Impact of rise and fall phases of shear on platelet activation and aggregation using microfluidics.

Blood flow disorders are often the result of the non-physiological narrowing of blood arteries caused by atherosclerosis and thrombus. The blood then proceeds through rising-peak-decreasing phases as it passes through the narrow area. Although abnormally high shear is known to activate platelets, the shear process that platelets undergo in small arteries is complex. Thus, understanding how each shear phase affects platelet activation can be used to improve antiplatelet therapy and decrease the risk of side effects like bleeding. Blood samples were sheared (68.8 ms,5200 s-1) in vitro by the microfluidic technique, and platelet activation levels (P-selectin and integrin αIIbß3) and von Willebrand factor (vWF) binding to platelets were analyzed by flow cytometry. Post-stenosis platelet aggregation was dynamically detected using microfluidic technology. We studied TXA2, P2Y12-ADP, and integrin αIIbß3-fibrinogen receptor pathways by adding antiplatelet drugs, such as acetylsalicylic acid (ASA, an active ingredient of aspirin that inhibits platelet metabolism), ticagrelor (hinders platelet activation), and tirofiban (blocks integrin αIIbß3 receptor) in vitro, respectively, to determine platelet activation function mediated by transient non-physiological high shear rates. We demonstrated that platelets can be activated under transient pathological high shear rates. The shear rise and fall phases influenced shear-induced platelet activation by regulating the binding of vWF to platelets. The degree of platelet activation and aggregation increased with multiple shear rise and fall phases. ASA did not inhibit shear-mediated platelet activation, but ticagrelor and tirofiban effectively inhibited shear-mediated platelet activation. Our data demonstrated that the shear rise and fall phases play an important role in shear-mediated platelet activation and promote platelet activation and aggregation in a vWF-dependent manner. Blocking integrin αIIbß3 receptor and hindering P2Y12-ADP were beneficial to reducing shear-mediated platelet activation.

Evaluating the impact of transient shear stress on platelet activation, adhesion, and aggregation with microfluidic chip technique.

BACKGROUND: When nonphysiological stenosis occurs, the transient high shear stress formed in vessels increases the risk of thrombosis and is a potential factor for cardiovascular diseases. But the platelet adhesion and aggregation behavior at nonphysiological post-stenosis and its affecting factors are not fully understood yet. METHODS: In this experiment, platelet aggregation on collagen and fibrinogen at different shear stresses and different hematocrits were observed by microfluidic technology. Platelet activation (P-selectin, glycoprotein IIb/IIIa) and monocyte-platelet aggregate (MPA) levels under different shear stresses were analyzed by flow cytometry. RESULTS: On fibrinogen, platelets aggregate more at higher shear stress conditions. While on collagen, it becomes more difficult for platelets to form stable aggregation at higher shear stress conditions. If platelets adhere initially at low shear stress, stable platelet aggregation can be formed at subsequent high shear stress. Moreover, when the shear stress increases, platelet activity markers (P-selectin, glycoprotein IIb/IIIa and MPAs) increase significantly. Hematocrit affects the degree of platelet aggregation, and the influence of hematocrit is obvious at high shear stress. CONCLUSION: Transient high shear stress (46 ms) can effectively activate platelets. Platelet aggregation behavior was different for coated fibrinogen and collagen protein. Stable platelet adhesion at post-stenosis is more dependent on fibrinogen and platelet aggregation is stable on both fibrinogen and collagen. Hematocrit can significantly affect the formation of platelet aggregation.

The rapid change of shear rate gradient is beneficial to platelet activation.

Fluid shear plays a key role in hemostasis and thrombosis, and the purpose of this study was to investigate the effect of shear gradient change rate (SGCR) on platelet reactivity and von Willebrand factor (vWF) activity and its mechanism. In this study, we developed a set of microfluidic chips capable of generating different shear gradients and simulated the shear rate distribution in the flow field by COMSOL Multiphysics software. Molecular markers of platelet activation (P-selectin, activated GPIIb/IIIa, phosphatidylserine exposure, and monocyte-platelet aggregate formation) were analyzed by flow cytometry. Platelet aggregation induced by shear gradient was studied by a microfluidic experimental platform, and plasma vWF ristocetin cofactor (vWF: RCO) activity was investigated by flow cytometry. The expression of p-Akt was studied by Western blotting. The results showed that the faster the SGCR, the higher the expression of platelet p-Akt, and the stronger the platelet reactivity and vWF activity. This indicates that fluid shear stress can activate platelets and vWF in a shear gradient-dependent manner through the PI3K/AKT signal pathway, and the faster the SGCR, the more significant the activation effect.

