A microfluidic model of hemostasis sensitive to platelet function and coagulation.
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ABSTRACT:
SUBMITTER: Schoeman RM
PROVIDER: S-EPMC5435378 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background: Untreated hypothyroidism is associated with acquired von Willebrand syndrome, and hyperthyroidism is associated with increased thrombosis risk. However, the causal effects of thyroid function on hemostasis, coagulation, and fibrinolysis are unknown. Methods: In a two-sample Mendelian randomization (MR) study with genome-wide association variants, we assessed causality of genetically predicted hypothyroidism (N = 134,641), normal-range thyrotropin (TSH; N = 54,288) and free thyroxine (fT4) (N = 49,269), hyperthyroidism (N = 51,823), and thyroid peroxidase antibody positivity (N = 25,821) on coagulation (activated partial thromboplastin time, von Willebrand factor [VWF], factor VIII [FVIII], prothrombin time, factor VII, fibrinogen) and fibrinolysis (D-dimer, tissue plasminogen activator [TPA], plasminogen activator inhibitor-1) from the CHARGE Hemostasis Consortium (N = 2583-120,246). Inverse-variance-weighted random effects were the main MR analysis followed by sensitivity analyses. Two-sided p < 0.05 was nominally significant, and p < 0.0011[ = 0.05/(5 exposures × 9 outcomes)] was Bonferroni significant for the main MR analysis. Results: Genetically increased TSH was associated with decreased VWF [β(SE) = -0.020(0.006), p = 0.001] and with decreased fibrinogen [β(SE) = -0.008(0.002), p = 0.001]. Genetically increased fT4 was associated with increased VWF [β(SE) = 0.028(0.011), p = 0.012]. Genetically predicted hyperthyroidism was associated with increased VWF [β(SE) = 0.012(0.004), p = 0.006] and increased FVIII [β(SE) = 0.013(0.005), p = 0.007]. Genetically predicted hypothyroidism and hyperthyroidism were associated with decreased TPA [β(SE) = -0.009(0.024), p = 0.024] and increased TPA [β(SE) = 0.022(0.008), p = 0.008], respectively. MR sensitivity analyses showed similar direction but lower precision. Other coagulation and fibrinolytic factors were inconclusive. Conclusions: In the largest genetic studies currently available, genetically increased TSH and fT4 may be associated with decreased and increased synthesis of VWF, respectively. Since Bonferroni correction may be too conservative given the correlation between the analyzed traits, we cannot reject nominal associations of thyroid traits with coagulation or fibrinolytic factors.
| S-EPMC8558080 | biostudies-literature
Project description:Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin alphaIIbbeta3-mediated platelet aggregation and beta1 integrin-mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet beta1 and beta3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian alpha2beta1 and alphaIIbbeta3 integrins in vivo.
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Project description:To reveal if coagulopathies relate to the course of COVID-19, we examined 255 patients with moderate and severe COVID-19, receiving anticoagulants and immunosuppressive drugs. Coagulopathy manifested predominantly as hypercoagulability that correlated directly with systemic inflammation, disease severity, comorbidities, and mortality risk. The prolonged clotting tests in about ¼ of cases were associated with high levels of C-reactive protein and antiphospholipid antibodies, which impeded coagulation in vitro. Contraction of blood clots was hindered in about ½ of patients, especially in severe and fatal cases, and correlated directly with prothrombotic parameters. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with platelet dysfunction. Clots with impaired contraction were porous, had a low content of compressed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin, suggesting that the uncompacted intravital clots are more obstructive but patients could also be prone to bleeding. The absence of consumption coagulopathy suggests the predominance of local and/or regional microthrombosis rather than disseminated intravascular coagulation. The results obtained (i) confirm the importance of hemostatic disorders in COVID-19 and their relation to systemic inflammation; (ii) justify monitoring of hemostasis, including the kinetics of blood clot contraction; (iii) substantiate the active prophylaxis of thrombotic complications in COVID-19.
| S-EPMC8357814 | biostudies-literature
Project description:Objective Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5β1, a receptor for fibronectin. The aim of this study was to characterize the role of α5β1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis.Approach and results We generated a mouse strain deficient for integrin α5β1 on megakaryocytes and platelets (PF4Cre-α5-/-). PF4Cre-α5-/- mice were viable, fertile, and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocyte morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5-/- platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5-/- platelets under shear flow on fibrinogen, laminin, or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5-/- platelets displayed a marked decrease in adhesion, activation, and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5-/- mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta, or laser-induced injury of mesenteric vessels.Conclusion In summary, this study shows that platelet integrin α5β1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.
