The (im)Balance of NETosis, Platelets, VWF, and ADAMTS13 During Immunothrombosis

Posted on: December 10, 2024

-Contributed by Abi Kasberg, PhD

Immunothrombosis defines the delicate intersection of innate immunity and coagulation. This complex bidirectional interaction between the immune system and clotting components activate each other, leading to abnormal clotting events when unbalanced. A central component to immunothrombosis is the NETosis response of activated neutrophils that result in the expulsion of neutrophil extracellular traps (NETs). NETs are found in thrombi and colocalize with platelets, von Willebrand factor (VWF), and components of the intrinsic and extrinsic coagulation pathways (Thakur et al. 2023). In particular, the complex interactions of NETs, VWF, platelets, and ADAMTS13 highlight the responsibility of these immune, vascular, and coagulation systems to maintain balance under normal conditions and drive pathological immunothrombosis when disrupted.

NETosis: A process where neutrophils release DNA-based structures called neutrophil extracellular traps (NETs) to capture and kill pathogens. While NETs play a key role in immunity, excessive formation of NETs can promote harmful inflammation and thrombosis.

Platelets: Key mediators of primary hemostasis, clot formation, and immune responses. Platelets adhere to VWF at sites of vascular injury to form aggregates and initiate clotting. Aberent platelet activation or enhanced interaction with VWF can contribute to pathological clotting.

VWF (von Willebrand Factor): A multimeric protein essential for clot formation. VWF acts like glue, tethering platelets to injured blood vessels. Elevated levels or abnormal forms of VWF are linked to pro-thrombotic conditions.

ADAMTS13: A protease responsible for regulating VWF activity. ADAMTS13 cleaves ultra-large VWF (UL-VWF) multimers into smaller, less adhesive forms, preventing excessive clot formation. Reduced levels of ADAMTS13 are associated with pro-coagulant conditions.

The Interconnection

NETs, VWF, and Platelets: A Trifecta in Thrombosis

The trio of NETs, VWF, and platelets form a powerful pro-thrombotic partnership, driving clot formation and amplifying inflammation in a coordinated yet potentially pathological manner. They do so in the following ways (Fig 1):

1. NETs function as a Scaffold for VWF and Platelets

NETs, composed of DNA, histones, antimicrobial proteins, and enzymes, serve as a structural framework that contribute to clot formation during immunothrombotic contexts. NETs capture VWF multimers via electrostatic force and VWF provides binding sites for platelets (Yang et al. 2020). Together, NETs create the scaffold for VWF, plasma proteins, and platelets to adhere to, functioning to stabilize and amplify thrombus formation. The adhesive properties of NETs enhance the recruitment of platelets to areas of vascular injury and inflammation.

2. NETS Activate Platelets

NET components, particularly histones and neutrophil elastase, can directly activate platelets, causing them to change shape, release granules, and aggregate. Activated platelets, in turn, induce NETosis by releasing platelet-derived soluble factors (Colicchia et al. 2022). NET components not only bind platelets, but they also activate platelets, leading to the release of thrombotic mediators and recruitment of additional platelets.

3. VWF Release

NET components such as histones stimulate endothelial cells to release ultra-large VWF (UL-VWF) multimers (Lam et al. 2016). These UL-VWF structures are highly adhesive and serve as a bridge between the endothelial surface and circulating platelets, tethering platelets to the vessel wall (Yang et al. 2020).

4. Platelet Recruitment to VWF

Platelets bind to UL-VWF through the glycoprotein Ib (GPIb) receptor, initiating the formation of platelet-VWF aggregates (Denorme et al. 2019). These aggregates anchor platelets to damaged endothelium, amplifying clot growth.

5. The Cycle of Thrombosis and NETosis

The interaction between NETs, VWF, and platelets creates a self-perpetuating cycle. Activated platelets release molecules that stimulate neutrophils to undergo NETosis (Yang et al. 2020). NETs in turn activate platelets and interact with VWF to enhance clot formation. This feedback loop exacerbates thrombus formation and inflammation when the axis of NETs, VWF, and platelet function is dysregulated.

