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 α_IIbβ₃. Further experiments showed that integrin α_IIbβ₃ 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.

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, α_IIbβ₃ (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 α_Mβ₂), which can associate with activated α_IIbβ₃ 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 α_Lβ₂) 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, α_IIbβ₃. Activated α_IIbβ₃ 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 α_IIbβ₃. 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).

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).

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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).

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Platelet binding to VWF under flow induces intraplatelet signaling and activation of α_IIbβ₃

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, α_IIbβ₃, 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 α_IIbβ₃ 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 α_IIbβ₃ was blocked (Figure 2b–e). These results demonstrate that the A1-GPIbα interaction leads to activation of α_IIbβ₃, 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).

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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 α_IIbβ₃, but minimal α-granule release, and represents part of the tunable response of platelets.

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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’.

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As VWF-‘primed’ platelets present activated α_IIbβ₃, we hypothesized that ‘outside-in’ integrin signaling might be important for platelet-leukocyte interactions to occur (Durrant et al., 2017). Contrary to this, we observed a significant (~2 fold) increase in the number of leukocytes interacting with the VWF-bound platelets in plasma-free conditions (Figure 3b). Moreover, addition of purified fibrinogen to plasma-free blood to 50% normal plasma concentration significantly reduced platelet-leukocyte interactions (Figure 3b) suggesting that leukocytes and fibrinogen compete for binding ‘primed’ platelets. Blocking α_IIbβ₃ (Figure 3a–b & Figure 3—figure supplement 1a) also significantly decreased platelet-leukocyte interactions irrespective of whether platelets were captured on FL-VWF or A1*, or whether experiments were performed in whole blood or plasma-free blood (Figure 3a–c).

To explore the role of activated α_IIbβ₃ in binding leukocytes, platelets were captured onto anti-PECAM-1 coated channels and an anti-β₃ antibody (ligand-induced binding site – LIBS) that induces activation of α_IIbβ₃ applied (Du et al., 1993). Antibody-mediated activation of α_IIbβ₃ caused a significant increase in the number of leukocytes binding in a manner that could be blocked with GR144053 (Figure 3d).

The best characterized platelet-leukocyte interaction is mediated by P-selectin on activated platelets binding to PSGL-1 on leukocytes (Vandendries et al., 2004). Although we detected little/no P-selectin on the surface of VWF-‘primed’ platelets, this did not formally exclude a role for P-selectin in leukocyte adhesion. Therefore, we first established the efficacy of P-selectin blockade through the marked reduction of leukocyte binding to collagen captured/activated platelets (Figure 3—figure supplement 1b–c). However, blockade of P-selectin on FL-VWF-bound platelets from whole blood or plasma-free blood had no effect upon the number of leukocytes interacting with the platelet surface, suggesting that the recruitment of leukocytes is independent of P-selectin (Figure 3e). Leukocytes rolled faster over platelet surfaces after blocking P-selectin in plasma-free blood (Figure 3f and Video 3) or whole blood (Figure 3—figure supplement 1d), suggesting that whereas leukocyte capture is highly dependent on activated α_IIbβ₃ (and not P-selectin), once recruited, small amounts of P-selectin on the platelet surface may slow leukocyte rolling.

Leukocytes bind directly to activated α_IIbβ₃

To more specifically test the leukocyte interaction with activated α_IIbβ₃ (and to exclude other platelet receptors), purified α_IIbβ₃ was covalently coupled to microchannels and, thereafter, activated with Mn²⁺ (Litvinov et al., 2005). Isolated peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) were perfused through α_IIbβ₃-coated channels at 50 s⁻¹. Cells from both PBMCs and PMNs (Figure 4ai–ii & and b) directly attached to the activated α_IIbβ₃ surface. This binding was significantly diminished (>70%) by adding either purified fibrinogen or eptifibatide to the PMNs (Figure 4aiv-v and b).

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We also captured purified α_IIbβ₃ to flow channel surfaces using the activating anti-β₃ (LIBS) antibody. Leukocytes were again efficiently captured to this surface in a manner that could be inhibited (~70%) by blocking α_IIbβ₃ (Figure 4b).

