Free hemoglobin increases von Willebrand factor–mediated platelet adhesion in vitro: implications for circulatory devices

Abstract

• 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...

Acquired von Willebrand syndrome associated with left ventricular assist device

“…96 Hemoglobin is also reported to sterically hinder ADAMTS-13-VWF interaction. 97 These findings are relevant because hemolysis often occurs in patients on LVAD support and is associated with thrombotic events.…”

Elevated free hemoglobin and decreased haptoglobin levels are associated with adverse clinical outcomes, unfavorable physiologic measures, and altered inflammatory markers in pediatric cardiac surgery patients

“…Hod and colleagues have suggested a name for these observations specifically related to blood transfusions: “the iron hypothesis.” Increased circulating cell‐free Hb, heme, and iron and decreased haptoglobin concentrations have been associated with morbidity and mortality in both animal models and patients . There are in vitro and animal model data linking increased free Hb, heme, and iron to inflammation, infection, platelet (PLT) activation, vasculopathy, and thrombosis . Therapeutically, use of Hb or heme‐binding proteins such as haptologlobin and hemopexin can mitigate these effects in animal models …”

Creating adequate pulsatility with a continuous flow left ventricular assist device

“…Similarly to the above discussion on ventricular unloading, this phenomenon is not a direct consequence of low pulsatile flow but, rather, of continuous flow LVAD design, which employs a rotor mechanism. Although the exact pathophysiology of device thrombosis has not been elucidated, over the past few years a number of important observations have been made: (1) during transit through the rotors, blood elements are exposed to a shear stress well above that at which hemolysis occurs [41]; (2) as a result, there is a chronic low level hemolysis in all continuous flow LVADs, which appears to be greater in axial than in centrifugal pumps [42]; and (3) hemolysis, in addition to being a marker of device thrombosis, likely contributes to its development as a prothrombotic factor [43,44]. In this regard, pulsatility may be important for the pump itself by providing an intermittent 'wash out' of the rotors to prevent adherence of red blood cells and in turn decrease levels of hemolysis.…”