effect upon either leukocyte recruitment or thrombus formation in murine DVT (von Brühl et al., 2012). Leukocytes can also indirectly interact with platelets through Mac-1 (integrin αMβ2), which can associate with activated αIIbβ3 via fibrinogen (Weber and Springer, 1997), or directly via GPIbα (Simon et al., 2000). Interactions are also possible through lymphocyte function-associated antigen 1 (LFA-1/integrin αLβ2) that can bind intercellular adhesion molecule 2 (ICAM-2) on platelets (Damle et al., 1992; Diacovo et al., 1994). In both instances though, leukocyte activation is necessary to activate Mac-1 or LFA-1 integrins before interactions can occur. Although it is often assumed that only activated platelets bind leukocytes, recent studies have revealed that platelets captured under flow by VWF released from activated endothelial cells can recruit leukocytes (Doddapattar et al., 2018; Zheng et al., 2015). If VWF-GPIbα-dependent signaling is capable of promoting leukocyte binding, this may be highly relevant to the non-hemostatic platelet functions (particularly when other agonists are not available/abundant), but may also provide major mechanistic insights into the early recruitment of leukocytes during the initiation of DVT. Genome wide association studies (GWAS) on venous thromboembolism (VTE) have identified a panel of genes (ABO, F2, F5, F11, FGG, PROCR) with well-described influences upon coagulation and thrombotic risk, as well as those with well-established causative links (e.g. PROS, PROC, SERPINC1) (Germain et al., 2015; Germain et al., 2011; Rosendaal and Reitsma, 2009). This is consistent with the efficacy of therapeutic targeting of coagulation to protect against DVT with anticoagulants (Chan et al., 2016). However, although the use of anticoagulants is effective, dosing and efficacy are limited by the increase in the risk of bleeding in treated individuals (Chan et al., 2016; Schulman et al., 2014; Schulman et al., 2009; Schulman et al., 2013; Wolberg et al., 2015). Therefore, alternative targets that inhibit DVT disease processes, but that do not modify bleeding risk may provide new adjunctive therapies to further protect against the development or recurrence of DVT. GWAS studies have also identified additional risk loci for VTE, but with no known role in coagulation (Apipongrat et al., 2019; Germain et al., 2015; Hinds et al., 2016). This provides encouragement that alternative therapeutic targets may exist with the potential to modify the disease process without affecting bleeding risk. These loci include SLC44A2 and TSPAN15 genes (Apipongrat et al., 2019; Germain et al., 2015; Hinds et al., 2016). Despite the identification of these loci, the function of these cell surface proteins with respect to their involvement in the pathogenesis of venous thrombosis remains unclear. Using microfluidic flow channels to enable analysis of the phenotypic effects of VWF-GPIbα signaling under flow, we confirm the rapid activation of the platelet integrin, αIIbβ3. Activated αIIbβ3 is capable of binding directly to neutrophils via a direct interaction with SLC44A2. Under flow, this interaction transduces a signal into neutrophils capable of driving NETosis. A single-nucleotide polymorphism (SNP; rs2288904-A) in SLC44A2 (minor allele frequency 0.22) that is protective against VTE (Germain et al., 2015) encodes a R154Q substitution in the first extracellular loop of the receptor that markedly reduces neutrophil-platelet binding via activated αIIbβ3. These results provide a functional explanation for the protective effects of the rs2288904-A SNP and highlight the potential of SLC44A2 as an adjunctive therapeutic target in DVT (Constantinescu-Bercu et al., 2020). Results To explore the influence of platelet binding to VWF under flow upon platelet function, full length (FL-) human VWF was adsorbed directly onto microfluidic microchannel surfaces, or the isolated recombinant VWF A1 domain, or an A1 domain variant (Y1271C/C1272R, termed A1*) that exhibits a 10-fold higher affinity for GPIbα (Blenner et al., 2014), were captured via their 6xHis tag. Fresh blood anticoagulated with D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) and labeled with DiOC6, was perfused through channels at 1000 s−1 for 3.5 min. On FL-VWF, A1 or A1*, a similar time-dependent increase in platelet recruitment/surface coverage was observed (Figure 1a and Figure 1—figure supplements 1–2). Figure 1 with 2 supplements see all Download asset Open asset Platelet rolling and attachment to VWF under flow. (a) Vena8 microchannels were coated with either full-length VWF (FL-VWF; i-iii), VWF A1 (iv-vi) or A1* (vii-ix). Whole blood