Desmopressin, Misoprostol, nor Carboprost Affect Platelet Aggregability Following Traumatic Brain Injury and Aspirin.

INTRODUCTION: Desmopressin (DDAVP) has been utilized clinically in patients taking aspirin (ASA) to improve drug-induced platelet dysfunction. Misoprostol and carboprost, prostaglandin analogs commonly used for postpartum hemorrhage, may also induce platelet aggregation. The aim of this study was to determine the effects of DDAVP, misoprostol, and carboprost administration on platelet aggregability following traumatic brain injury (TBI) in mice treated with ASA. METHODS: Male C57BL/6 mice were randomized into seven groups (n = 5 each): untouched, ASA only, Saline/TBI, ASA/TBI, ASA/TBI/DDAVP 0.4 µg/kg, ASA/TBI/misoprostol 1 mg/kg, and ASA/TBI/carboprost 100 µg/kg. TBI was induced via a weight drop model 4-h after ASA (50 mg/kg) gavage. Mice were given an intraperitoneal injection of DDAVP, misoprostol, or carboprost 10 minutes after TBI. In vivo testing was completed utilizing tail vein bleed. Mice were sacrificed 30-min posttreatment and blood was collected via cardiac puncture. Whole blood was analyzed via Multiplate impedance aggregometry, rotational thromboelastometry, and TEG6s. RESULTS: Mice receiving misoprostol after ASA/TBI demonstrated decreased tail vein bleeding times compared to ASA only treated mice. However, mice treated with misoprostol following ASA and TBI demonstrated decreased platelet aggregability compared to untouched mice and TBI only mice within the arachidonic acid agonist pathway. By contrast, DDAVP and carboprost did not significantly change platelet aggregability via adenosine diphosphate or arachidonic acid following ASA and TBI. However, DDAVP did decrease the platelet contribution to clot via rotational thromboelastometry. CONCLUSIONS: Reversal of medication-induced platelet inhibition has become increasingly controversial after TBI. Based on these results, DDAVP, misoprostol, nor carboprost consistently improve platelet aggregability following TBI in those also treated with ASA.

Genetic and nongenetic drivers of platelet reactivity in healthy Tanzanian individuals.

BACKGROUND: Platelets play a key role in hemostasis, inflammation, and cardiovascular diseases. Platelet reactivity is highly variable between individuals. The drivers of this variability in populations from Sub-Saharan Africa remain largely unknown. OBJECTIVES: We aimed to investigate the nongenetic and genetic determinants of platelet reactivity in healthy adults living in a rapidly urbanizing area in Northern Tanzania. METHODS: Platelet activation and reactivity were measured by platelet P-selectin expression and the binding of fibrinogen in unstimulated blood and after ex vivo stimulation with adenosine diphosphate and PAR-1 and PAR-4 ligands. We then analyzed the associations of platelet parameters with host genetic and nongenetic factors, environmental factors, plasma inflammatory markers, and plasma metabolites. RESULTS: Only a few associations were found between platelet reactivity parameters and plasma inflammatory markers and nongenetic host and environmental factors. In contrast, untargeted plasma metabolomics revealed a large number of associations with food-derived metabolites, including phytochemicals that were previously reported to inhibit platelet reactivity. Genome-wide single-nucleotide polymorphism genotyping identified 2 novel single-nucleotide polymorphisms (rs903650 and rs4789332) that were associated with platelet reactivity at the genome-wide level (P < 5 × 10-8) as well as a number of variants in the PAR4 gene (F2RL3) that were associated with PAR4-induced reactivity. CONCLUSION: Our study uncovered factors that determine variation in platelet reactivity in a population in East Africa that is rapidly transitioning to an urban lifestyle, including the importance of genetic ancestry and the gradual abandoning of the traditional East African diet.

A computational investigation of occlusive arterial thrombosis.

