Neutrophil Structural and Functional Alterations After High Mechanical Shear Stress Exposure.

Patients on mechanical circulatory support are prone to infections, increasing morbidity and mortality. These circulatory support devices generate high mechanical shear stress (HMSS) that can causes trauma to blood. When leukocytes become damaged, their immune response function may be impaired or weakened, leading to increased infection vulnerability. This study examined neutrophil structural and functional alterations after exposure to 75, 125, and 175 Pa HMSS for 1 second. Human blood was exposed to three levels of HMSS using a blood shearing device. Neutrophil morphological alteration was characterized by examining blood smears. Flow cytometry assays were used to analyze expression levels of CD62L and CD162 receptors, activation level (CD11b), and aggregation (platelet-neutrophil aggregates). Neutrophil phagocytosis and rolling were examined via functional assays. The results show neutrophil structure (morphology and surface receptors) and function (activation, aggregation, phagocytosis, rolling) were significantly altered after HMSS exposure. These alterations include cell membrane damage, loss of surface receptors (CD62L and CD162), initiation of activation and aggregation, upregulation of phagocytic ability and increased rolling speed. The alterations were the most severe after 175 Pa exposure. HMSS caused damage and activation of neutrophils, potentially impairing normal neutrophil function, leading to weakened immune defense and increasing a patient's vulnerability to infections.

Acquired platelet defects are responsible for nonsurgical bleeding in left ventricular assist device recipients.

BACKGROUND: Left ventricular assist devices (LVADs) have been used as a standard treatment option for patients with advanced heart failure. However, these devices are prone to adverse events. Nonsurgical bleeding (NSB) is the most common complication in patients with continuous flow (CF) LVADs. The development of acquired von Willebrand syndrome (AVWS) in CF-LVAD recipients is thought to be a key factor. However, AVWS is seen across a majority of LVAD patients, not just those with NSB. The purpose of this study was to examine the link between acquired platelet defects and NSB in CF-LVAD patients. METHODS: Blood samples were collected from 62 CF-LVAD patients at pre- and 4 post-implantation timepoints. Reduced adhesion receptor expression (GPIbα and GPVI) and activation of platelets (GPIIb/IIIa activation) were used as markers for acquired platelet defects. RESULTS: Twenty-three patients experienced at least one NSB episode. Significantly higher levels of platelet activation and receptor reduction were seen in the postimplantation blood samples from bleeders compared with non-bleeders. All patients experienced the loss of high molecular weight monomers (HMWM) of von Willebrand Factor (vWF), but no difference was seen between the two groups. Multivariable logistic regression showed that biomarkers for reduced platelet receptor expression (GPIbα and GPVI) and activation (GPIIb/IIIa) have more predictive power for NSB, with the area under curve (AUC) values of 0.72, 0.68, and 0.62, respectively, than the loss of HMWM of vWF (AUC: 0.57). CONCLUSION: The data from this study indicated that the severity of acquired platelet defects has a direct link to NSB in CF-LVAD recipients.

Neutrophil dysfunction due to continuous mechanical shear exposure in mechanically assisted circulation in vitro.

OBJECTIVE: Leukocytes play an important role in the body's immune system. The aim of this study was to assess alterations in neutrophil phenotype and function in pump-assisted circulation in vitro. METHODS: Human blood was circulated for four hours in three circulatory flow loops with a CentriMag blood pump operated at a flow of 4.5 L/min at three rotational speeds (2100, 2800, and 4000 rpm), against three pressure heads (75, 150, and 350 mm Hg), respectively. Blood samples were collected hourly for analyses of neutrophil activation state (Mac-1, CD62L, CD162), neutrophil reactive oxygen species (ROS) production, apoptosis, and neutrophil phagocytosis. RESULTS: Activated neutrophils indicated by both Mac-1 expression and decreased surface expression of CD62L and CD162 receptors increased with time in three loops. The highest level of neutrophil activation was observed in the loop with the highest rotational speed. Platelet-neutrophil aggregates (PNAs) progressively increased in two loops with lower rotational speeds. PNAs peaked at one hour after circulation and decreased subsequently in the loop with the highest rotational speed. Neutrophil ROS production dramatically increased at one hour after circulation and decreased subsequently in all three loops with similar levels and trends. Apoptotic neutrophils increased with time in all three loops. Neutrophil phagocytosis capacity in three loops initially elevated at one hour after circulation and decreased subsequently. Apoptosis and altered phagocytosis were dependent on rotational speed. CONCLUSIONS: Our study revealed that the pump-assisted circulation induced neutrophil activation, apoptosis, and functional impairment. The alterations were strongly associated with pump operating condition and duration.

