scholarly journals Ex vivo anticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping

2021 ◽  
Author(s):  
Laura Sachs ◽  
Jan Wesche ◽  
Lea Lenkeit ◽  
Andreas Greinacher ◽  
Markus Bender ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
High Throughput ◽  
Heavy Chain ◽  
Ex Vivo ◽  
Unmet Need ◽  
Bleeding Risk ◽  
Blood Platelet ◽  
Human Platelets ◽  
Analytical Variable ◽  
Cellular Components

AbstractInherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used for the collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

Pathological shear stress directly regulates platelet αIIbβ3signaling

2006 ◽  
Vol 291 (6) ◽  
pp. C1346-C1354 ◽  
Author(s):  
Shuju Feng ◽  
Xin Lu ◽  
Julio C. Reséndiz ◽  
Michael H. Kroll
Keyword(s):  
Shear Stress ◽  
Ligand Binding ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Tyrosine Kinases ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Mechanical Forces

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its β-subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin αIIbβ3is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on αIIbβ3, we examined αIIbβ3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that α-actinin, myosin heavy chain, and Syk coimmunoprecipitate with αIIbβ3in resting platelets and that 120 dyn/cm2shear stress leads to their disassociation from αIIbβ3. Shear-induced disassociation of α-actinin and myosin heavy chain from the β3tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to αIIbβ3. Syk's disassociation from β3is inhibited when VWF binding to either GpIb-IX-V or αIIbβ3is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to αIIbβ3but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing αIIbβ3with β3truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates αIIbβ3function and suggest that shear-induced αIIbβ3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the β3-tail.

Tempol prevents cardiac oxidative damage and left ventricular dysfunction in the PPAR-α KO mouse

2013 ◽  
Vol 304 (11) ◽  
pp. H1505-H1512 ◽  
Author(s):  
Aziz Guellich ◽  
Thibaud Damy ◽  
Marc Conti ◽  
Victor Claes ◽  
Jane-Lise Samuel ◽  
...  
Keyword(s):  
Glutathione Peroxidase ◽  
Glutathione Reductase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Papillary Muscle ◽  
Ex Vivo ◽  
Left Ventricular ◽  
Lv Function ◽  
Wild Type ◽  
Contractile Parameters

Peroxisome proliferator-activated receptor (PPAR)-α deletion induces a profound decrease in MnSOD activity, leading to oxidative stress and left ventricular (LV) dysfunction. We tested the hypothesis that treatment of PPAR-α knockout (KO) mice with the SOD mimetic tempol prevents the heart from pathological remodelling and preserves LV function. Twenty PPAR-α KO mice and 20 age-matched wild-type mice were randomly treated for 8 wk with vehicle or tempol in the drinking water. LV contractile parameters were determined both in vivo using echocardiography and ex vivo using papillary muscle mechanics. Translational and posttranslational modifications of myosin heavy chain protein as well as the expression and activity of major antioxidant enzymes were measured. Tempol treatment did not affect LV function in wild-type mice; however, in PPAR-α KO mice, tempol prevented the decrease in LV ejection fraction and restored the contractile parameters of papillary muscle, including maximum shortening velocity, maximum extent of shortening, and total tension. Moreover, compared with untreated PPAR-α KO mice, myosin heavy chain tyrosine nitration and anion superoxide production were markedly reduced in PPAR-α KO mice after treatment. Tempol also significantly increased glutathione peroxidase and glutathione reductase activities (∼ 50%) in PPAR-α KO mice. In conclusion, these findings demonstrate that treatment with the SOD mimetic tempol can prevent cardiac dysfunction in PPAR-α KO mice by reducing the oxidation of contractile proteins. In addition, we show that the beneficial effects of tempol in PPAR-α KO mice involve activation of the glutathione peroxidase/glutathione reductase system.

scholarly journals Development of dilated cardiomyopathy in Bmal1-deficient mice

2012 ◽  
Vol 303 (4) ◽  
pp. H475-H485 ◽  
Author(s):  
Mellani Lefta ◽  
Kenneth S. Campbell ◽  
Han-Zhong Feng ◽  
Jian-Ping Jin ◽  
Karyn A. Esser
Keyword(s):  
Circadian Clock ◽  
Dilated Cardiomyopathy ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Clock Gene ◽  
Ex Vivo ◽  
Chain Gene ◽  
Circadian Clock Gene ◽  
Myosin Heavy Chain Isoform ◽  
Wide Range

