Platelet Receptor Shedding Research Articles

In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

Multi-Method Investigation of Blood Damage Induced By Blood Pumps in Different Clinical Support Modes.

To investigate the effects of blood pumps operated in different modes on nonphysiologic flow patterns, cell and protein function, and the risk of bleeding, thrombosis, and hemolysis, an extracorporeal blood pump (CentriMag) was operated in three clinical modalities including heart failure (HF), venous-venous (V-V) extracorporeal membrane oxygenation (ECMO), and venous-arterial (V-A) ECMO. Computational fluid dynamics (CFD) methods and coupled hemolysis models as well as recently developed bleeding and thrombosis models associated with changes in platelet and von Willebrand factor (vWF) function were used to predict hydraulic performance and hemocompatibility. The V-A ECMO mode had the highest flow losses and shear stress levels, the V-V ECMO mode was intermediate, and the HF mode was the lowest. Different nonphysiologic flow patterns altered cell/protein morphology and function. The V-A ECMO mode resulted in the highest levels of platelet activation, receptor shedding, vWF unfolding, and high molecular weight multimers vWF (HMWM-vWF) degradation, leading to the lowest platelet adhesion and the highest vWF binding capacity, intermediate in the V-V ECMO mode, and opposite in the HF mode. The V-A ECMO mode resulted in the highest risk of bleeding, thrombosis, and hemolysis, with the V-V ECMO mode intermediate and the HF mode lowest. These findings are supported by published experimental or clinical statistics. Further studies found that secondary blood flow passages resulted in the highest risk of blood damage. Nonphysiologic blood flow patterns were strongly associated with cell and protein function changing, blood damage, and complications.

Investigation of the role of von Willebrand factor in shear-induced platelet activation and functional alteration under high non-physiological shear stress.

von Willebrand factor (vWF) plays a crucial role in physiological hemostasis through platelet and subendothelial collagen adhesion. However, its role in shear-induced platelet activation and functional alteration under non-physiological conditions common to blood-contacting medical devices (BCMDs) is not well investigated. Fresh healthy human blood was treated with an anti-vWF antibody to block vWF-GPIbα interaction. Untreated blood was used as a control. They were exposed to three levels of non-physiological shear stress (NPSS) (75, 125, and 175 Pa) through a shearing device with an exposure time of 0.5 s to mimic typical shear conditions in BCMDs. Flow cytometric assays were used to measure the expression levels of PAC-1 and P-Selectin and platelet aggregates for platelet activation and the expression levels of GPIbα, GPIIb/IIIa, and GPVI for receptor shedding. Collagen/ristocetin-induced platelet aggregation capacity was characterized by aggregometry. The levels of platelet activation and aggregates increased with increasing NPSS in the untreated blood. More receptors were lost with increasing NPSS, resulting in a decreased capacity of collagen/ristocetin-induced platelet aggregation. In contrast, the increase in platelet activation and aggregates after exposure to NPSS, even at the highest level of NPSS, was significantly lower in treated blood. Nevertheless, there was no notable difference in receptor shedding, especially for GPIIb/IIIa and GPVI, between the two blood groups at the same level of NPSS. The block of vWF exacerbated the decreased capacity of collagen/ristocetin-induced platelet aggregation. High NPSS activates platelets mainly by enhancing the vWF-GPIbα interaction. Platelet activation and receptor shedding induced by high NPSS likely occur through different pathways.