Activated (rather than resting) leukocytes can interact with platelets via Mac-1 (α_Mβ₂) (either directly through GPIbα or via fibrinogen bridge with activated α_IIbβ₃) or LFA-1 (α_Lβ₂) via ICAM-2 (Damle et al., 1992; Diacovo et al., 1994; Simon et al., 2000; Weber and Springer, 1997). However, blocking β₂ suggested no role for either of these activated integrins in leukocyte binding to VWF-‘primed’ platelets (Figure 4c). In summary, we show leukocytes bind α_IIbβ₃ directly dependent upon its RGD-binding groove, but in a manner that is independent of Mac-1 or LFA-1.

T-cells and neutrophils interact with VWF-‘primed’ platelets via activated α_IIbβ₃

We found no evidence of either CD14⁺ monocytes or CD19⁺ B-cells in PBMCs interacting with activated α_IIbβ₃. T-cells were the only cell type amongst the PBMCs capable of binding activated α_IIbβ₃ or VWF-‘primed’ platelets (Figure 4d–e).

Using isolated PMNs, we found that cells stained with anti-CD16 bound to activated α_IIbβ₃-coated channels and also to VWF-‘primed’ platelets (Figure 4f and g). Based on multi-lobulated segmented nuclear morphology (Figure 4f), these cells were indicative of CD16⁺ neutrophils. Neutrophils scanned the platelet- or α_IIbβ₃-coated surfaces (Figure 4g and Video 4) suggesting that the binding of neutrophils to α_IIbβ₃ under flow may itself initiate signaling events within neutrophils. In line with this, PMNs bound to VWF-‘primed’ platelets (Figure 5a) or activated α_IIbβ₃ (Figure 5b) surfaces exhibited similar intracellular Ca²⁺ release (Video 5) that reached a maximum after 200–300 s (Figure 5c–d).

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Binding of neutrophils to α_IIbβ₃ under flow induces Nox- and Ca²⁺-dependent NETosis

Platelets assist in the targeting of intravascular bacterial pathogens through stimulation of the release of NETs (Brinkmann et al., 2004; Gaertner et al., 2017; Wong et al., 2013; Yeaman, 2014). However, the physiological agonists or mechanisms that drive NETosis are not fully resolved (Nauseef and Kubes, 2016). We therefore examined whether the binding of neutrophils to α_IIbβ₃ might induce NETosis. Isolated PMNs were perfused over either activated α_IIbβ₃ or anti-CD16 (negative control) at 50 s⁻¹ for 10 min and NETosis was subsequently analyzed under static conditions (Figure 6a and Video 6). Nuclear decondensation was evident after ~60 min, and Sytox Green fluorescence, indicative of cell permeability that precedes NETosis, was detected from ~85 min. Nuclear decondensation, increased cell permeability and positive staining with a cell impermeable DNA fluorophore do not specifically identify NETosis. Therefore, an anti-citrullinated histone H3 antibody was perfused through the channels after 90 min to more specifically identify NETs. The introduction of flow at this point caused the DNA to form extended mesh-like NETs that were stained positively by Hoechst and the anti-citrullinated histone H3 antibody (Figure 6b). Very similar results were obtained with PMN bound to either α_IIbβ₃ (captured by the activating anti-β₃ antibody), or to platelets ‘primed’ by A1* or FL-VWF, which all exhibited a similar proportion of neutrophils undergoing NETosis within the 2 hr timeframe, suggesting that interaction with α_IIbβ₃ alone can promote NETosis and that any platelet releasate present does not appreciably augment this process under these conditions. On activated α_IIbβ₃, 69% ± 14% of neutrophils through the entire channel formed NETs after 2 hr, compared to minimal NETosis events (8 ± 8%) when neutrophils were captured by anti-CD16 (Figure 6c). When neutrophils were captured on α_IIbβ₃ in the absence of flow, neutrophils attached, but NETosis was significantly reduced by fourfold, with only 17% of neutrophils exhibiting signs of NETosis (Figure 6c). This suggested that the signaling mechanism from the platelet to the neutrophil is mechano-sensitive and does not require other platelet receptors or releasate components.