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Project description:ObjectiveUsing 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal.ConclusionsThis study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.
| S-EPMC7837688 | biostudies-literature
Project description:Platelet aggregation and adhesion are critically involved in both normal hemostasis and thrombosis during vascular injury. Before any surgery, it is important to identify the number of platelets and their functionality to reduce the risk of bleeding; therefore, platelet function testing is a requirement. We introduce a novel evaluation method of assessing platelet function with laser speckle contrast imaging. The speckle decorrelation time (SDT) of the blood flowing through a microfluidic channel chip provides a quantitative measure of platelet aggregation. We compared SDTs of whole blood and platelet-poor blood, i.e., whole blood stripped of its buffy coat region, and found a marked reduction in decorrelation time for platelet-poor blood. The measured SDT of platelet-poor blood was 1.04 ± 0.21 ms, while that of whole blood was 2.64 ± 0.83 ms. To further characterize the sensitivity of our speckle decorrelation time-based platelet function testing (SDT-PFT), we added various agonists involved in platelet aggregation, including adenosine diphosphate (ADP), epinephrine (EPI), and arachidonic acid (AA). In this study, the results show that whole blood with ADP resulted in the largest SDT, followed by whole blood with AA, whole blood with EPI, whole blood without agonist, and platelet-poor blood with or without agonist. These findings show that SDT-PFT has the potential for rapid screening of bleeding disorders and monitoring of anti-platelet therapies with only a small volume of blood.
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Project description:In patients with diabetes, metabolic disorders disturb the physiological balance of coagulation and fibrinolysis, leading to a prothrombotic state characterized by platelet hypersensitivity, coagulation disorders and hypofibrinolysis. Hyperglycemia and insulin resistance cause changes in platelet number and activation, as well as qualitative and/or quantitative modifications of coagulatory and fibrinolytic factors, resulting in the formation of fibrinolysis-resistant clots in patients with diabetes. Other coexisting factors like hypoglycemia, obesity and dyslipidemia also contribute to coagulation disorders in patients with diabetes. Management of the prothrombotic state includes antiplatelet and anticoagulation therapies for diabetes patients with either a history of cardiovascular disease or prone to a higher risk of thrombus generation, but current guidelines lack recommendations on the optimal antithrombotic treatment for these patients. Metabolic optimizations like glucose control, lipid-lowering, and weight loss also improve coagulation disorders of diabetes patients. Intriguing, glucose-lowering drugs, especially cardiovascular beneficial agents, such as glucagon-like peptide-1 receptor agonists and sodium glucose co-transporter inhibitors, have been shown to exert direct anticoagulation effects in patients with diabetes. This review focuses on the most recent progress in the development and management of diabetes related prothrombotic state.
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Project description:BackgroundNovel crystalloid solutions containing polyethylene glycol polymers (PEG-20k) produce dramatic resuscitation effects but dose-dependently produce a hypocoagulative state. The objective of this study was to examine possible mechanisms of this effect. Based on previous thromboelastography data, we hypothesize the effect is largely due to platelet interactions with the polymers.MethodsWhole citrated blood from healthy volunteers was diluted ex-vivo 10% with crystalloids and tested for coagulation and platelet function. The specific tests included prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and von Willebrand factor (vWf) activity, thrombin generation, thromboelastography with and without platelet mapping, platelet flow cytometry, and erythrocyte sedimentation rate.FindingsFibrinogen and vWF activities, PT, and aPTT were not affected by PEG-20k dilutions. Thrombin activity was mildly suppressed with PEG-20k (TTP- 20%). Platelet mapping demonstrated significantly greater % inhibition of both ADP and arachidonic acid-induced platelet aggregation with PEG-20k, but direct ADP-activated gpIIa/IIIb (PAC1) and P-selectin (CD62P) binding site expression was not altered. Mild dose-dependent suppression of TEG-MA was seen with PEG-20k using platelet poor plasma. Erythrocyte Sedimentation Rates (ESR) were dramatically accelerated after dilution with 10% PEG-20k, which was competitively blocked by smaller PEG polymers, suggesting nonspecific PEG-20k cell binding effects.ConclusionsPEG-20k creates a mild hypocoagulative state in whole blood at concentrations ≥10%, which may be due to platelet-PEG interactions at the IIb/IIIa interface with lesser effects on fibrin polymerization. This interaction may cause a functional thrombasthenia induced by nonspecific platelet surface passivation by the PEG polymer.
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Project description:We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.
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Project description:Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase-derived hydroxyeicosatetraenoic acid-phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.
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