What is the context? Recent studies have shown that fluid shear stress plays a key role in platelet activation and thrombosis. However, its mechanism and effect have not been fully elucidated.The development of microfluidic chip technology enables people to study platelet function in a precisely controlled flow field environment.Previous studies have shown that the PI3K-AKT signal pathway may be a mechanically sensitive signal transduction pathway.What is new?In this study, we designed a microfluidic model with different narrow geometry, and controlled the injection pump to perfuse fluid at the same flow rate, so that the platelets flowing through the model experienced the flow field environment of different shear gradients.We studied the activities of platelets and von Willebrand factor in different flow fields and explored their signal transduction pathways.What is the impact? Our results suggest that vascular stenosis does increase platelet activity and the risk of thrombosis. However, its ability to activate platelets is not only related to the peak shear rate and shear time, but also closely related to the decreasing rate of shear gradient. Even if the peak shear rate at the stenosis is the same, the faster the shear rate decreases, the higher the reactivity of platelets and von Willebrand factor, which may be mediated by the PI3K-AKT signal pathway. This study not only helps clinicians to judge the risk of thrombosis in patients with atherosclerosis or percutaneous coronary intervention, but also helps us to better understand the mechanism of shear-induced platelet activation.

[Microfluidic Chip and Flow Cytometry for Examination of the Antiplatelet Effect of Ticagrelor].

Objective To examine the antiplatelet effect of ticagrelor by microfluidic chip and flow cytometry under shear stress in vitro. Methods Microfluidic chip was used to examine the effect of ticagrelor on platelet aggregation at the shear rates of 300/s and 1500/s.We adopted the surface coverage of platelet aggregation to calculate the half inhibition rate of ticagrelor.The inhibitory effect of ticagrelor on ADP-induced platelet aggregation was verified by optical turbidimetry.Microfluidic chip was used to construct an in vitro vascular stenosis model,with which the platelet reactivity under high shear rate was determined.Furthermore,the effect of ticagrelor on the expression of fibrinogen receptor (PAC-1) and P-selectin (CD62P) on platelet membrane activated by high shear rate was analyzed by flow cytometry. Results At the shear rates of 300/s and 1500/s,ticagrelor inhibited platelet aggregation in a concentration-dependent manner,and the inhibition at 300/s was stronger than that at 1500/s (both P<0.001).Ticagrelor at a concentration ≥4 µmol/L almost completely inhibited platelet aggregation.The inhibition of ADP-induced platelet aggregation by ticagrelor was similar to the results under flow conditions and also in a concentration-dependent manner.Ticagrelor inhibited the expression of PAC-1 and CD62P. Conclusion We employed microfluidic chip to analyze platelet aggregation and flow cytometry to detect platelet activation,which can reveal the responses of different patients to ticagrelor.

Effect of pathological high shear exposure time on platelet activation and aggregation.