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Neutrophils captured by an anti-CD16 antibody and stimulated with phorbol 12-myristate 13-acetate (PMA) for 2 hr led to 100 ± 0.5% of neutrophils releasing NETs (Figure 6d). PMA-induced NETosis was not significantly inhibited in the presence of TMB-8 (an antagonist of intracellular Ca²⁺ release; 90 ± 9%), but was effectively inhibited in by DPI (NADPH oxidase inhibitor; 16 ± 13%), similar to previous reports (Gupta et al., 2014). NETosis of neutrophils captured by α_IIbβ₃ under flow was significantly inhibited by TMB-8 (17 ± 9%) and DPI (25 ± 10%) (Figure 6e). This highlights the dependency of both intracellular Ca²⁺ release and NADPH oxidase signaling pathways in NETosis in response to binding α_IIbβ₃ under flow.

‘Primed’ platelets interact with SLC44A2 receptor on neutrophils

Our data point to the presence of a specific receptor on the surface of neutrophils (and T-cells) that is not present on B cells or monocytes and that is capable of binding to activated α_IIbβ₃ and transducing a signal into the cell. To identify this leukocyte counter-receptor, we analyzed RNA sequencing data from different leukocyte populations, selecting genes that are expressed at higher levels in neutrophils (or in CD4⁺ T-cells) than in monocytes (Adams et al., 2012; Grassi et al., 2019; Figure 7). We further limited the candidate search by selecting those genes that code for transmembrane proteins. Using this approach, we identified 93 candidate genes. Of these, 33 genes were excluded as they are primarily associated with intracellular membranes. An additional 16 genes were also excluded due to the presence of short extracellular regions/domains (<30 a.a.) that would unlikely be capable of facilitating interactions with an extracellular binding partner (Figure 7). We then analyzed proteomic data to verify the preferential expression of the remaining candidates in neutrophils as opposed to monocytes (Rieckmann et al., 2017). These data suggested that the protein product of 14 of the remaining genes appeared to be detected in higher abundance in monocytes, which we used as a further exclusion criterion (Figure 7—figure supplement 1a). From the remaining 30 candidate genes, the SLC44A2 gene was selected for validation due to its recent identification as a risk locus for both DVT and stroke (Germain et al., 2015; Hinds et al., 2016), both of which are pathologies associated with described contributions of platelet-leukocyte interactions. SLC44A2 is a cell surface receptor with 10 membrane-spanning domains and five extracellular loops of 178a.a., 38a.a., 72a.a., 38a.a. and 18a.a. in length, respectively (Nair et al., 2016). We sourced antibodies against SLC44A2 that specifically recognize amino acid sequences within the first and second extracellular loops. Published proteomic profiling confirmed the preferential expression of SLC44A2 in neutrophils (Figure 7—figure supplement 1a; Rieckmann et al., 2017). Western blotting of isolated granulocyte lysates revealed two bands representing SLC44A2 (glycosylated and nascent/non-glycosylated SLC44A2) (Figure 7—figure supplement 1b). Perfusing human neutrophils over immobilized activated α_IIbβ₃ in the presence of the first anti-SLC44A2 antibody (anti-SLC44A2 #1) that recognizes the second extracellular loop revealed a dose-dependent blockade of neutrophil binding when compared to no antibody or control rabbit IgG (Figure 8a–b). A second anti-SLC44A2 antibody (anti-SLC44A2 #2) that recognizes the first extracellular loop region of SLC44A2 confirmed these findings (Figure 8a). The anti-SLC44A2 #2 almost completely blocked neutrophil binding to activated α_IIbβ₃ suggesting that this antibody more effectively blocks the neutrophil binding to the integrin than anti-SLC44A2 #1. This may suggest that the first and longest extracellular loop is involved in interaction with an extracellular ligand.

