Unraveling the Mechanism of Platelet Aggregation Suppression by Monoterpenoids.
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ABSTRACT:
SUBMITTER: Nikitina LE
PROVIDER: S-EPMC8773274 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.
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Project description:Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet-poor plasma (containing von Willebrand factor, vWF) and fluorochrome-labeled platelets were subjected to a range of shear rates (0-10 000 s-1 ) for 0, 5, 10, and 15 minutes using a custom-built blood-shearing device. Formed sizes of platelet aggregates under a range of shear-controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13-ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s-1 ). We found that the average size of platelet aggregates formed under physiological shear rates of 360-3000 s-1 (200-300 μm2 ) was significantly larger compared to those sheared at >6000 s-1 (50-100 μm2 ). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s-1 in order to maintain the optimal size of platelet aggregates to "plug off" the injury site and stop bleeding.
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Project description:Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1?, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies.
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Project description:In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin alpha(IIb)beta(3) demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin alpha(IIb)beta(3) mediated cell arrest. Platelet aggregation under flow appears to be a multistep process involving: (a) exposure of vWf on the surface of immobilized platelets; (b) a reversible phase of platelet aggregation mediated by the binding of GPIbalpha on the surface of free-flowing platelets to vWf on the surface of immobilized platelets; and (c) an irreversible phase of aggregation dependent on integrin alpha(IIb)beta(3). Studies of platelet thrombus formation in vivo demonstrate that this multistep adhesion mechanism is indispensable for platelet aggregation in arterioles and also appears to promote platelet aggregate formation in venules. Together, our studies demonstrate an important role for platelet vWf in initiating the platelet aggregation process under flow and challenge the currently accepted view that the vWf-GPIbalpha interaction is exclusively involved in initiating platelet aggregation at elevated shear rates.
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Project description:BackgroundPosttraumatic venous thromboembolism (VTE) remains prevalent in severely injured patients despite chemoprophylaxis. Importantly, although platelets are central to thrombosis, they are not routinely targeted in prevention of posttraumatic VTE. Furthermore, platelets from injured patients show ex vivo evidence of increased activation yet impaired aggregation, consistent with functional exhaustion. However, the relationship of this platelet functional phenotype with development of posttraumatic VTE is unknown. We hypothesized that, following injury, impaired ex vivo platelet aggregation (PA) is associated with the development of posttraumatic VTE.MethodsWe performed a secondary analysis of 133 severely injured patients from a prospective observational study investigating coagulation and inflammation (2011-2019). Platelet aggregation in response to stimulation with adenosine diphosphate (ADP), collagen, and thrombin was measured at presentation (preresuscitation) and 24 hours (postresuscitation). Viscoelastic clot strength and lysis were measured in parallel by thromboelastography. Multivariable regression examined relationships between PA at presentation, 24 hours, and the change (δ) in PA between presentation and 24 hours with development of VTE.ResultsThe 133 patients were severely injured (median Injury Severity Score, 25), and 14% developed VTE (all >48 hours after admission). At presentation, platelet count and PA were not significantly different between those with and without incident VTE. However, at 24 hours, those who subsequently developed VTE had significantly lower platelet counts (126 × 10 9 /L vs. 164 × 10 9 /L, p = 0.01) and lower PA in response to ADP ( p < 0.05), collagen ( p < 0.05), and thrombin ( p = 0.06). Importantly, the magnitude of decrease in PA (δ) from presentation to 24 hours was independently associated with development of VTE (adjusted odds ratios per 10 aggregation unit decrease: δ-ADP, 1.31 [ p = 0.03]; δ-collagen, 1.36 [ p = 0.01]; δ-thrombin, 1.41 [ p < 0.01]).ConclusionSeverely injured patients with decreasing ex vivo measures of PA despite resuscitation have an increased risk of developing VTE. This may have implications for predicting development of VTE and for studying platelet targeted chemoprophylaxis regimens.Level of evidencePrognostic/Epidemiological; Level III.
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Project description:Despite the clinical success and growth in the utilization of continuous flow ventricular assist devices (cfVADs) for the treatment of advanced heart failure, hemolysis and thrombosis remain major limitations. Inadequate and/or ineffective anticoagulation regimens, combined with high pump speed and non-physiological flow patterns, can result in hemolysis which often is accompanied by pump thrombosis. An unexpected increase in cfVADs thrombosis was reported by multiple major VAD implanting centers in 2014, highlighting the association of hemolysis and a rise in lactate dehydrogenase (LDH) presaging thrombotic events. It is well established that thrombotic complications arise from the abnormal shear stresses generated by cfVADs. What remains unknown is the link between cfVAD-associated hemolysis and pump thrombosis. Can hemolysis of red blood cells (RBCs) contribute to platelet aggregation, thereby, facilitating prothrombotic complications in cfVADs? Herein, we examine the effect of RBC-hemolysate and selected major constituents, i.e., lactate dehydrogenase (LDH) and plasma free hemoglobin (pHb) on platelet aggregation, utilizing electrical resistance aggregometry. Our hypothesis is that elements of RBCs, released as a result of shear-mediated hemolysis, will contribute to platelet aggregation. We show that RBC hemolysate and pHb, but not LDH, are direct contributors to platelet aggregation, posing an additional risk mechanism for cfVAD thrombosis.
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Project description:AimThe aim of this study was to evaluate the in vitro effect of coumarin and 15 monosubstituted derivatives on the inhibition of human platelet aggregation induced by various proaggregatory agonists, particularly by epinephrine.BackgroundThe emergence of residual platelet reactivity during the use of conventional antiplatelet agents (acetylsalicylic acid and clopidogrel) is one of the main causes of double therapy´s therapeutic failure. Platelet adrenoceptors participate in residual platelet reactivity. Therefore, it is necessary to develop new antiplatelet agents that inhibit epinephrine-induced platelet aggregation as a new therapeutic strategy. Information on the antiplatelet activity of coumarins in inhibiting epinephrine-induced aggregation is limited.ObjectiveThe objective of this study was to establish the structure-activity relationship (SAR) of coumarin derivatives with hydroxy, methoxy, and acetoxy groups in different positions of the coumarin nucleus to identify the most active molecules. Moreover, this study aimed to use in silico studies to suggest potential drug targets to which the molecules bind to produce antiplatelet effects.MethodsThe platelet aggregation was performed using a Lumi-aggregometer; the inhibitory activity of 16 compounds were evaluated by inducing the aggregation of human platelets (250 × 103/μl) with epinephrine (10 μM), collagen (2 μg/ml) or ADP (10 μM). The aggregation of control platelets was considered 100% of the response for each pro-aggregatory agonist.ResultsEleven molecules inhibited epinephrine-induced aggregation, with 3-acetoxycoumarin and 7-methoxycoumarin being the most active. Only coumarin inhibited collagen-induced platelet aggregation, but no molecule showed activity when using ADP as an inducer.ConclusionsIn silico studies suggest that most active molecules might have antagonistic interactions in the α2 and β2 adrenoceptors. The antiplatelet actions of these coumarins have the potential to reduce residual platelet reactivity and thus contribute to the development of future treatments for patients who do not respond adequately to conventional agents.
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