A mathematical model for assessing shear induced bleeding risk

PurposeThe objective of this study is to develop a bleeding risk model for assessing device-induced bleeding risk in patients supported with blood contact medical devices (BCMDs). MethodsThe mathematical model for evaluating bleeding risk considers the effects of shear stress on von Willebrand factor (vWF) unfolding, high molecular weight multimers-vWF (HMWM-vWF) degradation, platelet activation and receptor shedding and platelet-vWF binding ability. Functions of the effect of shear stress on the above factors are fitted/employed and solved by the Eulerian transport equation. An axial flow-through Couette device and two clinical VADs which are HeartWare Ventricular Assist Device (HVAD) and HeartMate II (HM II) blood pump were employed to perform the simulation to evaluate platelet receptor shedding (GPIbα and GPIIb/IIIa), loss of HWMW-vWF, platelet-vWF binding ability and bleeding risk for validating the accuracy of our model. ResultsThe platelet-vWF binding ability after being subjected to high shear region in the axial flow-through Couette device predicted by our bleeding model was highly consistent with reported experimental data. As indicated by our CFD simulation results in the axial flow-through Couette device, it can find that an increase in shear stress led to a decrease in the adhesion ability of platelets on vWF, while the binding ability of vWF with platelets first increase and then decrease as shear stress elevates gradually beyond a threshold. The factor of exposure time can enhance the effect of shear stress. Additionally, the shear-induced bleeding risk predicted by our model increases with increasing shear stress and exposure time in an axial flow-through Couette device. As indicated by our numerical model, the bleeding risk in HVAD was higher than HMII, which is highly consistent with the meta-analysis based on clinical statistics. Our simulation investigations in these two clinical VADs also found that HVAD caused a higher rate of platelet receptor shedding and lower damage to HWMW-vWF than HeartMate II. The high shear stress generated in the narrow and turbulent regions of both VADs was the underlying cause of device-induced bleeding. ConclusionIn this study, the shear-induced bleeding risk predicted by our bleeding model in axial flow-through Couette device and two clinical VADs is consistent or highly correlated with experimental and clinical findings, which proves the accuracy of our bleeding model. Our bleeding model can be used to aid the development of new BCMDs with improved functional characteristics and biocompatibility, and help to reduce risk of device-induced adverse events in patients.

Role of thrombin to non-physiological shear stress induced platelet activation and function alternation

ObjectiveNon-physiological shear stress (NPSS) and thrombin have two distinct mechanisms for activating platelets. NPSS in mechanically assisted circulation (MAC) devices can cause platelet dysfunction, e.g., by shedding its key receptors. In addition, patients with heart failure have increased levels of thrombin generation, which may further affect the NPSS-induced platelet dysfunction, resulting in device-associated complications. This study aimed to assess the combined effect of NPSS and thrombin in platelet activation, expression of adhesion receptors on the platelet surface, and alterations of platelet aggregation. MethodsFresh human blood from healthy donors was divided into two groups; one group was treated by adding 0.01 U/mL thrombin, and another group not treated with thrombin served as a control comparison. They were then pumped through a novel blood shearing device which produces similar shear stress conditions to those in the MAC devices. Three levels of NPSS (i.e., 75, 125, and 175 Pa) with a 1.0 s exposure time were selected for the shearing conditions. Expression of platelet activation markers (PAC-1, activated GPIIb/IIIa and CD62P, platelet surface P-selectin) were investigated along with the shedding of platelet receptors (GPIb, GPIIb/IIIa, and GPVI), generation of platelet microparticles, and Phosphatidylserine (PS)-positive platelets detected by flow cytometry. Platelet aggregation (induced by collagen/ristocetin) was measured by Lumi-aggregometry. ResultsPlatelet receptors were shed after exposure to NPSS showing a positive correlation with the level of shear stress. The generation of platelet microparticles and PS-positive platelets also increased with greater NPSS. Elevated NPSS decreased the platelet aggregation capacity. Platelet activation level increased with greater NPSS. Being treated by thrombin can further exacerbate these characteristics under same level of NPSS, except that platelet activation level drastically dropped after the exposure to 175 Pa NPSS in the thrombin-treated blood. ConclusionAfter being treated by thrombin, platelets became more susceptible to NPSS, resulting in more receptor shedding, platelet microparticles, and PS-positive platelets, thus limiting platelet aggregation capacity after exposure to NPSS. Platelet activation, in terms of PAC-1 and P-selectin, is an interim status competing between the expression and shedding of these makers/receptors. When platelets have reached a saturation level of activation, exposure to excessive NPSS can potentially impair activation.