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Based on these results, we transfected HEK293T cells with an expression vector for human SLC44A2 fused to turbo green fluorescent protein (tGFP) at the intracellular C-terminus. Transfected cells were perfused through activated α_IIbβ₃ coated channels and cell binding was quantified. Transfected cells bound to these surfaces in a manner that could be blocked by GR144053 (that blocks α_IIbβ₃) or by the anti-SLC44A2 #1 antibody (Figure 8c).

The SNP in SLC44A2 identified by GWAS studies that is protective against VTE and stroke (rs2288904-A) causes a missense mutation (R154Q) in the first 178a.a. extracellular loop of SLC44A2 (Germain et al., 2015). Based on this, we hypothesized that this substitution might exert a functional influence upon the ability of SLC44A2 to interact with α_IIbβ₃. Consistent with this hypothesis, HEK293T cells transfected with the SLC44A2 (R154Q)-tGFP expression vector exhibited reduced ability to interact with immobilized α_IIbβ₃ (Figure 8c). Western blot analysis of transfected HEK293T cells revealed that expression of the SLC44A2(R154)-tGFP and SLC44A2(Q154)-tGFP was similar and that the tGFP remained uniformly associated with the fusion protein (Figure 8c inset).

To further explore the potential interaction between SLC44A2 and activated α_IIbβ₃ on platelets, we first captured and ‘primed’ platelets over VWF-coated surfaces and, thereafter, perfused SLC44A2-tGFP-transfected HEK293T cells. Again, these cells bound to VWF-‘primed’ platelets in a manner that could be blocked completely with GR144053 (to block α_IIbβ₃) or the anti-SLC44A2 #1 antibody (Figure 8d–e). Consistent with the previous results, HEK293T cells transfected with SLC44A2(R154Q) exhibited markedly reduced binding to VWF-‘primed’ platelets (Figure 8d–e). A previous report suggested that SLC44A2 might bind directly to VWF (Bayat et al., 2015). However, when SLC44A2-tGFP-transfected HEK293T cells were perfused of VWF surfaces, in the absence of platelets, no binding was detected (Figure 8d). Similarly, isolated neutrophils also failed to interact directly with VWF-coated surfaces, demonstrating the absolute dependence of platelets in facilitating cell capture under flow.

Neutrophils homozygous for the rs2288904-A SNP exhibit reduced binding to activated α_IIbβ₃

The rs2288904-A SNP in SLC44A2 has a minor allele frequency of 0.22 and is protective against VTE (Germain et al., 2015). It is therefore the common/wild-type allele, rs2288904-G, that is the risk allele for VTE with an odds ratio of 1.2–1.3. The frequency of individuals homozygous for the protective rs2288904-A allele amongst VTE cases is 30–50% lower than in healthy controls, which perhaps provides a better indication to its protective phenotype. Given its prevalence, we genotyped a group of healthy volunteers to identify individuals homozygous for the major allele (rs2288904-G/G), SLC44A2 (R154/R154), and for the protective allele (rs2288904-A/A), SLC44A2 (Q154/Q154) (Figure 7—figure supplement 1c). SLC44A2 (R154/R154) neutrophils interacted with VWF-‘primed’ platelets as before (Figure 8f and Video 7). Consistent with the previous blocking experiments, this binding was partially blocked with anti-SLC44A2 #1, and almost completely blocked by anti-SLC44A2 #2 (Figure 8f and Video 7). Furthermore, and consistent with the transfection studies, neutrophils homozygous for the protective allele, SLC44A2 (Q154/Q154), exhibited markedly reduced (~75%) binding to VWF-‘primed’ platelets (Figure 8f and Video 7) demonstrating a functional consequence of the rs2288904-A polymorphism on this neutrophil-platelet interaction. Consistent with this, neutrophils heterozygous form the polymorphism SLC44A2 (R154/Q154) exhibited a trend toward intermediate binding to platelets (Figure 8f), although this did not reach statistical significance when compared to the SLC44A2 (R154/R154) (p=0.07) or SLC44A2 (Q154/Q154) (p=0.69) genotypes.