The generation of occlusive thrombi in stenotic arteries involves the rapid deposition of millions of circulating platelets under high shear flow. The process is mediated by the formation of molecular bonds of several distinct types between platelets; the bonds capture the moving platelets and stabilize the growing thrombi under flow. We investigated the mechanisms behind occlusive thrombosis in arteries with a two-phase continuum model. The model explicitly tracks the formation and rupture of the two types of interplatelet bonds, the rates of which are coupled with the local flow conditions. The motion of platelets in the thrombi results from competition between the viscoelastic forces generated by the interplatelet bonds and the fluid drag. Our simulation results indicate that stable occlusive thrombi form only under specific combinations for the ranges of model parameters such as rates of bond formation and rupture, platelet activation time, and number of bonds required for platelet attachment.

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.

Tumor cell-induced platelet aggregation accelerates hematogenous metastasis of malignant melanoma by triggering macrophage recruitment.

BACKGROUND: Tumor cell-induced platelet aggregation (TCIPA) is not only a recognized mechanism for paraneoplastic thrombocytosis but also a potential breakthrough alternative for a low response to immune checkpoint inhibitors (ICIs) in hematogenous metastasis of malignant melanoma (MM). However, there is no TCIPA-specific model for further investigation of the relationship among TCIPA, the tumor immune microenvironment (TIME), and metastasis. METHODS: We developed a TCIPA metastatic melanoma model with advanced hematogenous metastasis and enhanced TCIPA characteristics. We also investigated the pathway for TCIPA in the TIME. RESULTS: We found that TCIPA triggers the recruitment of tumor-associated macrophages (TAMs) to lung metastases by secreting B16 cell-educated platelet-derived chemokines such as CCL2, SDF-1, and IL-1ß. Larger quantities of TAMs in the TCIPA model were polarized to the M2 type by B16 cell reprocessing, and their surface programmed cell death 1 ligand 1 (PD-L1) expression was upregulated, ultimately assisting B16 cells in escaping host immunity and accelerating MM hematogenous metastasis. CONCLUSIONS: TCIPA accelerates MM lung metastasis via tumor-educated platelets (TEPs), triggering TAM recruitment, promoting TAM polarization (M2), and remodeling the suppressive TIME in lung metastases.

Image-based flow simulation of platelet aggregates under different shear rates.

Hemodynamics is crucial for the activation and aggregation of platelets in response to flow-induced shear. In this paper, a novel image-based computational model simulating blood flow through and around platelet aggregates is presented. The microstructure of aggregates was captured by two different modalities of microscopy images of in vitro whole blood perfusion experiments in microfluidic chambers coated with collagen. One set of images captured the geometry of the aggregate outline, while the other employed platelet labelling to infer the internal density. The platelet aggregates were modelled as a porous medium, the permeability of which was calculated with the Kozeny-Carman equation. The computational model was subsequently applied to study hemodynamics inside and around the platelet aggregates. The blood flow velocity, shear stress and kinetic force exerted on the aggregates were investigated and compared under 800 s-1, 1600 s-1 and 4000 s-1 wall shear rates. The advection-diffusion balance of agonist transport inside the platelet aggregates was also evaluated by local Péclet number. The findings show that the transport of agonists is not only affected by the shear rate but also significantly influenced by the microstructure of the aggregates. Moreover, large kinetic forces were found at the transition zone from shell to core of the aggregates, which could contribute to identifying the boundary between the shell and the core. The shear rate and the rate of elongation flow were investigated as well. The results imply that the emerging shapes of aggregates are highly correlated to the shear rate and the rate of elongation. The framework provides a way to incorporate the internal microstructure of the aggregates into the computational model and yields a better understanding of the hemodynamics and physiology of platelet aggregates, hence laying the foundation for predicting aggregation and deformation under different flow conditions.

INVESTIGATION INTO P2Y RECEPTOR FUNCTION IN PLATELETS FROM PATIENTS WITH SEPSIS.