labeled with DiOC6 was perfused at 1000 s−1. Representative images (n = 3) of platelets (green) after 30, 90 and 180 s are shown. Scale; 50 μm (see also Video 1). (b) Experiments performed as in a), bound platelets (blue) were tracked (depicted by multi-colored lines) representing distance travelled in the first 30 s of flow. Scale bar; 50 μm. (c) Platelet rolling velocity on channels coated with A1 and A1*. Data plotted are median ±95% CI. n = 3562 platelets from 3 different experiments (A1) and n = 4047 platelets from 3 different experiments (A1*). Data were analyzed using the Mann-Whitney test. Figure 1—source data 1 Platelet rolling source data. https://cdn.elifesciences.org/articles/53353/elife-53353-fig1-data1-v2.xlsx Download elife-53353-fig1-data1-v2.xlsx Platelets rolled prior to attaching more firmly on all VWF channels. However, median initial platelet rolling velocity on VWF A1 was 1.76 μms−1, whereas on A1* this was significantly slower (median 0.23μms−1) (Figure 1b & c), reflective of its 10-fold higher affinity for GPIbα (Video 1). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Platelet capture on FL-VWF, VWF A1 or VWF A1*. Vena8 microchannels were coated with either full-length VWF (FL-VWF), VWF A1 or A1*. Whole blood labelled with DiOC6 was perfused at 1000 s−1 for 3 min. Note the reduced rate of platelet rolling on microchannels coated with A1* when compared to FL VWF or A1. Platelet binding to VWF under flow induces intraplatelet signaling and activation of αIIbβ3 Platelets bound to either FL-VWF, A1 or A1* formed small aggregates after about 2 min (Figure 2ai) due to activation of the platelet integrin, αIIbβ3, and its binding to plasma fibrinogen. Consistent with this, when plasma-free blood (i.e. RBCs, leukocytes and platelets resuspended in plasma-free buffer) was used, platelets remained as a uniform monolayer, and did not form microaggregates (Figure 2aii). Similarly, when activated αIIbβ3 was blocked in whole blood with eptifibatide or GR144053, aggregation was also inhibited (Figure 2aiii and iv). Irrespective of the surface (VWF, A1 or A1*), platelet aggregation was markedly reduced if plasma-free blood was used, or if αIIbβ3 was blocked (Figure 2b–e). These results demonstrate that the A1-GPIbα interaction leads to activation of αIIbβ3, which is consistent with previous reports (Goto et al., 1995; Kasirer-Friede et al., 2004). In support of this, fluorescent fibrinogen bound to platelets tethered via FL-VWF, but not to platelets captured to channel surfaces using an anti-PECAM-1 antibody (Figure 2—figure supplement 1a). Figure 2 with 1 supplement see all Download asset Open asset Platelet binding to VWF under flow induces αIIbβ3-dependent aggregation. (a) Vena8 microchannels were coated with A1* via its 6xHis tag. (i) Whole blood (WB) or ii) plasma-free blood (PFB), iii) WB containing eptifibatide or iv) WB containing GR144053 were perfused through channels at 1000 s−1. Representative images acquired after 3 min. Scale bar; 50 μm. (b) Graph measuring platelet aggregation over time in WB perfused through channels coated with A1 (red, n = 3), A1* (purple, n = 4) and FL-VWF (blue, n = 3), WB pre-incubated with eptifibatide (orange, n = 3) or GR144053 (green, n = 4) over channels coated with A1* and PFB over channels coated with A1* (magenta, n = 3) or FL-VWF (teal, n = 3). Data plotted are mean ± SD. (c–e) Bar charts comparing area under the curve (AUC) of the data presented in b). (c) WB perfused over A1 or A1* with or without eptifibatide or GR144053. (d) WB or PFB perfused over A1*. (e) WB or PFB perfused over FL VWF. Data presented are mean ± SD, n = 3 or 4 as indicated in b). Data were analyzed using the Mann-Whitney test. Figure 2—source data 1 Platelet aggregation source data. https://cdn.elifesciences.org/articles/53353/elife-53353-fig2-data1-v2.xlsx Download elife-53353-fig2-data1-v2.xlsx To investigate the effect of A1-GPIbα-dependent signaling, platelets were preloaded with the Ca2+-sensitive fluorophore, Fluo-4 AM. Platelets bound to A1* under flow exhibited repeated transient increases in fluorescence, corresponding to Ca2+ release from platelet intracellular stores in response to A1-GPIbα binding under flow (Video 2; Kasirer-Friede et al., 2004; Mu et al., 2010). Despite intracellular Ca2+ release, this did not lead to appreciable P-selectin exposure (i.e. α-granule release) (Figure 2—figure supplement 1b). Intraplatelet Ca2+ release was not detected when platelets were captured under flow using an anti-PECAM1 antibody. We therefore propose that flow-dependent VWF-GPIbα signaling 'primes', rather than activates, platelets. This 'priming' is characterized by activation of αIIbβ3, but minimal α-granule release, and represents part of the tunable response of platelets. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg VWF A1-GpIbα interaction induces intraplatelet Ca2+ release under flow. Platelets were pre-loaded with Fluo-4 AM prior to perfusion through VWF A1*-coated microchannels at 1000 s−1 for 5 min. Increases in platelet fluorescence corresponds to platelet intracellular Ca2+ release following attachment to VWF A1*. Note the repeated transient increases in fluorescence under flow, indicative of sustained and repeated signaling stimuli. Platelets 'primed' by VWF interact with leukocytes To explore the influence of platelet 'priming' upon their ability to interact with leukocytes, platelets were captured and 'primed' on VWF for 3 min at 1000 s−1. Thereafter, leukocytes in whole blood (also labeled with DiOC6) that were perfused at 50 s−1, rolled on the platelet-covered surface (Video 3). Leukocytes did not interact with platelets captured via an anti-PECAM-1 antibody (Figure 3a), demonstrating the dependency on prior A1-GPIbα-mediated platelet 'priming'. Figure 3 with 1 supplement see all Download asset Open asset Leukocytes bind to VWF-bound platelets under flow (see also Video 3). (a) Graph of the number of leukocytes/minute in WB interacting with platelets bound to FL-VWF in the absence (n = 12) or presence of eptifibatide/GR144053 (n = 5) or binding to platelets bound to anti-PECAM-1 antibody (n = 3). (b) Graph of the number of leukocytes/minute in WB or PFB interacting with platelets bound to FL-VWF in the absence (n = 12) or presence of 1.3 mg/ml fibrinogen (n = 3) or eptifibatide/GR144053 (n = 4), or binding to platelets bound to anti-PECAM-1 antibody (n = 3). (c) Graph of the number of leukocytes/minute in PFB interacting with platelets bound to A1* in the absence (n = 11) or presence of fibrinogen (n = 3) or eptifibatide/GR144053 (n = 4). (d) Graph of the number of leukocytes/minute in PFB interacting with platelets bound to anti-PECAM-1 antibody in the absence (n = 5) or presence of LIBS/anti-β3 activating antibody (n = 5)±GR144053 (n = 5). (e) Graph of the number of leukocytes/minute in WB or PFB, as shown, interacting with platelets bound to FL-VWF in the absence or presence of a blocking anti-P-selectin antibody. (f) Graph of leukocyte rolling velocity on platelets bound to FL-VWF in PFB in the absence or presence of a blocking anti-P-selectin antibody. Data shown are individual leukocyte rolling velocities (n=121 and 178, respectively) for three separate experiments. In all graphs, data plotted are mean ± SD. Data were analyzed using unpaired, two-tailed Student's t test; ns not significant. Figure 3—source data 1 Leukocytes bind to VWF-bound platelets source data. https://cdn.elifesciences.org/articles/53353/elife-53353-fig3-data1-v2.xlsx Download elife-53353-fig3-data1-v2.xlsx Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Leukocytes bind/roll on platelets. Whole blood or plasma-free blood labeled with DiOC6 was perfused through channels coated with VWF A1* at for 3.5 min to capture platelets in the presence or absence of either a blocking anti-P-selectin antibody or eptifibatide prior to the of The rate was reduced to 50 s−1 to leukocyte rolling or attaching in Leukocytes on platelets in whole plasma-free blood and plasma-free blood containing anti-P-selectin blocking but not in the presence of eptifibatide The use of the anti-P-selectin blocking antibody increase the rolling velocity of leukocytes over the platelet As platelets present activated αIIbβ3, we that integrin signaling be important for platelet-leukocyte interactions to occur et al., to this, we observed a increase in the number of leukocytes interacting with the VWF-bound platelets in plasma-free (Figure addition of fibrinogen to plasma-free blood to normal plasma significantly reduced platelet-leukocyte interactions (Figure that leukocytes and fibrinogen for binding 'primed' platelets. αIIbβ3 (Figure & Figure supplement also significantly platelet-leukocyte interactions of whether platelets were captured on FL-VWF or A1*, or whether experiments were performed in whole blood or plasma-free blood (Figure To explore the role of activated αIIbβ3 in binding leukocytes, platelets were captured onto anti-PECAM-1 coated channels and an antibody binding site – that induces activation of αIIbβ3 et al., activation of αIIbβ3 caused a increase in the number of leukocytes binding in a that could be blocked with GR144053 (Figure The characterized platelet-leukocyte interaction is mediated by P-selectin on activated platelets binding to on leukocytes et al., 