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Although the ability of platelet GPIbα binding to VWF to mediate intraplatelet signaling events has been known for many years, the role that this signaling fulfils remains poorly understood (Goto et al., 1995). We demonstrate that under flow GPIbα-A1 binding ‘primes’, rather than activates, platelets, based on the rapid activation of α_IIbβ₃, but the lack of appreciable surface P-selectin exposure (Figure 2 and Figure 2—figure supplement 1b). Some studies have reported that GPIbα-VWF-mediated signaling can induce modest α-granule release. However, the use of static conditions and processing of platelets may explain those observations. Despite this, when compared to other platelet agonists, degranulation and P-selectin exposure induced by VWF binding are both very low (de Witt et al., 2014; Deng et al., 2016).

That platelet binding to VWF under flow ‘primes’, rather than activates, platelets is consistent with in vivo observations. At sites of vessel damage, VWF is important for platelet accumulation through all layers of the hemostatic plug (Joglekar et al., 2013; Lei et al., 2014; Verhenne et al., 2015). All platelets within a thrombus/hemostatic plug likely form interactions with VWF. Despite this, it is only the platelets in the ‘core’ of the thrombus that become P-selectin-positive, procoagulant platelets, whereas the more loosely bound platelets that form the surrounding ‘shell’ remain essentially P-selectin-negative (Welsh et al., 2014). If VWF-binding alone were sufficient to fully activate platelets, the differential platelet characteristics of the ‘core’ and ‘shell’ would not be observed.

Although it is frequently implied that VWF is only important for platelet capture under high shear conditions, murine models of venous thrombosis with no collagen exposure have repeatedly revealed an important role for VWF-mediated platelet accumulation (Bergmeier et al., 2008; Brill et al., 2011; Chauhan et al., 2007). Platelet binding to VWF occurs most efficiently at arterial shear rates, but still occurs under lower linear venous shear (Miyata and Ruggeri, 1999; Yago et al., 2008; Zheng et al., 2015). However, linear channels do not mimic the distorted and branched paths of the vascular system that cause more disturbed flow patterns, particularly around valves. Using channels with changing geometry under lower shear conditions, we and others have noted that VWF captures platelets appreciably more efficiently in areas of disturbed flow (Zheng et al., 2015). Indeed, at venous flow rates through bifurcated channels (Figure 9a), we detected platelet capture on FL-VWF with concomitant ‘priming’ and leukocyte binding (Figure 9b). This was appreciably augmented at bifurcation points where disturbed flow exists. Consistent with our earlier findings, leukocyte binding was almost completely inhibited when α_IIbβ₃ was blocked (Figure 9c). This implies that VWF can function in platelet recruitment within the venous system, particularly in areas of turbulence (e.g. branch sites, valves), which are frequently the nidus for thrombus formation in DVT.

Figure 9

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In venous thrombosis, the thrombus generally forms over the intact endothelium, in the absence of vessel damage. This poses the question of how VWF might contribute to DVT if subendothelial collagen is not exposed. It is likely that this reflects the function of newly secreted ultra-large VWF released from endothelial cells. Under low disturbed flow, released ultra-large VWF may tangle to form strings/cables over the surface of the endothelium. Tangled VWF strings/cables are appreciably more resistant to ADAMTS13 proteolysis than VWF that is simply unraveled. In the murine stenosis model of DVT, complete VWF-deficiency prevents platelet binding over the endothelium (Bergmeier et al., 2008; Brill et al., 2011; Chauhan et al., 2007). Similarly, blocking GPIbα binding to VWF also completely blocks platelet accumulation and thrombus formation in the stenosis model of DVT. Thus, when platelets bind to VWF under flow in such settings, platelets may become ‘primed’ facilitating both aggregation and neutrophil binding through activated α_IIbβ₃, but without activating them into procoagulant platelets.