ABSTRACT: Key underlying pathological mechanisms contributing to sepsis are hemostatic dysfunction and overwhelming inflammation. Platelet aggregation is required for hemostasis, and platelets are also separately involved in inflammatory responses that require different functional attributes. Nevertheless, P2Y receptor activation of platelets is required for this dichotomy of function. The aim of this study was to elucidate whether P2YR-dependent hemostatic and inflammatory functions were altered in platelets isolated from sepsis patients, compared with patients with mild sterile inflammation. Platelets from patients undergoing elective cardiac surgery (20 patients, 3 female) or experiencing sepsis after community-acquired pneumonia (10 patients, 4 female) were obtained through the IMMunE dysfunction and Recovery from SEpsis-related critical illness in adults (IMMERSE) Observational Clinical Trial. In vitro aggregation and chemotaxis assays were performed with platelets after stimulation with ADP and compared with platelets isolated from healthy control subjects (7 donors, 5 female). Cardiac surgery and sepsis both induced a robust inflammatory response with increases in circulating neutrophil counts with a trend toward decreased circulating platelet counts being observed. The ability of platelets to aggregate in response to ex vivo ADP stimulation was preserved in all groups. However, platelets isolated from patients with sepsis lost the ability to undergo chemotaxis toward N -formylmethionyl-leucyl-phenylalanine, and this suppression was evident at admission through to and including discharge from hospital. Our results suggest that P2Y 1 -dependent inflammatory function in platelets is lost in patients with sepsis resulting from community-acquired pneumonia. Further studies will need to be undertaken to determine whether this is due to localized recruitment to the lungs of a platelet responsive population or loss of function as a result of dysregulation of the immune response.

In-vitro antiplatelet effect of melatonin in healthy individuals and patients with type 2 diabetes mellitus.

PURPOSE: The incidence of acute myocardial infarctions (AMI) shows circadian variation typically peaking during morning hours with a decline at night. However, this variation does not occur in patients with diabetes mellitus (DM). The night's decline of AMI may be partially explained by melatonin-related platelet inhibition. Whether this effect is absent in diabetic patients is unknown. The aim was to study the effect of melatonin on in-vitro platelet aggregation in healthy individuals and patients with type 2 DM. METHODS: Platelet aggregation was measured in blood samples from healthy individuals (n = 15) and type 2 DM patients (n = 15) using multiple electrode aggregometry. Adenosine diphosphate (ADP), arachidonic acid (ASPI) and thrombin (TRAP) were used as agonists. Aggregability for each subject was tested after adding melatonin in two concentrations. RESULTS: In healthy individuals, melatonin inhibited platelet aggregation in both higher (10-5 M) and lower concentrations (10-9 M) induced by ADP, ASPI, and TRAP (p < 0.001, p = 0.002, p = 0.029, respectively). In DM patients, melatonin did not affect platelet aggregation in both concentrations induced by ADP, ASPI, and TRAP. Melatonin decreased platelet aggregation induced by ADP, ASPI, and TRAP significantly more in healthy individuals compared to patients with DM. (p = 0.005, p = 0.045 and p = 0.048, respectively). CONCLUSION: Platelet aggregation was inhibited by melatonin in healthy individuals. In-vitro antiplatelet effect of melatonin in type 2 DM patients is significantly attenuated.

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.

Platelet Function is Preserved After Moderate Cardiopulmonary Bypass Times But Transiently Impaired After Protamine.

OBJECTIVES: Previous studies have described impaired platelet function after cardiopulmonary bypass (CPB). Whether this is still valid in contemporary cardiac surgery is unclear. This study aimed to quantify changes in function and number of platelets during CPB in a present-day cardiac surgery cohort. DESIGN: Prospective, controlled clinical study. SETTING: A single-center university hospital. PARTICIPANTS: Thirty-nine patients scheduled for coronary artery bypass graft surgery with CPB. INTERVENTIONS: Platelet function and numbers were measured at 6 timepoints in 39 patients during and after coronary artery bypass graft surgery; at baseline before anesthesia, at the end of CPB, after protamine administration, at intensive care unit (ICU) arrival, 3 hours after ICU arrival, and on the morning after surgery. MEASUREMENTS AND MAIN RESULTS: Platelet function was assessed with impedance aggregometry and flow cytometry. Platelet numbers are expressed as actual concentration and as numbers corrected for dilution using hemoglobin as a reference marker. There was no consistent impairment of platelet function during CPB with either impedance aggregometry or flow cytometry. After protamine administration, a decrease in platelet function was seen with impedance aggregometry and for some markers of activation with flow cytometry. Platelet function was restored 3 hours after arrival in the ICU. During CPB (85.0 ± 21 min), the number of circulating platelets corrected for dilution increased from 1.73 ± 0.42 × 109/g to 1.91 ± 0.51 × 109/g (p < 0.001). CONCLUSIONS: During cardiac surgery with moderate CPB times, platelet function was not impaired, and no consumption of circulating platelets could be detected. Administration of protamine transiently affected platelet function.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbß3 integrin.

