Endothelial activation and microvascular thrombosis are hallmarks of thrombotic microangiopathy—a group of life-threatening disorders that includes thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Activated endothelial cells release von Willebrand factor (VWF), which can form long strands under flow that remain attached to the endothelium until they are cleaved off by the metalloprotease ADAMTS13. Failure to remove these strands, either because of ADAMTS13 deficiency or oxidation of its cleavage site on VWF, results in microvascular thrombosis.
Until now, studies of VWF strands under flow have been performed either in flow chambers with cultured endothelial cells, which does not account for either vessel caliber or geometry, or in live mice, in which it is impossible to study individually the contributions of the various blood components. Recently, we developed a technique to engineer microvessels in vitro that enables us to precisely control several vessel parameters, including lumen diameter and branching architecture, flow patterns, and applied shear stresses, in addition to being able to test individual components of the blood in a system with only human components (PNAS 2012, 109:9342–9347). In the current study, we used this system to examine the effects of a number of variables on the formation of VWF strands from the endothelium of stimulated vessels. We found that VWF fibers can extend across the vessel lumen and attach to opposite sides of the vessel wall in agonist-treated microvessels of up to 200 μm in diameter. Depending on flow conditions, smaller strands can self-associate to form longer and thicker cables. The VWF cables produced solely from VWF contributed by the vessel wall reached lengths up to 5 cm, and became so thick as to be visible, unstained, by light microscopy. When plasma or recombinant VWF was perfused over the VWF cables, the fluid-phase VWF associated with the vessel-bound cables, further thickening them and sometimes inducing web-like structures. The location and structure of the VWF fibers were dependent on vessel geometry and flow pattern; secondary flows that developed at bends or bifurcations in the vessel induced circular clumping of the VWF strands. When whole blood was perfused into the vessels, the transluminal VWF fiber webs caught flowing platelets and leukocytes to form aggregates in the middle of blood stream that sometimes occluded the vessels. The region where the vessel is most likely to occlude also depends on geometry. After this type of trapping, leukocytes were seen to transmigrate across the endothelium. The structure and size of the cables also depended on the agonist employed to stimulate VWF release from the endothelium. Phorbol myristate acetate and shiga-like toxin–2 both produced thicker cables than histamine did, and these were more resistant to ADAMTS13 cleavage. This difference is potentially a result of the former agonists stimulating an endothelial respiratory burst and oxidation of the ADAMTS13 cleavage site on VWF.
In summary, our data show that VWF secreted from activated endothelial cells can form transluminal fibers and cables in small vessels. Some of the fibers or cables are resistant to ADAMTS13 cleavage, a likely consequence of their thickness and possibly, oxidation. The webs of VWF fibers or cables in the lumen of small vessels obstruct blood flow by binding to circulating platelets and leukocytes, and are also capable of shredding erythrocytes as they flow past. These findings provide insights into the mechanisms of microangiopathy, and raise the possibility that VWF cables alone, even in the absence of bound platelets, may be capable of occluding small blood vessels and produce many of the characteristic signs of thrombotic microangiopathy.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.