Device-Induced Hemostatic Disorders in Mechanically Assisted Circulation

Mechanically assisted circulation (MAC) sustains the blood circulation in the body of a patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) or on ventricular assistance with a ventricular assist device (VAD) or on extracorporeal membrane oxygenation (ECMO) with a pump-oxygenator system. While MAC provides short-term (days to weeks) support and long-term (months to years) for the heart and/or lungs, the blood is inevitably exposed to non-physiological shear stress (NPSS) due to mechanical pumping action and in contact with artificial surfaces. NPSS is well known to cause blood damage and functional alterations of blood cells. In this review, we discussed shear-induced platelet adhesion, platelet aggregation, platelet receptor shedding, and platelet apoptosis, shear-induced acquired von Willebrand syndrome (AVWS), shear-induced hemolysis and microparticle formation during MAC. These alterations are associated with perioperative bleeding and thrombotic events, morbidity and mortality, and quality of life in MCS patients. Understanding the mechanism of shear-induce hemostatic disorders will help us develop low-shear-stress devices and select more effective treatments for better clinical outcomes.

Loss of GPVI and GPIbα contributes to trauma-induced platelet dysfunction in severely injured patients

AbstractTrauma-induced coagulopathy (TIC) is a complex, multifactorial failure of hemostasis that occurs in 25% of severely injured patients and results in a fourfold higher mortality. However, the role of platelets in this state remains poorly understood. We set out to identify molecular changes that may underpin platelet dysfunction after major injury and to determine how they relate to coagulopathy and outcome. We performed a range of hemostatic and platelet-specific studies in blood samples obtained from critically injured patients within 2 hours of injury and collected prospective data on patient characteristics and clinical outcomes. We observed that, although platelet counts were preserved above critical levels, circulating platelets sampled from trauma patients exhibited a profoundly reduced response to both collagen and the selective glycoprotein VI (GPVI) agonist collagen-related peptide, compared with those from healthy volunteers. These responses correlated closely with overall clot strength and mortality. Surface expression of the collagen receptors GPIbα and GPVI was reduced on circulating platelets in trauma patients, with increased levels of the shed ectodomain fragment of GPVI detectable in plasma. Levels of shed GPVI were highest in patients with more severe injuries and TIC. Collectively, these observations demonstrate that platelets experience a loss of GPVI and GPIbα after severe injury and translate into a reduction in the responsiveness of platelets during active hemorrhage. In turn, they are associated with reduced hemostatic competence and increased mortality. Targeting proteolytic shedding of platelet receptors is a potential therapeutic strategy for maintaining hemostatic competence in bleeding and improving the efficacy of platelet transfusions.

Modeling Clot Formation of Shear-Injured Platelets in Flow by a Dissipative Particle Dynamics Method.

The regions with high non-physiological shear stresses (NPSS) are inevitable in blood-contacting medical devices (BCMDs) used for mechanically assisted circulatory support. NPSS can cause platelet activation and receptor shedding potentially resulting in the alteration of hemostatic function. In this study, we developed a dissipative particle dynamics model to characterize clot formation (platelet-collagen and inter-platelet adhesion) of NPSS-traumatized blood at a vascular injury site. A rectangular tube of 50 × 50 × 200µm with an 8 × 8µm collagen-coated area was modeled as a small blood vessel and perfusion with blood. Clot formation dynamics during perfusion was simulated. NPSS-traumatized blood was modeled to have more activated platelet and fewer adhesion receptors with weakened inter-platelet binding. Computational results showed that clots grew at a faster rate while the structure of the clots was less stable and collapsed more frequently for NPSS-traumatized blood compared with normal blood. The finding that NPSS-traumatized platelets could result in quicker but more easily breakable blood clots at injury sites may explain why increased risks of thrombotic and bleeding complications occurred concurrently in patients implanted with BCMDs.

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 Patients after Left Ventricular Assist Device Implantation