BACKGROUND: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbß3, thereby compromising platelet binding to fibrinogen and thrombus stability. METHODS: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. RESULTS: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbß3 and sEng and molecular modeling showed a good fitting between αIIbß3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbß3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. CONCLUSIONS: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbß3, suggesting its involvement in primary hemostasis control.

Artificial cerebrospinal fluid restores aspirin-inhibited physiological hemostasis through recovery of platelet aggregation function.

BACKGROUND: Optimal hemostasis provides safety and reliability during neurosurgery which improves surgical outcomes. Previously, artificial cerebrospinal fluid (aCSF) and its component sodium bicarbonate were found to facilitate physiological hemostasis by amplifying platelet aggregation. This study aimed to verify whether aCSF amplifies platelet-dependent hemostasis in the presence of antiplatelet agents. METHODS: We prepared platelet-rich plasma (PRP) or washed platelets using aspirin (acetylsalicylic acid, (ASA)) or normal saline (NS). We evaluated samples treated with a commercially available aCSF solution or NS for amplification of aggregation, activation of integrin αIIbß3, phosphatidylserine (PS) exposure, P-selectin (CD62P) expression, and formation of microparticles (MPs). We assessed the effect of aCSF on in vivo hemostasis in the presence of ASA by measuring the tail bleeding time in ASA-or NS-injected C57BL/6 N mice. RESULTS: Compared with NS, aCSF amplified ASA-inhibited platelet aggregation by recovering platelet activation including PS exposure, MP release, CD62P expression, and integrin αIIbß3 activation. When using washed platelets, aCSF almost completely counteracted the inhibition of platelet aggregation by ASA. Prolonged bleeding time from the amputated tail of ASA-injected mice was significantly shortened by the treatment with aCSF compared to NS. Sodium bicarbonate also directly amplified ASA-inhibited platelet aggregation. CONCLUSIONS: aCSF and sodium bicarbonate facilitate physiological hemostasis through the recovery of inhibited platelet aggregation even in the presence of ASA. The utilization of aCSF in the operative field may be advantageous for facilitating hemostasis in patients with impaired platelet function and contribute to improving outcomes of neurosurgery.

Development of the whole tomato and olive-based food supplement enriched with anti-platelet aggregating nutrients.

BACKGROUND: Platelet dysfunctions are shared by cardiovascular diseases and a wide range of inflammatory diseases. AIMS: To determine the ability of a new whole tomato-based food supplement (WTBFS) containing carotenoid and olive polyphenols to inhibit platelet aggregation. METHODS: Aggregation was evaluated in platelet-rich plasma using microtiter plates and a plate reader. RESULTS: Platelets treated with WTBFS showed a >70% reduction of 5 µM adenosine diphosphate (ADP)-induced platelet aggregation; at 10 µM of ADP, the inhibitory effect of WTBFS was reduced of about 50%. Similarly, 78% and 48% reduction were obtained using 5 µg/mL and 10 µg /mL of collagen as an agonist. CONCLUSION: Since the compounds in WTBFS share the ability to inhibit STAT3, the inhibition of its signaling pathway may represent the mechanism underlying the antiplatelet activities. The activity of a lipophilic solution prepared from WTBS was in vitro tested on the platelet aggregation in response to ADP agonists and Collagen.

Gelsolin Modulates Platelet Dense Granule Secretion and Hemostasis via the Actin Cytoskeleton.