Our study reveals, that T-cells and neutrophils can bind directly to activated α_IIbβ₃ on platelets or that has been coupled to microchannel surfaces (Figure 3a–d & Figure 4a–b). In both cases, the interaction can be inhibited by eptifibatide and GR144053 suggesting that both cell types may share the same receptor, in a manner that is dependent upon the RGD binding groove of activated α_IIbβ₃.

Previous studies have identified roles for β₂ integrins, Mac-1 (α_Mβ₂) and LFA-1 (α_Lβ₂), on leukocytes in mediating interactions with platelets. It should be recognized that the interactions of these molecules are dependent upon the integrins first being activated (and therefore also the cell), which is not the case in our system and is in contrast to previous studies implicating Mac-1 and LFA-1. However, we provide evidence that Mac-1 (α_Mβ₂) and LFA-1 (α_Lβ₂) are not involved by: 1) Leukocytes do not bind to ‘unprimed’ platelets captured by anti-PECAM-1. As Mac-1 and LFA-1 bind to GPIbα and ICAM-2, respectively, both of which are constitutively presented on the platelet surface (Kuijper et al., 1998; Simon et al., 2000), if Mac-1 and LFA-1 were the receptors involved binding would have been observed in these experiments (Figure 3a–b). 2) Mac-1 on leukocytes can bind indirectly to activated α_IIbβ₃ via a fibrinogen bridge (Weber and Springer, 1997). However, we demonstrate that fibrinogen competes for leukocyte binding to bind to activated α_IIbβ₃ (Figure 3b–c & Figure 4b). If fibrinogen were required, removal of fibrinogen from our perfusion system would have diminished the number of leukocyte interactions with primed platelets/α_IIbβ₃ if Mac-1 were involved. 3) Antibody-mediated blocking of β₂ integrins did not reduce VWF-‘primed’ platelet-leukocyte interactions (Figure 4c). 4) Only neutrophils and T cells interact with the ‘primed’ platelets, whereas Mac-1 and LFA-1 are also highly expressed in monocytes which do not bind (Figure 4d–f).

We also excluded a role for P-selectin-PSGL-1 for the platelet-leukocyte interaction that we observe as; 1) we detected little/no P-selectin on VWF-‘primed’ platelets (Figure 2—figure supplement 1), suggestive of minimal degranulation occurring; this also provides indirect evidence for the lack of CD40L on the platelet surface. 2) P-selectin blockade had no effect upon the number of leukocytes binding (Figure 3e), and 3) only T-cells and neutrophils bind VWF-‘primed’ platelets (Figure 4) - given that all leukocytes express PSGL-1,(Laszik et al., 1996) and CD40, if the capture of leukocytes were entirely P-selectin or CD40L-mediated, such cell-type selectivity would not be observed. We did, however, measure an influence of P-selectin upon the rolling speed of leukocytes over VWF-primed platelets (Figure 3f and Figure 3—figure supplement 1d). This suggests that although low levels of P-selectin present on the platelet surface is insufficient to facilitate leukocyte capture, it may synergize to slow rolling of leukocytes that are first captured by α_IIbβ₃.

There are several studies that provide support for P-selectin-independent interactions of neutrophils and T-cells with platelets. Guidotti et al demonstrated the interaction of T-cells with small intrasinusoidal platelet aggregates in the liver during hepatotropic viral infections was independent of both P-selectin and CD40L in platelets (Guidotti et al., 2015). Using a murine model of peritonitis, Petri et al demonstrated that neutrophil recruitment and extravasation was highly dependent upon VWF, GPIbα, and platelets, but largely independent of P-selectin (Petri et al., 2010). Two further studies also corroborate the contention that VWF/GPIbα-bound platelets are capable of promoting neutrophil recruitment/extravasation in murine models of ischemia/reperfusion via P-selectin-independent mechanisms (Gandhi et al., 2012; Khan et al., 2012). These studies support the idea that both VWF and platelets can function beyond hemostasis to fulfil a role in leukocyte recruitment at sites of inflammation.

