Correction: Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor.

[This corrects the article DOI: 10.1371/journal.pone.0173077.].

Stasis Promotes Erythrocyte Adhesion to von Willebrand Factor.

OBJECTIVE: Venous thromboembolism is a major contributor to global disease burden. Leukocytes and platelets initiate thrombogenesis on blood stasis and initiate the formation of a fibrin, VWF (von Willebrand factor), and neutrophil extracellular trap scaffold for erythrocytes. However, there is little knowledge on how erythrocytes become stably incorporated into this scaffold. Recently, we described the adhesion of calcium-loaded erythrocytes to endothelial-derived VWF strings. Because VWF is part of the scaffold of venous thrombi, we questioned whether reduced flow or stasis promotes the adhesion of normal erythrocytes to VWF and whether venous thrombi show evidence of erythrocyte-VWF interactions. APPROACH AND RESULTS: In the present work, we perfused, under controlled shear conditions, washed, normal erythrocytes over surface-immobilized plasma and extracellular matrix proteins and showed that normal erythrocytes specifically bind to VWF. The interaction between erythrocytes and VWF significantly increased when the wall shear stress was reduced. Next, we investigated whether erythrocyte-VWF interactions support the structure of venous thrombi. High-resolution immunofluorescence imaging of human venous thrombi showed a striking pattern between erythrocytes, VWF, and fibrin, which suggests that VWF plays a supporting role, linking erythrocytes to fibrin in the thrombus. CONCLUSIONS: Our data suggest that erythrocyte retention in venous thrombi is mediated by erythrocyte-VWF or erythrocyte-VWF-fibrin interactions. Targeting erythrocyte retention could be a new strategy in the treatment or prevention of venous thrombosis.

Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor.

Adhesion of erythrocytes to endothelial cells lining the vascular wall can cause vaso-occlusive events that impair blood flow which in turn may result in ischemia and tissue damage. Adhesion of erythrocytes to vascular endothelial cells has been described in multiple hemolytic disorders, especially in sickle cell disease, but the adhesion of normal erythrocytes to endothelial cells has hardly been described. It was shown that calcium-loaded erythrocytes can adhere to endothelial cells. Because sickle erythrocyte adhesion to ECs can be enhanced by ultra-large von Willebrand factor multimers, we investigated whether calcium loading of erythrocytes could promote binding to endothelial cells via ultra-large von Willebrand factor multimers. We used (immunofluorescent) live-cell imaging of washed erythrocytes perfused over primary endothelial cells at venular flow rate. Using this approach, we show that calcium-loaded erythrocytes strongly adhere to histamine-stimulated primary human endothelial cells. This adhesion is mediated by ultra-large von Willebrand factor multimers. Von Willebrand factor knockdown or ADAMTS13 cleavage abolished the binding of erythrocytes to activated endothelial cells under flow. Platelet depletion did not interfere with erythrocyte binding to von Willebrand factor. Our results reveal platelet-independent adhesion of calcium-loaded erythrocytes to endothelium-derived von Willebrand factor. Erythrocyte adhesion to von Willebrand factor may be particularly relevant for venous thrombosis, which is characterized by the formation of erythrocyte-rich thrombi.

F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries and veins.

OBJECTIVE: Vascular endothelial-cadherin- and integrin-based cell adhesions are crucial for endothelial barrier function. Formation and disassembly of these adhesions controls endothelial remodeling during vascular repair, angiogenesis, and inflammation. In vitro studies indicate that vascular cytokines control adhesion through regulation of the actin cytoskeleton, but it remains unknown whether such regulation occurs in human vessels. We aimed to investigate regulation of the actin cytoskeleton and cell adhesions within the endothelium of human arteries and veins. APPROACH AND RESULTS: We used an ex vivo protocol for immunofluorescence in human vessels, allowing detailed en face microscopy of endothelial monolayers. We compared arteries and veins of the umbilical cord and mesenteric, epigastric, and breast tissues and find that the presence of central F-actin fibers distinguishes the endothelial phenotype of adult arteries from veins. F-actin in endothelium of adult veins as well as in umbilical vasculature predominantly localizes cortically at the cell boundaries. By contrast, prominent endothelial F-actin fibers in adult arteries anchor mostly to focal adhesions containing integrin-binding proteins paxillin and focal adhesion kinase and follow the orientation of the extracellular matrix protein fibronectin. Other arterial F-actin fibers end in vascular endothelial-cadherin-based endothelial focal adherens junctions. In vitro adhesion experiments on compliant substrates demonstrate that formation of focal adhesions is strongly induced by extracellular matrix rigidity, irrespective of arterial or venous origin of endothelial cells. CONCLUSIONS: Our data show that F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries from veins. We conclude that the biomechanical properties of the vascular extracellular matrix determine this endothelial characteristic.

Plasmin cleavage of von Willebrand factor as an emergency bypass for ADAMTS13 deficiency in thrombotic microangiopathy.

BACKGROUND: Von Willebrand factor (VWF) multimer size is controlled through continuous proteolysis by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13). This prevents spontaneous platelet agglutination and microvascular obstructions. ADAMTS13 deficiency is associated with thrombotic thrombocytopenic purpura, in which life-threatening episodes of microangiopathy damage kidneys, heart, and brain. Enigmatically, a complete ADAMTS13 deficiency does not lead to continuous microangiopathy. We hypothesized that plasmin, the key enzyme of the fibrinolytic system, serves as a physiological backup enzyme for ADAMTS13 in the degradation of pathological platelet-VWF complexes. METHODS AND RESULTS: Using real-time microscopy, we determined that plasmin rapidly degrades platelet-VWF complexes on endothelial cells in absence of ADAMTS13, after activation by urokinase-type plasminogen activator or the thrombolytic agent streptokinase. Similarly, plasmin degrades platelet-VWF complexes in platelet agglutination studies. Plasminogen directly binds to VWF and its A1 domain in a lysine-dependent manner, as determined by enzyme-linked immunosorbent assay. Plasma levels of plasmin-α2-antiplasmin complexes increase with the extent of thrombocytopenia in patients with acute episodes of thrombotic thrombocytopenic purpura, independent of ADAMTS13 activity. This indicates that plasminogen activation takes place during microangiopathy. Finally, we show that the thrombolytic agent streptokinase has therapeutic value for Adamts13(-/-) mice in a model of thrombotic thrombocytopenic purpura. CONCLUSIONS: We propose that plasminogen activation on endothelial cells acts as a natural backup for ADAMTS13 to degrade obstructive platelet-VWF complexes. Our findings indicate that thrombolytic agents may have therapeutic value in the treatment of microangiopathies and may be useful to bypass inhibitory antibodies against ADAMTS13 that cause thrombotic thrombocytopenic purpura.