Non-surgical bleeding (NSB) is one of the major clinical complications for expanding usage of left ventricular assist devices (LVAD) in patients with end-stage heart failure. It has been reported that non-physiological shear stress caused by LVADs could induce platelet receptor shedding which leads to bleeding disorders caused by dysfunctions in the coagulation system. Therefore, we focused on the characterization of platelets to compare patients with bleeding complications and patients without coagulation disorder after LVAD implantation. Blood samples were obtained from LVAD patients with postoperative NSB (bleeder group, n=20) and compared to LVAD patients without NSB (non-bleeder group, n=20). Platelet receptors (platelet endothelial cell adhesion molecule-1, protease-activated receptor-1, GPIbα), platelet activation (P-selectin, CD63 and GPIIb/IIIa) and platelet reactive oxygen species (ROS) production was quantified by flow cytometry. Aggregation capacity induced by adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP) was evaluated by aggregometry. The surface expression level of GPIIb/IIIa, P-selectin and GPIbα on platelets was decreased in bleeders (GPIIb/IIIa: 32662 ± 1316 U; P-selectin: 475 ± 69 U; GPIbα: 393 ± 44 U) compared to non-bleeders (GPIIb/IIIa: 37247 ± 1599 U, p = 0.03; P-selectin: 845 ± 116 U, p = 0.09; GPIbα: 538 ± 53 U, p=0.04). Additionally, the mean fluorescence intensity of both ADP-activated P-selectin and CD31 expressing platelets was reduced in bleeders (P-selectin: 970 ± 81 U; CD31: 6889 ± 371 U) compared to non-bleeders (P-selectin: 1311 ± 130 U, p = 0.03; CD31: 8558 ± 664 U, p = 0.03). Bleeders showed a higher content of ROS formation in platelets (88.7 ± 2.5%) than non-bleeders (81.5 ± 2.1%, p = 0.03). The findings of this study suggested that changes in platelet receptor pattern of three different platelet receptors (GPIIb/IIIa, P-selectin and GPIbα) and elevated oxidative stress levels may play a role in patients with bleeding complications following LVAD implantation. These results might help to understand the altered hemostatic function as an important cause for NSB in patients on LVAD support.

Ibrutinib, but not zanubrutinib, induces platelet receptor shedding of GPIb-IX-V complex and integrin αIIbβ3 in mice and humans

AbstractThe Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has proven to be efficacious in the treatment of B-cell chronic lymphocytic leukemia (B-CLL) and related diseases. However, a major adverse side effect of ibrutinib is bleeding, including major hemorrhages. The bleeding associated with ibrutinib use is thought to be due to a combination of on-target irreversible Btk inhibition, as well as off-target inhibition of other kinases, including EGFR, ITK, JAK3, and Tec kinase. In this study, we investigated the effects of ibrutinib vs zanubrutinib (a more selective Btk inhibitor) on platelet activation, glycoprotein expression, and thrombus formation. Ibrutinib, but not zanubrutinib, induced a time- and dose-dependent shedding of GPIb-IX complex and integrin αIIbβ3, but not of GPVI and GPV, from the platelet surface. The shedding of GPIbα and GPIX was blocked by GM6001 and TAPI-2, an ADAM17 inhibitor but not ADAM10 inhibitor. Ibrutinib but not zanubrutinib treatment of human platelets increased ADAM17 activation. Pretreatment of C57BL/6 mice with ibrutinib (10 mg/kg), but not zanubrutinib (10 mg/kg), inhibited ex vivo and in vivo thrombus growth over time. Platelets from ibrutinib-treated patients with CLL showed reduced GPIb-IX complex and integrin αIIbβ3 surface expression and reduced ex vivo thrombus formation under arterial flow, which was not observed in zanubrutinib-treated patients. In mice, ibrutinib, but not zanubrutinib, led to increased soluble GPIbα and soluble αIIb levels in plasma. These data demonstrate that ibrutinib induces shedding of GPIbα and GPIX by an ADAM17-dependent mechanism and integrin αIIbβ3 by an unknown sheddase, and this process occurs in vivo to regulate thrombus formation.

The impact of shear stress on device-induced platelet hemostatic dysfunction relevant to thrombosis and bleeding in mechanically assisted circulation.