The Regulatory Role of ADAMTS13 on Clot Formation

ADAMTS13 acts as a critical counterbalance to the pro-thrombotic effects of VWF. ADAMTS13 cleaves UL-VWF into smaller fragments, reducing the platelet-binding capacity of VWF and limiting clot formation. However, this delicate balance can be disrupted in the context of NETosis and immunothrombosis (Fig 1):

6. NET inhibition of ADAMTS13

Neutrophils and NET components damage or inhibit ADAMTS13, impairing its ability to regulate VWF activity (Yang et al. 2020). For example, NETosis-derived oxidation of ADAMTS13 decreases ADAMTS13 activity while NET component PAD4 mediates citrullination of ADAMTS13, reducing its enzymatic activity (Yang et al. 2020). These and other NETosis-mediated effects on ADAMTS13 activity leads to elevated UL-VWF levels and increased VWF-platelet aggregate formations.

7. Unregulated VWF Activity

Without sufficient ADAMTS13 activity, UL-VWF multimers persist in circulation, enhancing platelet aggregation and the formation of pathological thrombi. This is particularly dangerous in small blood vessels, where microvascular thrombosis can lead to tissue ischemia and organ damage.

Figure 1: The interactions and feedback of NETs, Platelets, ultra-large von Willebrand factor (UL-VWF), and ADAMTS13 during immunothrombosis.

Beyond Thrombosis: The Role of Inflammation

The connections between NETosis, VWF, platelets, and ADAMTS13 extend beyond thrombosis into the realm of inflammation, creating a complex interplay that drives both protective and pathological processes (Fig. 2).

Pro-inflammatory NETs

While NETs effectively trap and neutralize pathogens, their excessive formation contributes to tissue damage and chronic inflammation. This is due in part to the release of histones during NETosis. When found in the extracellular space, histones are damage-associated molecular pattern (DAMP) proteins. DAMP proteins activate the immune system and are highly cytotoxic (Silk et al. 2017). Thus, NET-released histones are damaging to the vascular endothelium, increase vascular permeability, promote platelet activation, and correlate with coagulopathy (Esmon 2011; Silk et al. 2017). This creates a feedback loop that sustains both clotting and inflammation.

Pro-inflammatory Activated Platelets

Platelets interact with NETs and NET-associated molecules via platelet toll-like receptors and integrins. These interactions amplify inflammatory and thrombotic signaling, linking NETosis to platelet activation (Lam et al. 2015). Furthermore, activated platelets release pro-inflammatory molecules, such as platelet factor 4 (PF4), which recruit and activate neutrophils, amplifying immune response and inflammation (Lam et al. 2015).

Pro-Inflammatory VWF

VWF is not only essential for platelet adhesion and clot formation but also actively participates in inflammatory responses. By recruiting platelets to the injured endothelium, VWF contributes to platelet-mediated recruitment of leukocytes to inflamed tissues (Denorme et al. 2019). The pro-inflammatory actions of increased levels of VWF exacerbate vascular inflammation, procoagulation, and endothelial activation through VWF-mediated platelet adhesion mechanisms (Manz et al. 2022).

The Regulatory Role of ADAMTS13 on Inflammation

ADAMTS13 attempts to balance excessive inflammation through the cleavage of UL-VWF multimers. This prevents excessive platelet adhesion and aggregation, thus mitigating the pro-inflammatory effects of platelets and VWF. ADAMTS13 also indirectly reduces endothelial injury by reducing the accumulation of UL-VWF, platelets, and NETs on the vascular surface.

Figure 2: The interactions between neutrophil extracellular traps (NETs), platelets, von Willebrand factor (VWF), histones, and ADAMTS13 affect inflammation and clot formation. NETs directly or indirectly impact inflammation by promoting vascular permeability, vascular inflammation, platelet activation, and neutrophil recruitment and activation. ADAMTS13 functions in a regulatory capacity by cleaving ultra-large VWF (UL-VWF) into VWF multimers.

Pathological Implications

Dysregulated interactions between NETosis, VWF, platelets, and ADAMTS13 underlie various inflammatory and thrombotic diseases.