Circulating platelets are sometimes exposed to high shear rate environments due to vascular stenosis, and the effect of transiently elevated pathological high shear rates on platelet activation and aggregation function has not been clarified. The aim of this study was to investigate the effect of pathological high shear rate (8302s-1) exposure time (3.16-25.3 ms) on platelet activation and aggregation function. In addition, by adding active ingredients of antiplatelet drugs such as ASA (an active ingredient of aspirin), Ticagrelor, Tirofiban and GP1BA (platelet membrane protein GPIb inhibitor) in vitro, we studied TXA2, P2Y12-ADP, GPIIb/IIIa-fibrinogen and GPIb /IX/V-vWF receptor pathways to determine platelet activation function mediated by pathological high shear rate. In this study, we designed a set of microfluidic chips with stenosis lengths of 0.5 mm, 1 mm, 2 mm, 3 mm, and 4 mm, all with 80% stenosis, to generate pathological high shear forces that can act at different times. The whole blood flowing through the microchannels was collected by perfusion of sodium citrate anticoagulated whole blood at a physiological arterial shear rate (1500 s-1), and the expression levels of platelet surface activation markers (P-selectin and GP IIb/IIIa) and the degree of platelet aggregation were analyzed by flow cytometry; platelet aggregation patterns were observed by microscopic examination of blood smears. The results showed that shearing significantly increased platelet activation and aggregation levels compared to un-sheared whole blood, and the activation and aggregation levels increased with increasing duration of pathological high shear rate. In vitro inhibition studies showed that ASA barely inhibited the expression of P-selectin and PAC-1 on the platelet surface; Ticagrelor effectively inhibited the expression of both P-selectin and PAC-1; Tirofiban significantly inhibited the expression of PAC-1 on the platelet surface and slightly inhibited the expression of P-selectin; GP1BA significantly inhibited the expression of both. Our results suggest that transient pathological high shear rate (8302s-1) exposure can induce platelet activation in a time-dependent manner; however, the mechanism is more complex and may be due to the following reasons: transient elevated pathological high shear rate activates platelets through the GPIb/IX/V-vWF receptor pathway, and after platelet activation, its surface membrane protein GPIIb/IIIa receptors activate platelets through fibrinogen to form platelet-platelet aggregates, and further activation of active substances such as ADP and TXA2 released by platelet alpha particles, which contribute to the formation of irreversible platelet aggregation.

Novel Antiplatelet Activity of Ginsenoside Re Through the Inhibition of High Shear Stress-Induced Platelet Aggregation.

ABSTRACT: Bleeding is one of the most serious side effects of antiplatelet drugs. Efforts have been made to find new antiplatelet agents without bleeding complications. Shear-induced platelet aggregation (SIPA) occurs only under pathological conditions and is a promising target for overcoming bleeding problems. This work demonstrates that the ginsenoside Re selectively inhibits platelet aggregation induced by high shear stress. Human platelets were exposed to high shear stress using microfluidic chip technology, and aggregation, activation, and phosphatidylserine (PS) exposure were measured. The Von Willebrand Ristocetin Cofactor (vWF:RCo) assay and western blot were used to evaluate the effect of the vWF-GPⅠb/PI3K/Akt signal pathway. The coagulation and bleeding risk were evaluated by measuring the coagulation parameters PT, APTT, TT, and thromboelastography. The 3-dimensional morphology of platelet aggregates was observed by a microscopic 3-dimensional imaging. Re was a potent inhibitor of SIPA, with an IC 50 of 0.071 mg/mL. It effectively blocked shear stress-induced platelet activation without any significant toxicity. It was highly selective against SIPA, effectively inhibiting vWF-GPIb and the downstream PI3K/Akt signaling pathway. Most importantly, Re did not affect normal blood coagulation and did not increase the risk of bleeding. In conclusion, Re inhibits platelet activation through the inhibition of the vWF-GPIb/PI3K/Akt pathway. Thus, it might be considered as a new antiplatelet drug in the prevention of thrombosis without increasing the risk of bleeding.

Application of microfluidic chip technology to study the inhibitory effect of tetramethylpyrazine on platelet aggregation, activation, and phosphatidylserine exposure mediated by pathological high shear rate.

OBJECTIVE: In order to study the antithrombotic effect and mechanism of tetramethylpyrazine (TMA). METHODS: In this study, we developed a microfluidic chip model that can mimic normal arteries and stenotic arterial vessels, and studied the inhibitory effects of TMA on platelet aggregation, activation (P-selectin, GPIIb/IIIa, monocyte-platelet aggregates) and phosphatidyl serine (PS) exposure. In addition, we also investigated the effect of TMA on ADP and ristocetin-induced platelet aggregation by turbidimetry. RESULTS: The results showed that TMA significantly inhibited the platelet aggregation, activation and PS exposure induced by pathological high shear rate. Under static conditions, TMA can inhibit ADP and ristocetin-induced platelet aggregation. CONCLUSION: The results indicated that TMA mainly inhibited platelet aggregation, activation and PS exposure by inhibiting the binding of von Willebrand factor (vWF) to the GPIb/IX/V complex, and partially inhibited platelet aggregation through the platelet P2Y 12 -ADP receptor pathway.