• Extracellular Hb alters the GPIba-VWF interaction.Intravascular hemolysis occurs in patients on extracorporeal membrane oxygenation. High levels of free acellular adult hemoglobin (free HbA) are associated with clotting in this mechanical device that can result in thrombotic complications. Adsorption of fibrinogen onto the surface of biomaterial correlates with platelet adhesion, which is mediated by von Willebrand factor (VWF). Because free Hb interacts with VWF, we studied the effect of hemoglobin (Hb) on platelet adhesion to fibrin(ogen) under conditions of different hydrodynamic forces. This effect was investigated using purified human HbA and fibrinogen, extracellular matrix, collagen, or purified plasma VWF as surface-coated substrates to examine flow-dependent platelet adhesion. Antibodies and VWF-deficient plasma were also used. Free Hb ( ‡50 mg/dL) effectively augmented platelet adhesion, and microthrombi formation on fibrin(ogen), extracellular matrix, and collagen at high shear stress. The effect of free Hb was effectively blocked by anti-glycoprotein Iba (GPIba) antibodies or depletion of VWF. Unexpectedly, free Hb also promoted firm platelet adhesion and stable microthrombi on VWF. Lastly, we determined that Hb interacts directly with the A1 domain. This study is the first to demonstrate that extracellular Hb directly affects the GPIba-VWF interaction in thrombosis, and describes another mechanism by which hemolysis is connected to thrombotic events. (Blood. 2015; 126(20):2338-2341 IntroductionThe excessive release of hemoglobin (Hb) from erythrocytes into the circulation of patients on mechanical circulatory support devices is a well-recognized major clinical complication.1 Increasing incidence of hemolysis and thrombosis is associated with morbidity and mortality in patients on extracorporeal membrane oxygenation (ECMO).2 Prevention of circuit clotting in ECMO can improve clinical outcome.von Willebrand factor (VWF) is a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation under high flow conditions. 3-7 Plasma VWF mediates platelet adhesion to surfaces coated with fibrin(ogen), 8,9 which is adsorbed onto surfaces of many materials used in biomedical instruments, including ECMO.10,11 Previously, we reported that free Hb interacts with the A2 domain of VWF 12 and, moreover, we and many others have described that the A2 domain regulates the binding of its neighboring A1 domain in VWF to platelet receptor glycoprotein Iba (GPIba).13-15 Thus, in this study, we examined the effect of the free Hb-VWF interaction on mediating platelet adhesion to immobilized fibrin(ogen) at high shear stress; a mechanism not previously investigated. Study design ReagentsPurified Hb and plasma VWF were obtained using established methods.13,16 Human collagen type III was purchased from Advanced BioMatrix, human fibrinogen from Calbiochem, and extracellular matrix (ECM) from Sigma-Aldrich. Anti-GPIba antibody 6D1 was a gift from Dr Barry Coller (The Rockefeller University, New Yor...

Leukocyte adhesion to vascular endothelium and platelets is an early step in the acute inflammatory response. The initial process is mediated through P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes binding to platelets adhered to endothelium and the endothelium itself via P-selectin. Although these interactions are generally beneficial, pathologic inflammation may occur in undesirable circumstances, such as in acute lung injury (ALI) and ischemia and reperfusion injury. Therefore, the development of novel therapies to attenuate inflammation may be beneficial. In this article, we describe the potential benefit of using a recombinant human vimentin (rhVim) on reducing human leukocyte adhesion to vascular endothelium and platelets under shear stress. The addition of rhVim to whole blood and isolated neutrophils decreased leukocyte adhesion to endothelial and platelet monolayers. Furthermore, rhVim blocked neutrophil adhesion to P-selectin-coated surfaces. Binding assays showed that rhVim binds specifically to P-selectin and not to its counterreceptor, PSGL-1. Finally, in an endotoxin model of ALI in C57BL/6J mice, treatment with rhVim significantly decreased histologic findings of ALI. These data suggest a potential role for rhVim in attenuating inflammation through blocking P-selectin-PSGL-1 interactions.

• Vimentin expressed on the platelet surface serves as adhesive receptor for VWF.The interaction between platelet receptor glycoprotein Iba and the A1 domain of von Willebrand factor (VWF) mediates tethering/translocation of platelets to sites of vascular injury. Unexpectedly, we observed platelets translocating over A1A2A3 domains protein slower than on A1 domain at high shear stress. This observation suggests an additional interaction between A domains and an adhesive receptor. We investigated vimentin because we have data showing the interaction of vimentin with the A2 domain of VWF. Moreover, vimentin is expressed on the platelet surface. This novel interaction was analyzed by using purified VWF, recombinant proteins, anti-vimentin antibodies, parallel flow chamber adhesion assays, flow cytometry, and vimentin-deficient murine platelets. The active form of VWF bound to vimentin, and the purified A2 domain blocked that binding. The interaction of a gain-of-function A1A2A3 mutant with platelet was reduced using anti-vimentin antibody. Platelet adhesion to wild-type (WT) A1A2A3 protein, collagen, and fibrin(ogen) was inhibited (32-75%) by anti-vimentin antibody under high shear stress. Compared with WT mice, platelets from vimentin-deficient mice had a reduced flow-dependent adhesion to both collagen and purified murine VWF. Last, the vimentin knockout mice had a prolonged tail bleeding time. The results describe that platelet vimentin engages VWF during platelet adhesion under high shear stress. (Blood. 2014;123(17):2715-2721 Introduction Atherothrombotic events, including acute coronary syndrome and stroke, are the result of platelet adhesion and activation on the ruptured atherosclerotic plaques. This platelet-mediated arterial thrombosis starts with the contact of the rapidly flowing platelets to components of the damaged blood vessel. von Willebrand factor (VWF), a multimeric plasma and subendothelial glycoprotein, is relevant in mediating platelet adhesion and activation at sites of lesions in the coronary arteries, where high shear conditions prevail.1,2 VWF captures the circulating platelets through its interaction with the platelet receptor glycoprotein (GP)Ib/IX/V complex. This interaction is responsible for the tethering, rolling, and activation of platelets that eventually become firmly adhered, leading to thrombus formation within a coronary artery. 3,4 Mature VWF consists of a 2050-residue subunit formed by domains arranged in the order of D9-D3-A1-A2-A3-D4-B-C. 5 The A1 domain contains the binding site for the platelet receptor GPIba 6 ; the cleavage site for the enzyme ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs)-13 is localized in the A2 domain, 7 and the A3 domain binds to collagen. 8 Unlike the A3 domain, both the A1 and A2 domains do not have access to their ligands until their domain structure is altered.9 This structural modification can be induced by mutations, 10 hydrodynamic forces, 11 immobilization on a surface, or artificially with the modulator ristocetin....