Neutrophil injury and function alterations induced by high mechanical shear stress with short exposure time.

High mechanical shear stresses (HMSS) can cause damage to blood, which manifests as morphologic changes, shortened life span, biochemical alterations, and complete rupture of blood cells and proteins, leading to the alterations of normal blood function. The aim of this study is to determine the state of neutrophil activation and function alterations caused by HMSS with short exposure time relevant to ventricular assist devices. Blood from healthy donors was exposed to three levels of HMSS (75Pa, 125Pa, and 175Pa) for a short exposure time (0.5 s) using our Couette-type blood-shearing device. Neutrophil activation (Mac-1, platelet-neutrophil aggregates) and surface expression levels of two key functional receptors (CD62L and CD162) on neutrophils were evaluated by flow cytometry. Neutrophil phagocytosis and transmigration were also examined with functional assays. Results showed that the expression of Mac-1 on neutrophils and platelet-neutrophil aggregates increased significantly while the level of CD62L expression on neutrophils decreased significantly after the exposure to HMSS. The Mac-1 expression progressively increased while the CD62L expression progressively decreased with the increased level of HMSS. The level of CD162 expression on neutrophils slightly increased after the exposure to HMSS, but the increase was not significant. The phagocytosis assay data revealed that the ability of neutrophils to phagocytose latex beads coated with fluorescently labeled rabbit IgG increased significantly with the increased level of HMSS. The transmigration ability of neutrophils slightly increased after the exposure to HMSS, but did not reach a significant level. In summary, HMSS with a short exposure time of 0.5 seconds could induce neutrophil activation, platelet-neutrophil aggregation, shedding of CD62L receptor, and increased phagocytic ability. However, the exposure to the three levels of HMSS did not cause a significant change in neutrophil transmigration capacity and shedding of CD162 receptor on neutrophils.

Impact of high mechanical shear stress and oxygenator membrane surface on blood damage relevant to thrombosis and bleeding in a pediatric ECMO circuit.

The roles of the large membrane surface of the oxygenator and the high mechanical shear stress (HMSS) of the pump in the extracorporeal membrane oxygenation (ECMO) circuit were examined under a pediatric support setting. A clinical centrifugal pump and a pediatric oxygenator were used to construct the ECMO circuit. An identical circuit without the oxygenator was constructed for comparison. Fresh human blood was circulated in the two circuits for 4 hours under the identical pump speed and flow. Blood samples were collected hourly for blood damage assessment, including platelet activation, generation of platelet-derived microparticles (PDMP), losses of key platelet hemostasis receptors (glycoprotein (GP) Ibα (GPIbα) and GPVI), and high molecular weight multimers (HMWM) of von Willebrand factor (VWF) and plasma free hemoglobin (PFH). Platelet adhesion on fibrinogen, VWF, and collagen was further examined. The levels of platelet activation and generation of PDMP and PFH exhibited an increasing trend with circulation time while the expression levels of GPIbα and GPVI receptors on the platelet surface decreased. Correspondingly, the platelets in the blood samples exhibited increased adhesion capacity to fibrinogen and decreased adhesion capacities on VWF and collagen with circulation time. Loss of HMWM of VWF occurred in both circuits. No statistically significant differences were found in all the measured parameters for blood damage and platelet adhesion function between the two circuits. The results indicate that HMSS from the pump played a dominant role in blood damage associated with ECMO and the impact of the large surface of the oxygenator on blood damage was insignificant.

The Role of a Disintegrin and Metalloproteinase Proteolysis and Mechanical Damage in Nonphysiological Shear Stress-Induced Platelet Receptor Shedding.

In order to explore the role of a disintegrin and metalloproteinase (ADAM) proteolysis and direct mechanical damage in non-physiologic shear stress (NPSS)-caused platelet receptor shedding, the healthy donor blood treated with/without ADAM inhibitor was exposed to NPSS (150 Pa). The expression of the platelet surface receptors glycoprotein (GP) Ibα and glycoprotein (GP) VI (GPVI) in NPSS-damaged blood was quantified with flow cytometry. The impact of ADAM inhibition on adhesion of NPSS-damaged platelets on von Willibrand factor (VWF) and collagen was explored with fluorescence microscopy. The impact of ADAM inhibition on ristocetin- and collagen-caused aggregation of NPSS-damaged platelets was examined by aggregometry. The results showed that ADAM inhibition could lessen the NPSS-induced loss of platelet surface receptor GPIbα (12%) and GPVI (9%), moderately preserve adhesion of platelets on VWF (7.4%) and collagen (8.4%), and partially restore the aggregation of NPSS-sheared platelets induced by ristocetin (18.6 AU*min) and collagen (48.2 AU*min). These results indicated that ADAM proteolysis played a role in NPSS-induced receptor shedding. However, the ADAM inhibition couldn't completely suppress the NPSS-caused loss of the platelet surface receptors (GPIbα and GPVI), only partially prevented the NPSS-induced reduction of platelet adhesion to VWF and collagen, and the agonist (ristocetin and collagen)-caused platelet aggregation. These results suggested that the direct mechanical damage is partially responsible for NPSS-induced receptor shedding in addition to the ADAM proteolysis. In conclusion, NPSS relevant to blood contacting medical devices can induce ADAM proteolysis and direct mechanical damage on the platelet receptor GPIbα and GPVI, leading to comprised hemostasis.

