Endothelial cells form the inner lining of blood vessels (Alberts et al., 2002). The endothelial glycocalyx is located at the apical side of these cells and it’s composed of proteins and carbohydrates responsible for regulating extracellular functions, such as cell signaling transduction, and intercellular interactions (Jin et al., 2021; Reitsma et al., 2007; Tarbell and Ebong, 2008) (Figure 1B). These proteins and carbohydrates are proteoglycans, glycosaminoglycans (GAGs), and glycoproteins as shown in Figure 1B. GAGs are characterized by distinct disaccharide units repeats that give rise to different components such as heparan sulphate, chondroitin sulphate, hyaluronic acid, and different forms of sialic acids (Figure 1B), (Fu and Tarbell, 2013; Weinbaum et al., 2021). The functions of glycocalyx are very dependent on the way GAGs are arranged (Weinbaum et al., 2007; Choi and Lillicrap, 2020). Heparan sulfate expression patterns are dependent on endothelial cell activation and stimulation, which are shear rate dependent (Reitsma et al., 2007; Zeng et al., 2012). Primarily binding to syndecans-1, heparan sulfate makes up the majority of the GAGs, binding 3–5 heparan sulfate per syndecan with larger syndecans also binding chondroitin sulfate (Zeng et al., 2022) (Figure 1B). It is a linear sulphated polysaccharide, formed from 40–300 sugar residues, approximately 20–150 nm in length, and is anchored to the apical core proteins, syndecans and glypicans (Giantsos-Adams et al., 2013; Ebong et al., 2014). The syndecan family comprises of different members: syndecan-1,2,3 and 4. Syndecan-1 is reported to be present on the apical glycocalyx, whiles syndecan-4 is mostly found in the basal membrane of the endothelial cells. Not much is known about the structure and functions of syndecan-2 and syndecan-3, and merits further investigation (Koo et al., 2013). Members of the glypican family include six members glypican-1,2,3,4,5, and 6. Among these members of the glypican family, only glypican-1 is expressed on endothelial cell glycocalyx (Tarbell, 2010). Hyaluronic acid is a non-sulphated GAG that is not covalently bound to a core protein. Hyaluronic acid is usually much longer than protein attached GAGs (Weinbaum et al., 2003; Dogné and Flamion, 2020). Long chains of hyaluronic acid, attached to endothelial membrane bound receptors, such as CD44, are presumed to intertwine through the glycocalyx and provide part of its structure (Curry and Adamson, 2012), (Figure 1B). Sialic acid, attached to other core proteins via the terminal ends is another component of the glycocalyx (Mensah et al., 2019). The most prominent salic acid residues that are expressed on the endothelial cells includes ∝-2,6-linked, ∝-2,3-linked, and ∝-2,8-linked residues which interact with the cells through recognition binding (Betteridge et al., 2017; Mensah et al., 2019). The other endothelium components such as glycoproteins which include selectins, integrins and immunoglobulin-like molecules are known to interact with the endothelial glycocalyx (Rai et al., 2015; S Reitsma et al., 2007). P-selectin and E-selectin are usually upregulated during inflammation (Rai et al., 2015; S Reitsma et al., 2007).

The endothelial glycocalyx plays a crucial role in regulating endothelial cell permeability, inflammation, signal transduction, and anticoagulation, contributing significantly to vascular homeostasis. Serving as the vascular gatekeeper, the glycocalyx controls the passage of water, proteins, and various molecules (Butler et al., 2020). Studies, such as the one conducted by Van Haaren et al. (2003), using different sizes of dextran to investigate rat myocardial capillaries, indicate that the glycocalyx acts as a barrier, restricting the passage of specific molecules through its layer. Degradation of the glycocalyx, as observed in studies by Kang et al. (2021) and van Haaren et al. (2003), allows the transport of water and low-density lipids into rat abdominal aorta cell membrane.

The degradation of the glycocalyx is recognized as a key indicator of inflammation. When the glycocalyx undergoes degradation, it exposes vascular adhesion receptors, facilitating the binding of immune cells (Kang et al., 2021). Fatal diseases such as sepsis and COVID-19 involve a systemic breakdown of the glycocalyx, believed to be initiated by specific enzymes and cytokines targeting the glycosaminoglycans (GAGs) within the glycocalyx. These enzymes lead to instability, reducing both the overall thickness and coverage of the glycocalyx (Uchimido et al., 2019). It is noteworthy that the comprehensive impact of inflammation and its underlying mechanistic pathways on the glycocalyx remains incompletely understood, warranting further investigation.