BACKGROUND AND OBJECTIVE: The mechanisms underlying platelet granule release are not fully understood. The actin cytoskeleton serves as the platelet's structural framework that is remodeled upon platelet activation. Gelsolin is a calcium-dependent protein that severs and caps existing actin filaments although its role in modulating platelet granule exocytosis is unknown. METHODS: The hemostatic function of wild-type (WT) and gelsolin null (Gsn-/- ) mice was measured ex vivo by rotational thromboelastometry analysis of whole blood. Platelets were purified from WT and Gsn-/- mouse blood and activated with thrombin. Platelet aggregation was assessed by light-transmission aggregometry. Clot retraction was measured to assess outside-in integrin signaling. Adenosine triphosphate (ATP) release and surface P-selectin were measured as markers of dense- and α-granule secretion, respectively. RESULTS: The kinetics of agonist-induced aggregation, clot retraction, and ATP release were accelerated in Gsn-/- platelets relative to WT. However, levels of surface P-selectin were diminished in Gsn-/- platelets. ATP release was also accelerated in WT platelets pretreated with the actin-depolymerizing drug cytochalasin D, thus mimicking the kinetics observed in Gsn-/- platelets. Conversely, ATP release kinetics were normalized in Gsn-/- platelets treated with the actin polymerization agonist jasplakinolide. Rab27b and Munc13-4 are vesicle-priming proteins known to promote dense granule secretion. Co-immunoprecipitation indicates that the association between Rab27b and Munc13-4 is enhanced in Gsn-/- platelets. CONCLUSIONS: Gelsolin regulates the kinetics of hemostasis by modulating the platelet's actin cytoskeleton and the protein machinery of dense granule exocytosis.

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

OBJECTIVE: Non-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. METHODS: Fresh 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. RESULTS: Platelet 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. CONCLUSION: After 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.

Aspirin Administration Mitigates Platelet Hyperaggregability After Splenectomy in a Murine Model.

INTRODUCTION: Patients who undergo splenectomy (SPLN) have an estimated 10%-35% risk of venous thromboembolic events; however, the underlying mechanism and strategy for prevention have yet to be identified. The goals of this study were to 1) investigate platelet aggregation after SPLN, 2) examine if aspirin administration could mitigate this effect, and 3) determine if concomitant hemorrhage would affect post-SPLN platelet function and response to aspirin. METHODS: Murine models of operative SPLN and submandibular bleed (SMB) were utilized. Mice were randomized to eight groups as follows: untouched, SPLN, sham (laparotomy only), SMB, SPLN + SMB, SPLN + aspirin (ASA), SMB + ASA, and SPLN + SMB + ASA. Aspirin (50 mg/kg) was administered on postoperative days (PODs) one and two via oral gavage. Mice were euthanized on POD 3, platelet counts were obtained, and blood samples were analyzed via rotational thromboelastometry and impedance aggregometry with adenosine diphosphate (ADP) and arachidonic acid (AA) as agonists. RESULTS: By POD 3, SPLN mice displayed a significant thrombocytosis compared to untouched, SMB, and sham SPLN mice. Clotting time and clot formation time were significantly decreased in SPLN and SPLN + SMB cohorts compared to untouched and sham controls with elevated mean clot firmness. SPLN mice also displayed a significant increase in ADP- and AA-mediated platelet aggregability compared to untouched controls, SMB, and SPLN + SMB. ASA significantly decreased platelet aggregation via both ADP and AA signaling in SPLN and SPLN + SMB cohorts without affecting viscoelastic coagulation testing. CONCLUSIONS: Platelet hyperaggregability after SPLN is mediated by both ADP and AA signaling. Early aspirin administration may prevent increased platelet aggregation exacerbated after polytrauma.

Pannexin-1 Activation by Phosphorylation Is Crucial for Platelet Aggregation and Thrombus Formation.