Device-induced platelet dysfunction in mechanically assisted circulation increases the risks of thrombosis and bleeding.

Thrombotic and bleeding complications are the major obstacles for expanding mechanical circulatory support (MCS) beyond the current use. While providing the needed hemodynamic support, those devices can induce damage to blood, particularly to platelets. In this study, we investigated device-induced alteration of three major platelet surface receptors, von Willebrand factor (VWF) and associated hemostatic functions relevant to thrombosis and bleeding. Fresh human whole blood was circulated in an extracorporeal circuit with a clinical rotary blood pump (CentriMag, Abbott, Chicago, IL, USA) under the clinically relevant operating condition for 4 hours. Blood samples were examined every hour for glycoprotein (GP) IIb/IIIa activation and receptor loss of GPVI and GPIbα on the platelet surface with flow cytometry. Soluble P-selectin in hourly collected blood samples was measured by enzyme linked immunosorbent assay to characterize platelet activation. Adhesion of device-injured platelets to fibrinogen, collagen, and VWF was quantified with fluorescent microscopy. Device-induced damage to VWF was characterized with western blotting. The CentriMag blood pump induced progressive platelet activation with blood circulating time. Particularly, GPIIb/IIIa activation increased from 1.1% (Base) to 11% (4 hours) and soluble P-selectin concentration increased from 14.1 ng/mL (Base) to 26.5 ng/mL (4 hours). Those device-activated platelets exhibited increased adhesion capacity to fibrinogen. Concurrently, the CentriMag blood pump caused progressive platelet receptor loss (GPVI and GPIbα) with blood circulating time. Specifically, MFI of the GPVI and GPIbα receptors decreased by 17.2% and 16.1% for the 4-hours sample compared to the baseline samples, respectively. The device-injured platelets exhibited reduced adhesion capacities to collagen and VWF. The high molecular weight multimers (HMWM) of VWF in the blood disappeared within the first hour of the circulation. Thereafter the multimeric patterns of VWF were stable. The change in the VWF multimeric pattern was different from the progressive structural and functional changes of platelets with the circulation time. This study suggested that the CentriMag blood pump could induce two opposite effects on platelets and associated hemostatic functions under a clinically relevant operating condition. The device-altered hemostatic function may contribute to thrombosis and bleeding simultaneously as occurring in patients supported by a rotary blood pump. Device-induced damage of platelets may be an important cause for bleeding in patients supported with rotary blood pump MCS systems relative to device-induced loss of HMWM-VWF.

Direct current stimulation of endothelial monolayers induces a transient and reversible increase in transport due to the electroosmotic effect.

We investigated the effects of direct current stimulation (DCS) on fluid and solute transport across endothelial cell (EC) monolayers in vitro. Our motivation was transcranial direct current stimulation (tDCS) that has been investigated for treatment of neuropsychiatric disorders, to enhance neurorehabilitation, and to change cognition in healthy subjects. The mechanisms underlying this diversity of applications remain under investigation. To address the possible role of blood-brain barrier (BBB) changes during tDCS, we applied direct current to cultured EC monolayers in a specially designed chamber that generated spatially uniform direct current. DCS induced fluid and solute movement across EC layers that persisted only for the duration of the stimulation suggesting an electroosmosis mechanism. The direction of induced transport reversed with DCS polarity - a hallmark of the electroosmotic effect. The magnitude of DCS-induced flow was linearly correlated to the magnitude of the applied current. A mathematical model based on a two-pore description of the endothelial transport barrier and a Helmholtz model of the electrical double layer describes the experimental data accurately and predicts enhanced significance of this mechanism in less permeable monolayers. This study demonstrates that DCS transiently alters the transport function of the BBB suggesting a new adjunct mechanism of tDCS.