The glycocalyx serves a crucial role beyond regulating permeability and contributing to inflammation—it acts as a mechanotransducer. According to Weinbaum et al. (2003), hydrodynamic forces acting on the apical side of the glycocalyx’s core proteins result in a bending moment that transduces fluid shear stress. The tension in GAGs induced by hydrodynamic drags, caused by blood flow through the glycocalyx layer, transmits fluid shear stress through the core proteins into the cell cytoskeleton (Weinbaum et al., 2007).

From a cellular perspective, the drag force experienced by the glycocalyx during blood flow in vessels translates mechanical forces through the core proteins into the cell cytoskeleton. Studies by Zeng and Tarbell (2014) delves into the dynamics of spatial redistribution of the actin cytoskeleton in response to shear stress. Under static conditions, dense peripheral actin bands were present at the cell periphery of rat fat pad endothelial cells. After 30 min of shear stress, noticeable polymerization and polarization of actin filaments occurred, with stress fibers oriented parallel to the nearest edge, and the emergence of lamellipodia and filopodia. Extended exposure to 24 h of shear stress further reinforced the polymerization and polarization of actin filaments, leading to the observation of prominent stress fibers (Zeng and Tarbell, 2014).

The glycocalyx is recognized for its anticoagulation properties, with components like proteoglycans and glycoproteins (Figure 1B) binding to anticoagulation mediators such as antithrombin III, heparin cofactor II, and thrombomodulin (Reitsma et al., 2007). Specifically, the interaction between antithrombin III and the heparan sulfate of the glycocalyx strengthens its anticoagulant properties. Additionally, thrombomodulin binds to the chondroitin sulfate of the glycocalyx, initiating specific anticoagulation pathways (Kozar and Pati, 2015). Diseases such as COVID-19, disseminated intravascular coagulation (DIC), sepsis, cancer and malaria are characterized by glycocalyx degradation (Chelazzi et al., 2015; Buijsers et al., 2020; Huang et al., 2021) and circulating levels of specific glycocalyx components such as syndecan, may be used as markers for endothelial dysfunction and diseases severity.

While endothelial extracellular proteins like von Willebrand factor and P-selectin are known to play a role in the inflammation and coagulation process, their direct interaction with the glycocalyx and the exact mechanism of participation remain unclear and is the focus of this review.

The von Willebrand Factor is a glycoprotein that plays an integral role in the maintenance of hemostasis (Peyvandi et al., 2011). It is composed of repeating subunits that form a long multimer (Cortes and El-Nakeep, 2023). The von Willebrand Factor is named after Erik von Willebrand, a Finnish doctor who documented a family with a seemingly hereditary bleeding disease, now known as von Willebrand disease (VWD) (Peyvandi et al., 2011) and was able to differentiate this disorder from hemophilia, despite similar symptoms (Favaloro, 2014). In the early 1960s, the combined deficiency of Factor VIII and an unknown protein was recognized as the primary cause of VWD. With advancements in ristocetin testing in the 1970s, von Willebrand Factor’s role in VWD was confirmed (Kreuz, 2008; Favaloro, 2014). The synthesis of von Willebrand Factor primarily happens in endothelial cells (Jaffe et al., 1974) and megakaryocytes (Sporn et al., 1985).

von Willebrand Factor has different conformations depending on the magnitude of shear stress that the glycoprotein is exposed to (Okhota et al., 2020). The original form of von Willebrand Factor is very similar to a folded protein, existing in a globular state. Unlike a folded protein, von Willebrand Factor does not have a sequence specific binding protein (Schneider et al., 2007). Therefore, the monomers can orient themselves in any order and in the direction of the shear stress field (Di Stasio and De Cristofaro, 2010). The changes to the orientation and state of the glycoproteins take place at a very small length scale, changing about 10 nm at a time (Singh et al., 2009). von Willebrand Factor structure is advantageous for capturing and binding to platelets in the bulk flow, which is essential in the regulation of hemostasis (Di Stasio and De Cristofaro, 2010).

Changes in von Willebrand Factor are dependent on the shear rate present in the cellular microenvironment. At low shear rates (10–1,000/s) von Willebrand Factor remains in a compact conformation (Schneider et al., 2007). The von Willebrand Factor experiences minimal alterations at shear rates below 3,000/s, with the overall size remaining unchanged, exhibiting only modifications at the domain level (Singh et al., 2009). The unfolding of von Willebrand Factor begins in a condensed arrangement when shear stress levels reach 30 dyne/cm^2. As the glycoprotein unfolds, it stretches and adopts a chain-like conformation, aligning itself in the overall direction of the shear stress field (Di Stasio and De Cristofaro, 2010).