Acute Ischemic Stroke is a condition where occlusion of a cerebral artery leads to a loss of blood flow to the brain. Immunothrombotic events mediated by increased NET-platelet-VWF interactions contribute to microvascular occlusion, worsening brain ischemia.

Antiphospholipid Syndrome is an autoimmune disorder characterized by autoantibodies that attack the body’s own blood phospholipids. The binding of these autoantibodies activate endothelial cells and platelets, which promotes NETosis-driven vascular injury, and the amplification of immunothrombosis.

Cancer-Associated Thrombosis is an increased risk of blood clot formation in patients with cancer. Cancer cells stimulate platelet activation and the release of coagulation factors that stimulate NETosis. Together, these events increase immunothrombotic risk due to a hypercoagulable state that is susceptible to being tipped into imbalance.

Deep Vein Thrombosis (DVT) is the occurrence of a blood clot within veins that have blood stagnancy and hypoxia. Activation of the vascular endothelium coupled with immune cell recruitment drives thrombus development in DVT (Budnik and Brill 2018). A complication of DVT can include pulmonary embolism, which together with DVT is known as venous thromboembolism (VTE).

Sepsis and COVID-19: Inflammatory conditions like sepsis and severe COVID-19 often exhibit heightened NETosis and elevated VWF levels, contributing to disseminated intravascular coagulation (DIC) and organ failure. NETs and VWF are key players in the hypercoagulable state observed in severe cases of sepsis and COVID-19.

Sickle Cell Disease is characterized by chronic inflammation, which primes neutrophils for NETosis. Hypoxia and endothelial damage stimulate excessive VWF release while reduced ADAMTS13 activity creates conditions for immunothromobosis and vaso-occlusive episodes.

Thrombotic Thrombocytopenic Purpura (TTP) is a type of thrombotic microangiopathy (TMA) characterized by small blood clots caused by severe ADAMTS13 deficiency and uncontrolled UL-VWF activity. NETs exacerbate TTP by directly inhibiting ADAMTS13 activity, amplifying the pro-thrombotic environment and microvascular clotting.

Therapeutic Approaches

The NETosis-VWF-platelet-ADAMTS13 axis exemplifies the complex interplay between immune response, vascular integrity, and thrombosis. Understanding and targeting these interconnected mechanisms can provide novel strategies to mitigate thrombo-inflammatory diseases. Approaches include:

Targeting NETs through NET inhibitors like DNAse or histone neutralization could reduce endothelial damage and vascular inflammation caused by aberrant NETosis.

Platelet-Targeted Therapies such as anti-platelet therapeutics to selectively inhibit platelet activation may reduce thromboinflammation while preserving hemostasis. Interventions targeting the binding of platelets to NETs would disrupt the inflammatory amplification loop seen in immunothrombosis. The pathogenic consequences of platelet-derived cytokines could be mitigated by targeting the secretion of platelet-derived cytokines that signal immune infiltration and inflammation.

VWF Modulation through therapeutics that target and limit VWF activity function to decrease platelet adhesion and aggregation. Drugs that interfere with the interaction between VWF and platelet receptors have potential to directly impact thrombus formation.

ADAMTS13 Augmentation enhances ADAMTS13 activity to prevent the accumulation of UL-VWF multimers to prevent thrombotic events in conditions with high levels of NETs and VWF. ADAMTS13 replacement therapies have been explored in TTP and sepsis to restore the balance between coagulation and immune response.

Conclusion: The Importance of Balance

The interconnection between NETosis, platelets, VWF, and ADAMTS13 illustrates the importance of maintaining equilibrium within the immune and coagulation systems. Infection control and vascular repair are critical pillars of hemostasis, however, once disrupted, imbalances can lead to devastating immunothrombotic consequences. Research exploring these relationships will grow a better understanding of the mechanisms driving these complex thrombo-inflammatory disorders. This highlights the need for targeted interventions aimed at restoring homeostatic balance in the NETosis-VWF-platelet-ADAMTS13 axis.

The (im)Balance of NETosis, Platelets, VWF, and ADAMTS13 During Immunothrombosis

The Interconnection

Pathological Implications

Therapeutic Approaches

Conclusion: The Importance of Balance

Further Reading