2004). Although we detected P-selectin on the surface of platelets, this did not a role for P-selectin in leukocyte Therefore, we first the efficacy of P-selectin through the of leukocyte binding to collagen platelets (Figure supplement However, of P-selectin on platelets from whole blood or plasma-free blood no effect upon the number of leukocytes interacting with the platelet that the recruitment of leukocytes is of P-selectin (Figure Leukocytes rolled over platelet surfaces after blocking P-selectin in plasma-free blood (Figure and Video 3) or whole blood (Figure supplement that whereas leukocyte capture is highly dependent on activated αIIbβ3 not small of P-selectin on the platelet surface may leukocyte Leukocytes bind directly to activated αIIbβ3 To more the leukocyte interaction with activated αIIbβ3 to other platelet αIIbβ3 was to microchannels activated with et al., blood cells and cells were perfused through channels at 50 s−1. from both and (Figure & and directly to the activated αIIbβ3 This binding was significantly by either fibrinogen or eptifibatide to the (Figure and b). Figure 4 Download asset Open asset Leukocytes bind to activated αIIbβ3 under flow. (a) αIIbβ3 or as were to microchannel surfaces by αIIbβ3 was activated using and Ca2+ in all (i) or labeled with DiOC6 were perfused through channels at 50 s−1 in the presence and absence of eptifibatide or 1.3 mg/ml fibrinogen leukocytes to are to and to from leukocytes in Scale bar; 50 μm. (b) of relative leukocyte binding to activated αIIbβ3 in the presence and absence of eptifibatide or 1.3 mg/ml or to after min of perfusion (n = 3), or to αIIbβ3 captured and activated by LIBS/anti-β3 activating antibody in the absence and presence of GR144053 (n = 3). Data plotted are mean ± SD. Data were analyzed using unpaired, two-tailed Student's t test. (c) Graph of the number of leukocytes/minute in PFB interacting with platelets bound to FL-VWF in the absence (n = 12) or presence of a blocking integrin antibody (n = 5) capable of blocking both LFA-1 or Mac-1 on (d) with and Representative of n = Scale bar; μm. (e) PFB with DiOC6 was perfused over FL-VWF at 1000 s−1 by 50 s−1. labeled with were to to 'primed' platelets with and Representative of n = Scale bar; μm (see also Video 4). a neutrophil with the 'primed' platelets with DiOC6 shown were and s after neutrophil attachment the of the neutrophil shown see also Video Figure data 1 Leukocytes bind to activated αIIbβ3 source data. Download We also captured αIIbβ3 to flow channel surfaces using the activating antibody. Leukocytes were captured to this surface in a that could be inhibited by blocking αIIbβ3 (Figure Activated than leukocytes can interact with platelets via Mac-1 directly through GPIbα or via fibrinogen with activated or LFA-1 via (Damle et al., 1992; Diacovo et al., et al., and Springer, However, blocking no role for either of these activated integrins in leukocyte binding to platelets (Figure In we leukocytes bind αIIbβ3 directly dependent upon its but in a that is of Mac-1 or and neutrophils interact with platelets via activated αIIbβ3 We found no of either or in interacting with activated αIIbβ3. were the only cell the capable of binding activated αIIbβ3 or platelets (Figure Using isolated we found that cells with bound to activated channels and also to platelets (Figure and on (Figure these cells were indicative of the or surfaces (Figure and Video 4) that the binding of neutrophils to αIIbβ3 under flow may signaling events within In with this, bound to platelets (Figure or activated αIIbβ3 (Figure surfaces exhibited similar intracellular Ca2+ release (Video 5) that a after s (Figure Figure 5 Download asset Open asset Binding to αIIbβ3 induces intracellular Ca2+ release in (a) Representative images of neutrophils pre-loaded with Fluo-4 AM bound to platelets captured (Video 5). are The a in which the fluorescence has increased in the For neutrophil shown, intracellular Ca2+ release is by of mean fluorescent over (b) As in neutrophils were perfused over activated αIIbβ3. increased for neutrophils and but not for neutrophil (c) Graph the change in as a function of time after neutrophil attachment to microchannels coated with activated αIIbβ3 (n = neutrophils from three different platelets (n = neutrophils from one or (n = neutrophils from different Data plotted are mean ± (d) the time between neutrophil attachment and of neutrophils binding to αIIbβ3 or platelets Data plotted are median ±95% Data were analyzed using the Mann-Whitney test. Figure data 1 Ca2+ release in neutrophils source data. Download Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg following interaction with