The aim of this study was to examine the impact of the nonphysiological shear stress (NPSS) on platelet hemostatic function relevant to thrombosis and bleeding in mechanically assisted circulation. Fresh human blood was circulated for four hours in in vitro circulatory flow loops with a CentriMag blood pump operated under a flow rate of 4.5L/min against three pressure heads (70 mmHg, 150 mmHg, and 350 mmHg) at 2100, 2800, and 4000 rpm, respectively. Hourly blood samples from the CentriMag pump-assisted circulation loops were collected and analyzed for glycoprotein (GP) IIb/IIIa activation and receptor shedding of GPVI and GPIbα on the platelet surface with flow cytometry. Adhesion of platelets to fibrinogen, collagen, and von Willebrand factor (VWF) of the collected blood samples was quantified with fluorescent microscopy. In parallel, mechanical shear stress fields within the CentriMag pump operated under the three conditions were assessed by computational fluid dynamics (CFD) analysis. The experimental results showed that levels of platelet GPIIb/IIIa activation and platelet receptor shedding (GPVI and GPIbα) in the blood increased with increasing the circulation time. The levels of platelet activation and loss of platelet receptors GPVI and GPIbα were consistently higher with higher pressure heads at each increasing hour in the CentriMag pump-assisted circulation. The platelet adhesion on fibrinogen increased with increasing the circulation time for all three CentriMag operating conditions and was correlated well with the level of platelet activation. In contrast, the platelet adhesion on collagen and VWF decreased with increasing the circulation time under all the three conditions and was correlated well with the loss of the receptors GPVI and GPIbα on the platelet surface, respectively. The CFD results showed that levels of shear stresses inside the CentriMag pump under all three operating conditions exceeded the maximum level of shear stress in the normal physiological circulation and were strongly dependent on the pump operating condition. The level of platelet activation and loss of key platelet adhesion receptors (GPVI and GPIbα) were correlated with the level of NPSS generated by the CentriMag pump, respectively. In summary, the level of NPSS associated with pump operating condition is a critical determinant of platelet dysfunction in mechanically assisted circulation.

Quantitative Characterization of Shear-Induced Platelet Receptor Shedding: Glycoprotein Ibα, Glycoprotein VI, and Glycoprotein IIb/IIIa.

The structural integrity of platelet receptors is essential for platelets to play the normal hemostatic function. The high non-physiologic shear stress (NPSS) commonly exists in blood-contacting medical devices and has been shown to cause platelet receptor shedding. The loss of platelet receptors may impair the normal hemostatic function of platelets. The aim of this study was to quantify NPSS-induced shedding of three key receptors on the platelet surface. Human blood was subjected to the matrix of well-defined shear stresses and exposure times, generated by using a custom-designed blood-shearing device. The expression of three key platelet receptors, glycoprotein (GP) Ibα, GPVI, and GPIIb/IIIa, in sheared blood was quantified using flow cytometry. The quantitative relationship between the loss of each of the three receptors on the platelet surface and shear condition (shear stress level and exposure time) was explored. It was found that these relationships followed well the power law functional form. The coefficients of the power law models for the shear-induced shedding of these platelet receptors were derived with coefficients of determination (R) of 0.77, 0.73, and 0.78, respectively. The power law models with these coefficients may be potentially used to predict the shear-induced platelet receptor shedding of human blood.

Abstract 532: Shear-induced Release of Platelet Receptors Contributes to Bleeding Outcomes in Patients With Lvads or Exposed to Ecmo

Despite advances in design and materials, as well as pharmacological prophylaxis, hemostatic complications continue to plague device recipients. Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. We examined blood samples from patients with heart failure or in receipt of high fluid shear mechanocirculatory support to assess whether loss of platelet receptors occurs in vivo , and the relationship with acquired von Willebrand syndrome (AVWS) and changes in inflammatory cytokines. Platelet counts, levels of inflammatory cytokines IL-1β, IL-6, TNFα, MCP-1, IL-17A and IFNγ, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 13 continuous flow VAD (CF-VAD), 14 ECMO, and 24 heart failure patients. Levels of platelet receptors were measured by flow cytometry or ELISA. The loss of high molecular weight VWF multimers was observed in 12 of 13 CF-VAD and 7 of 14 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Significant differences in levels of inflammatory cytokines monocyte-chemoattractant protein and tissue necrosis factor-a in a subset of patients with decompensated HF. These data link AVWS and increased platelet receptor shedding in patients with CFVADs or ECMO. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CFVAD and ECMO patients may be linked with extent of inflammation and may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions.

Intraplatelet reactive oxygen species (ROS) correlate with the shedding of adhesive receptors, microvesiculation and platelet adhesion to collagen during storage: Does endogenous ROS generation downregulate platelet adhesive function?