Pannexin-1 (PANX1) is a transmembrane protein that forms ion channels as hexamers on the plasma membrane. Electrophysiological studies prove that PANX1 has a high conductance for adenosine triphosphate (ATP), which plays an important role as a signal molecule in platelet activation. Recently, it was shown that PANX1 channels modulate platelet functions. To date, it remains unclear how PANX1 channels are activated and which signaling mechanisms are responsible for impaired hemostasis and thrombosis. Analysis of PANX1 phosphorylation at Tyr198 and Tyr308, and the impact on platelet activation and thrombus formation using genetically modified platelets or pharmacological inhibitors. Platelet activation via immunoreceptor tyrosine-based activation motif (ITAM) coupled, G Protein-Coupled Receptors (GPCR) and thromboxane receptor (TP)-mediated signaling pathways led to increased PANX1 phosphorylation at Tyr198 and Tyr308. We identified the Src-GPVI signaling axes as the main pathway inducing PANX1 activation, while PKC and Akt play a minor role. PANX1 channels function as ATP release channels in platelets to support arterial thrombus formation. PANX1 activation is regulated by phosphorylation at Tyr198 and Tyr308 following platelet activation. These results suggest an important role of PANX1 in hemostasis and thrombosis by releasing extracellular ATP to support thrombus formation.

Resveratrol Inhibits Metabolism and Affects Blood Platelet Function in Type 2 Diabetes.

Chronic hyperglycemia contributes to vascular complications in diabetes. Resveratrol exerts anti-diabetic and anti-platelet action. This study aimed to evaluate the effects of resveratrol on metabolism and the function of blood platelets under static and in in vitro flow conditions in patients with type 2 diabetes. Blood obtained from 8 healthy volunteers and 10 patients with type 2 diabetes was incubated with resveratrol and perfused over collagen-coated capillaries. Isolated blood platelets were incubated with resveratrol and activated by collagen to assess platelet function, metabolism, ATP release, TXA2 production, lipid peroxidation, and gluthatione content. In the type 2 diabetes group, plasma glucose and fructosamine concentrations were significantly higher than in the healthy group. In in vitro studies, collagen-induced thrombi formation in the blood of diabetic patients was 33% higher than in the healthy group. Resveratrol reduced thrombi by over 50% in the blood of healthy and diabetic patients. TXA2 production was 47% higher in diabetic platelets than in the healthy group. Resveratrol reduced TXA2 release by 38% in healthy platelets and by 79% in diabetic platelets. Resveratrol also reduced the activities of enzymes responsible for glycolysis and oxidative metabolism in the platelets of both groups. These data indicate that the resveratrol-induced inhibition of platelet metabolism and TXA2 release may lead to a reduction of platelet function and thrombus formation in patients with type 2 diabetes. Therefore, resveratrol may be beneficial to prevent vascular complications as a future complementary treatment in aspirin-resistant diabetic patients.

Canine Cancer Cells Activate Platelets via the Platelet P2Y12 Receptor.

In addition to their well-known functions in haemostasis, anucleated platelets have a critical role in cancer biology. Many human and non-human cancer types can directly interact with and activate platelets, promoting cancer malignancy and progression. Although naturally occurring canine neoplastic diseases mimic the biologically complex conditions of human cancers more closely than laboratory-bred mice, studies evaluating the relationship between cancer cells and platelets in dogs are scarce, and the effects of tumour cells on platelets in these animals are unknown. To evaluate whether cancer cells could activate canine platelets, we assessed the response of platelet-rich plasma to cultured canine cancer cells using light transmittance aggregometry. Similar to human and murine cancer cell research, we demonstrated that both canine osteosarcoma and mammary carcinoma cells activated canine platelets in vitro, resulting in platelet aggregation. The degree of aggregation was most pronounced at a cancer cell to platelet ratio of 1:200 for most cell lines. Mechanistic studies revealed that the platelet adenosine diphosphate (ADP) receptor P2Y12 is essential for canine platelet aggregation induced by canine cancer. ADP receptor blockage on platelets inhibited >50% of cancer cell-induced maximum platelet aggregation in all cell lines evaluated. As in other species, our results suggest that canine cancers may activate canine platelets in vivo. This mechanism is likely relevant for the biology and progression of cancer in the dog.