As T-cells and neutrophils (and not B-cells or monocytes) can bind platelets via activated α_IIbβ₃, this suggests that a specific receptor exists on these cells that is absent on B-cells or monocytes. Using transcriptomic and proteomic data, we identified 30 transmembrane candidates that were preferentially expressed in neutrophils (or T-cells) over monocytes. From this list, SLC44A2 stood out due to its recent identification as a risk locus for both VTE and stroke, but with as yet unknown functional association with these pathologies (Apipongrat et al., 2019; Germain et al., 2015; Hinds et al., 2016). As platelet-leukocyte interactions are involved in both of these thrombotic disorders, we hypothesized that SLC44A2 functions as the neutrophil counter receptor for activated α_IIbβ₃. The cellular function of SLC44A2 is not well-defined. It contains 10 transmembrane domains with five extracellular loops. The intracellular N-terminal tail contains several putative phosphorylation sites of unknown functional significance. As well as neutrophils, SLC44A2 expression has also been reported in endothelial cells and platelets. However, proteomic data suggest that levels in neutrophils are >300 fold greater in neutrophils that platelets (Rieckmann et al., 2017).

We provide several lines of evidence to support the direct interaction between SLC44A2 and activated α_IIbβ₃. 1) Two different anti-SLC44A2 antibodies that recognize extracellular loops of the receptor blocked the binding of neutrophils to both VWF-‘primed’ platelets and to activated α_IIbβ₃. 2) Recombinant expression of SLC44A2 in HEK293T cells imparted the ability of these cells to bind both VWF-primed platelets and activated α_IIbβ₃ under flow in a manner that can be blocked by either GR144053 or by anti-SLC44A2 antibodies. 3) Introduction of the rs2288904-A SNP in SLC44A2 that is protective against VTE resulted in markedly reduced binding of transfected HEK293T cells to both VWF-‘primed’ platelets and activated α_IIbβ₃. 4) Neutrophils homozygous for the rs2288904-A/A SNP exhibit significantly reduced binding to VWF-‘primed’ platelets.

Although NET production is an established mechanism through which neutrophils control pathogens (Brinkmann et al., 2004), many questions remain as to how NETosis is regulated (Nauseef and Kubes, 2016). Binding of platelets to Kupffer cells in the liver of mice following infection with B. cereus or S. aureus is mediated by VWF (Wong et al., 2013). This binding augments the recruitment of neutrophils, NET production and the control of infection (Kolaczkowska et al., 2015). Mice lacking VWF or GPIbα do not form these aggregates and so have diminished neutrophil recruitment and, therefore, decreased survival (Wong et al., 2013). How NETosis is initiated following platelet binding remains uncertain. Alone, lipopolysaccharide (LPS) is not a potent activator of NETosis (Clark et al., 2007). However, LPS-stimulated platelets, which bind of fibrinogen (i.e. α_IIbβ₃ is activated) and also robustly activate NETosis independent of P-selectin (Clark et al., 2007; Looney et al., 2009; Lopes Pires et al., 2017; McDonald et al., 2012).

We detected rapid release of intracellular Ca²⁺ (within minutes) in bound neutrophils (Figure 5 and Video 5) that preceded the release of NETs after 80–90 min (Figure 6). This process was dependent upon neutrophils being captured under flow, suggesting that signal transduction through binding of α_IIbβ₃ to SLC44A2 may be mechanosensitive, which is consistent with the recent report suggesting a major influence of shear upon NETosis in the presence of platelets (Yu et al., 2018). As NETosis can be induced following binding to purified α_IIbβ₃ under flow alone, this suggests that this process does not require a component of the platelet releasate (e.g. high mobility group box 1, platelet factor 4, RANTES and thromboxane A2) which have been reported to be capable of driving NETosis (Carestia et al., 2016). Naturally, if platelet degranulation occurs (in response to other agonists), these components may have the potential to further augment this process.