Platelets storage lesion is mainly orchestrated by platelet activating signals during storage. Reactive oxygen species (ROS) are being considered as important signaling molecules modulating platelet function while their production has also been shown to be augmented by platelet activation. This study investigated to what extent endogenous ROS generation during platelet storage could be correlated with platelet receptor shedding, microvesiculation and adhesive function.10 PRP-platelet concentrates were subjected to flow cytometry analysis to examine the generation of intraplatelet ROS on days 1, 5 and 7 after storage. In 5 day-stored platelets considering 40% of ROS generation as a cutoff point, samples were divided into two groups of those with higher or lower levels of ROS. The expression of adhesion receptors (GPVI, GPIbα), the amount of microparticles and phosphatidylserine exposure in each group were then examined by flow cytometry. Platelet receptor shedding and adhesion to collagen matrix were respectively measured by western blotting and microscopic assays.Our data showed lowered expression of GPIbα (p < 0.05) and GPVI in samples with ROS > 40% than those with ROS ≤ 40%, whereas receptors shedding and microvesiculation were (p < 0.05) elevated in platelets with higher levels of ROS. Functionally, we observed significantly (p < 0.05) lower levels of platelet adhesion to collagen matrix in samples with ROS generation more than 40%.Taken together, we showed correlations between intraplatelet ROS generation and either platelet receptors or microparticle shedding as well as platelet adhesive capacity to collagen. These findings suggest that augmented ROS generation during storage might be relevant to down-regulation of platelet adhesive function.

High shear induces platelet dysfunction leading to enhanced thrombotic propensity and diminished hemostatic capacity

Thrombosis and bleeding are devastating adverse events in patients supported with blood-contacting medical devices (BCMDs). In this study, we delineated that high non-physiological shear stress (NPSS) caused platelet dysfunction that may contribute to both thrombosis and bleeding. Human blood was subjected to NPSS with short exposure time. Levels of platelet surface GPIbα and GPVI receptors as well as activation level of GPIIb/IIIa in NPSS-sheared blood were examined with flow cytometry. Adhesion of sheared platelets on fibrinogen, von Willibrand factor (VWF), and collagen was quantified with fluorescent microscopy. Ristocetin- and collagen-induced platelet aggregation was characterized by aggregometry. NPSS activated platelets in a shear and exposure time-dependent manner. The number of activated platelets increased with increasing levels of NPSS and exposure time, which corresponded well with increased adhesion of sheared platelets on fibrinogen. Concurrently, NPSS caused shedding of GPIbα and GPVI in a manner dependent on shear and exposure time. The loss of intact GPIbα and GPVI increased with increasing levels of NPSS and exposure time. The number of platelets adhered on VWF and collagen decreased with increasing levels of NPSS and exposure time, respectively. The decrease in the number of platelets adhered on VWF and collagen corresponded well with the loss in GPIbα and GPVI on platelet surface. Both ristocetin- and collagen-induced platelet aggregation in sheared blood decreased with increasing levels of NPSS and exposure time. The study clearly demonstrated that high NPSS causes simultaneous platelet activation and receptor shedding, resulting in a paradoxical effect on platelet function via two distinct mechanisms. The results from the study suggested that the NPSS could induce the concurrent propensity for both thrombosis and bleeding in patients.

Constricted microfluidic devices to study the effects of transient high shear exposure on platelets.

Due to the critical roles that platelets play in thrombosis during many biological and pathological events, altered platelet function may be a key contributor to altered hemostasis, leading to both thrombotic and hemorrhagic complications. Platelet adhesion at arterial shear rates occurs through binding to von Willebrand Factor via the glycoprotein (GP) GPIb receptor. GPIb binding can induce platelet activation distinguishable by P-selectin (CD62P) surface expression and αIIbβ3 activation, resulting in platelet aggregation and formation of the primary hemostatic plug to stop bleeding. Previous studies have used cone and plate viscometers to examine pathologic blood flow conditions, applied shear rates that are relatively low, and examined exposure times that are orders of magnitude longer compared to conditions present in ventricular assist devices, mechanical heart valves, or pathologic states such as stenotic arteries. Here, we evaluate the effect of short exposure to high shear on granule release and receptor shedding utilizing a constricted microfluidic device in conjunction with flow cytometry and enzyme-linked immunosorbent assay. In this study, platelets were first perfused through microfluidic channels capable of producing shear rates of 80 000-100 000 s-1 for exposure times of 0-73 ms. We investigated platelet activation by measuring the expression level of CD62P (soluble and surface expressed), platelet factor 4 (PF4), and beta-thromboglobulin (βTG). In addition, we measured potential platelet receptor shedding of GPVI and GPIb using flow cytometry. The results showed that a single pass to high shear with short exposure times (milliseconds) had no effect on the levels of CD62P, GPVI and GPIb, or on the release of alpha granule content (PF4, βTG, and sP-selectin).