The role of von Willebrand Factor in hemostasis is spurred by its binding patterns to platelets and connective tissue (Peyvandi et al., 2011). The A1 domain holds the primary function of being the binding site for the platelet receptor protein, GP1b. Platelet aggregation occurs at injury sites, resulting in the introduction of other plasma proteins such as Factor VIII in addition to von Willebrand Factor leading to thrombi formation (Ruggeri et al., 2006; Zhang et al., 2009). von Willebrand Factor can exist in ultralong forms which are cleaved in a shear stress dependent manner by ADisintegrin and Metalloprotease with ThromboSpondin motif (ADAMTS13) to prevent unwanted coagulations (Zhang et al., 2009; Bartoli et al., 2015; Zheng, 2015). The anchorage of ultralong von Willebrand Factor to the endothelium is thought to be mediated by P-selectin expressed on the endothelial bed (Padilla et al., 2004), a mechanism that requires further investigation.

Under normal physiological conditions, the von Willebrand Factor will act according to the processes described above to successfully encourage platelet aggregation (Peyvandi et al., 2011), and the eventual cessation of bleeding. However, its function during a disease state is less concrete (Ladikou et al., 2020). Thrombotic incidents are prevalent amongst critically ill COVID-19 patients (Mei et al., 2021), an infection caused by SARS-Cov-2 virus (Li et al., 2020). This acute coagulation disorder is termed COVID-19-associated coagulopathy and the von Willebrand Factor potentially contributes to its development (Mei et al., 2021). Elevated levels of both von Willebrand Factor activity and antigens have been associated with clinically adverse outcomes for patients, leading to questions about its contribution to this pathophysiology. In addition to elevated von Willebrand Factor levels, decreased levels of the ADAMTS13 enzyme have been reported, meaning an increase amount of uncleaved von Willebrand Factor would be in circulating during this condition (Mei et al., 2021).

Von Willebrand Factor’s effects during COVID-19-associated coagulopathy does not appear unique to the virus, but rather to the presence of inflammation, as other conditions such as sepsis, DIC, cancer, and malaria also show marked effects on the von Willebrand Factor. Severe DIC outcomes are also associated with an increase in von Willebrand Factor levels and a decrease in ADAMTS13 (Habe et al., 2012). This same pattern is observed during the massive immune response responsible for the development of sepsis and septic shock (Feroz Azfar et al., 2017). The widespread inflammation and endothelial dysfunction in these conditions is indicative of the relationship between endothelial damage and thrombotic dysregulation (Patel et al., 2019).

P-selectin is expressed and produced on the endothelial bed and megakaryocytes and encoded by the SELP gene in humans. One of the numerous proteins attached to the glycocalyx is P-selectin (S Reitsma et al., 2007). This selectin is composed of an N terminal lectin domain, an epidermal growth factor, about nine repeating regulatory proteins, as well as transmembrane sections and a small intracytoplasmic end (Blann et al., 2003). P-selectin is stored in the Weibel–Palade bodies and can be localized in the ⍺-granules during exocytosis, bringing it to the cell surface when stimulated by thrombin. P-Selectin is expressed during endothelial activation and lasts for a short time and quickly internalized and destroyed within the cell (Merten and Thiagarajan, 2004; Reitsma et al., 2007; Tvaroška et al., 2020). P-selectin binds to heparan sulfate (Wang and Geng, 2003), using the P-selectin glycoprotein ligand 1 (PSGL-1) and the GP-IB-IX-V complex. The binding to the GP-IB-IX-V complex allows P-selectin to mediate platelet adhesion, furthermore, stabilizing the GPIIb/IIIa fibrin interaction mediates inter-platelet aggregation (Blann et al., 2003). P-selectin is important in the initial adhesion of platelets and leukocytes during injury and inflammation, hence plays a major role in homeostasis and thrombosis (Agrati et al., 2021). Conditions like COVID-19 which is known to cause the increased production of Tumor Necrosis Factor Alpha (TNF-alpha) results in the increased transcription of P-selectin (Tvaroška et al., 2020).

DIC and some cancers are pro-thrombotic events, manifesting as microthrombi events, the over expression of P-selectin promotes coagulation through the circulation of particles with its counter receptor, PSGL-1, that bring tissue factors to the platelet thrombus (Mosad et al., 2011; Agrati et al., 2021). The release of soluble P-selectin in the plasma of patients with DIC is reported to be an indicator for the severity of the disease (Wang et al., 2012). Other inflammatory associated disease such as sepsis is also associated with soluble P-selectin and the level of blood concentrations of P-selectin has been correlated with the severity of sepsis (Zonneveld et al., 2014). Expression of P-selectin can also be seen when the endothelium is damaged, activating the interactions between the leukocytes, platelets and endothelium. P-selectin’s role in inflammation and thrombotic events and location on the endothelium makes it an ideal biomarker for studying pro-thrombotic diseases and conditions (Perkins et al., 2019). While P-selectin is a great biomarker, the binding site is very short, it cannot be detected in healthy conditions and can only be discovered during glycocalyx degradation (Moore et al., 2021).