We propose a model in which platelets have a tunable response that can distinguish their roles in hemostasis and immune cell activation (Figure 10). The ‘priming’ of platelets by binding to VWF under flow (in the absence of other platelet agonists) may assist in the targeting of leukocytes to resolve pathogens or mediate vascular inflammatory response. The activated α_IIbβ₃ integrin can then mediate neutrophil recruitment through binding to SLC44A2 (Figure 10). We do not exclude a supporting role for P-selectin in maintaining T-cell/neutrophil recruitment, but this is not essential for initiating recruitment. Under flow SLC44A2 transduces a mechanical stimulus capable of promoting NETosis via a pathway involving synergy between NADPH oxidase and Ca²⁺ signaling (Figure 10). Homeostatically, this may be beneficial for immune responses. However, during chronic infection or vascular inflammation NET production may promote intravascular thrombosis. It remains to be determined whether the rs2288904-A SNP in SLC44A2 that is protective against VTE might also (negatively) influence the host response to infection, or certain routes of infection that are more reliant upon platelet immune cell function. This should determine the net selective pressure upon the SNP. However, as VTE is a comparatively recent selective pathology (in evolutionary terms) and rates of VTE are typically low during the most common child-bearing years, this positive selective pressure on rs2288904-A SNP may not strong. Indeed, the prevalence of this SNP in different global populations suggests that the SNP has not originated recently. Moreover, if the host response to infection is, at least in certain settings, also impaired in individuals carrying the rs2288904-A SNP, this would be predicted to slow the rate of positive selection. More work is needed to understand role of rs2288904-A SNP in VTE and infection risk and penetrance of SNP within large genomic datasets.

Figure 10

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This study identifies activated α_IIbβ₃ as a receptor and agonist for neutrophils through SLC44A2. This provides a previously uncharacterized mechanism of how platelet-neutrophil cross-talk is manifest in innate immunity; it also provides an explanation for how VWF and platelet-dependent neutrophil recruitment and NETosis may occur in thrombotic disorders such as DVT (Laridan et al., 2019), but also thrombotic microangiopathies like thrombotic thrombocytopenic purpura (Fuchs et al., 2012b). Identification of SLC44A2 as the counter-receptor for activated α_IIbβ₃ in conjunction with the prior identification of the protective rs2288904-A SNP in SLC44A2 that impairs the binding of neutrophils to platelets highlights SLC44A2 as a potential therapeutic target. Recent data reveal that SLC44A2-deficient mice exhibit normal hemostatic responses (Tilburg et al., 2018) but are protected against development of venous thrombosis (Tilburg et al., 2020), provide further encouragement for this strategy.

All data generated or analysed during this study are included in the manuscript and supporting files. The source data underlying Figs 1, 2, 3, 4, 5c, 6, 8, and Figure 1, 3 and 7 Supplements are provided in separate 'Source Data' files.

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Author details

1. Adela Constantinescu-Bercu

   1. Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom
   2. Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1274-2867

2. Luigi Grassi

   1. Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, United Kingdom
   2. National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
   3. National Institute for Health Research BioResource, Rare Diseases, Cambridge University Hospitals, Cambridge, United Kingdom

   Contribution

   Resources, Data curation, Formal analysis

   Competing interests

   No competing interests declared

3. Mattia Frontini

   1. Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, United Kingdom
   2. National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
   3. British Heart Foundation Centre of Excellence, Cambridge Biomedical Campus, Cambridge, United Kingdom

   Contribution

   Resources, Data curation, Formal analysis

   Competing interests

   No competing interests declared

4. Isabelle I Salles-Crawley

   Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Project administration

   Contributed equally with

   Kevin Woollard and James TB Crawley

   For correspondence

   i.salles@imperial.ac.uk

   Competing interests

   No competing interests declared

5. Kevin Woollard

   Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Project administration

   Contributed equally with

   Isabelle I Salles-Crawley and James TB Crawley

   For correspondence

   k.woollard@imperial.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9839-5463

6. James TB Crawley

   Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Project administration

   Contributed equally with

   Isabelle I Salles-Crawley and Kevin Woollard

   For correspondence

   j.crawley@imperial.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6723-7841

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