Mechanical circulatory support is associated with loss of platelet receptors glycoprotein Ibα and glycoprotein VI

Background Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. Objectives To investigate whether loss of platelet receptors occurs in vivo, and the relationship with acquired von Willebrand syndrome (AVWS). Methods Platelet counts, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 21 continuous flow VAD (CF-VAD), 20 ECMO, 12 heart failure and seven aortic stenosis patients. Levels of platelet receptors were measured by flow cytometry or ELISA. Results The loss of high molecular weight VWF multimers was observed in 18 of 19 CF-VAD and 14 of 20 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Conclusions These data link AVWS and increased platelet receptor shedding in patients with CF-VADs or ECMO for the first time. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CF-VAD and ECMO patients may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions.

GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion

ABSTRACTPlatelet storage is associated with deleterious changes leading to the loss of platelet reactivity and response. During storage, platelets experience increased expression and shedding of P-selectin and CD40L as specific markers of platelet activation, whereas GPIbα decreases due to ectodomain shedding. As an important adhesive receptor, GPVI contributes significantly to thrombus formation while its expression and shedding levels during storage of platelet products have not been well characterized yet. This study investigated the modulation of GPVI during platelet storage.For this study, samples obtained from 10 PRP-platelet concentrates (PCs) were subjected to flow-cytometry analysis to examine the expression of platelet activation markers and GPVI on days 1, 3, and 5 post-storage. To examine the levels of etcodomain shedding of these molecules, microparticle (MP)-free supernatants were also analyzed by either ELISA or Western blot methods.According to results, the expression levels of P-selectin and CD40L as well as the amounts of their soluble forms significantly increased during storage. The expression of GPIbα and GPVI decreased whereas their shedding significantly increased post-storage. The expression and shedding levels of these two receptors were significantly correlated. Negative correlations between the expressions of GPIbα or GPVI and P-selectin have been observed whereas their shedding levels were significantly relevant together. In a control study, the use of biotinylated platelet resuspended in Tyrode’s buffer in the presence of ionophore with/without EDTA, confirmed the role of calcium in receptors shedding. In citrated PRP-PCs, recalcification of platelets also enhanced shedding levels of both GPIbα and GPVI. Intriguingly, the shedding levels of GPVI in stored PRP-PCs were much higher than those of ionophore-treated controls obtained from washed platelets. The ratios of sGPVI in stored platelet to ionophore-treated controls were also at least six times higher than those of GPIbα during storage. In conclusion, here we showed significant decreases of GPVI expression associated with its increasing levels of shedding during storage, suggesting GPVI as a valid marker of platelet storage lesion. Importantly, we found higher levels of GPVI shedding in stored platelets than those of ionophore-treated non-stored control samples. This suggests whereas platelet receptor shedding is mainly modulated by calcium-dependent signals, either platelet–surface interactions with the container walls during storage or induced shear stress under long-term agitation, might be also involved in the excessive shedding of GPVI during the storage of PCs.

Bleeding and thrombosis in chronic ventricular assist device therapy: focus on platelets.

Left ventricular assist devices (LVADs) have markedly improved the survival for patients with advanced heart failure but are plagued with significant morbidity, including pump thrombosis and bleeding. Better understanding of the platelet, and its role in the balance of bleeding and thrombosis, stands to impact the frequency and treatment of these significant complications. In patients with LVADs, there is little consistency linking traditional biomarkers of platelet activation and clinical events. A number of innovative methods of assessing platelet functionality, including shedding of platelet receptors and formation of microparticle complexes as well as measuring mitochondrial membrane potentials, exist and appear to be clinically relevant. Acquired von Willebrand syndrome, while not explaining all bleeding events, is a central feature of mechanical support and offers a target for innovative therapies. Although the platelet is only one component of impacting thrombosis and bleeding in patients supported with LVADs, it plays a central role in mediating these two opposing forces. Innovations in understanding platelet physiology as well as manipulating genomic and receptor interactions for an individual patient will be critical if we are to decrease these serious adverse events in the future.