Circadian rhythms are approximate 24-h oscillations in physiology and behavior. Circadian rhythm disruption has been associated with increased incidence of hypertension, coronary artery disease, dyslipidemia, and other cardiovascular pathologies in both humans and animal models. Mice lacking the core circadian clock gene, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein ( Bmal1), are behaviorally arrhythmic, die prematurely, and display a wide range of organ pathologies. However, data are lacking on the role of Bmal1 on the structural and functional integrity of cardiac muscle. In the present study, we demonstrate that Bmal1 −/− mice develop dilated cardiomyopathy with age, characterized by thinning of the myocardial walls, dilation of the left ventricle, and decreased cardiac performance. Shortly after birth the Bmal1 −/− mice exhibit a transient increase in myocardial weight, followed by regression and later onset of dilation and failure. Ex vivo working heart preparations revealed systolic ventricular dysfunction at the onset of dilation and failure, preceded by downregulation of both myosin heavy chain isoform mRNAs. We observed structural disorganization at the level of the sarcomere with a shift in titin isoform composition toward the stiffer N2B isoform. However, passive tension generation in single cardiomyocytes was not increased. Collectively, these findings suggest that the loss of the circadian clock gene, Bmal1, gives rise to the development of an age-associated dilated cardiomyopathy, which is associated with shifts in titin isoform composition, altered myosin heavy chain gene expression, and disruption of sarcomere structure.

Shear Stress Directly Regulates Platelet αIIbβ3 Signaling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3570-3570
Author(s):  
Shuju Feng ◽  
Xin Lu ◽  
Julio C. Resendiz ◽  
Michael H. Kroll
Keyword(s):  
Shear Stress ◽  
Ligand Binding ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cho Cells ◽  
Tyrosine Kinases ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Mechanical Forces

Abstract Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced by an integrin’s ligand-bound extracellular domain through its β subunit’s cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin αIIbβ3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on αIIbβ3, we examined αIIbβ3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that α-actinin, myosin heavy chain and Syk co-immunoprecipitate with αIIbβ3 in resting platelets, and that 120 dynes/cm2 shear stress leads to their disassociation from αIIbβ3. Shear-induced disassociation of α-actinin and myosin heavy chain from the β3 tail is unaffected by blocking VWF binding to GpIb-IX-V but abolished by blocking VWF binding to αIIbβ3. Syk’s disassociation from β3 is inhibited when VWF binding to either GpIb-IX-V or αIIbβ3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine phosphorylated SLAP-130 are inhibited by blocking ligand binding to αIIbβ3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary (CHO) cells expressing human αIIbβ3 demonstrate calcium and shear-dependent (1 and 10 dynes/cm2) attachment to glass cover slips coated with 20 μg/ml VWF. CHO cells expressing αIIbβ3 with β3 truncated of its cytoskeletal binding domains at C-terminal residue 716 demonstrate decreased VWF-dependent adhesion and cohesion in response to 1 dyne/cm2 shear stress. These results support the hypothesis that shear stress directly modulates αIIbβ3 function, and suggest that shear-induced αIIbβ3-mediated signaling regulates platelet aggregation by directing the release of α-actinin and myosin heavy chain from the β3 tail.

Molecular basis of impaired extraocular muscle function in a mouse model of congenital myopathy due to compound heterozygous Ryr1 mutations

2020 ◽  
Vol 29 (8) ◽  
pp. 1330-1339
Author(s):  
Jan Eckhardt ◽  
Christoph Bachmann ◽  
Sofia Benucci ◽  
Moran Elbaz ◽  
Alexis Ruiz ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Extraocular Muscle ◽  
Calcium Release ◽  
Ex Vivo ◽  
Force Production ◽  
Myosin Heavy Chain Isoforms ◽  
Mutant Mice ◽  
Compound Heterozygous ◽  
Macromolecular Complex

Abstract Mutations in the RYR1 gene are the most common cause of human congenital myopathies, and patients with recessive mutations are severely affected and often display ptosis and/or ophthalmoplegia. In order to gain insight into the mechanism leading to extraocular muscle (EOM) involvement, we investigated the biochemical, structural and physiological properties of eye muscles from mouse models we created knocked-in for Ryr1 mutations. Ex vivo force production in EOMs from compound heterozygous RyR1p.Q1970fsX16+p.A4329D mutant mice was significantly reduced compared with that observed in wild-type, single heterozygous mutant carriers or homozygous RyR1p.A4329D mice. The decrease in muscle force was also accompanied by approximately a 40% reduction in RyR1 protein content, a decrease in electrically evoked calcium transients, disorganization of the muscle ultrastructure and a decrease in the number of calcium release units. Unexpectedly, the superfast and ocular-muscle-specific myosin heavy chain-EO isoform was almost undetectable in RyR1p.Q1970fsX16+p.A4329D mutant mice. The results of this study show for the first time that the EOM phenotype caused by the RyR1p.Q1970fsX16+p.A4329D compound heterozygous Ryr1 mutations is complex and due to a combination of modifications including a direct effect on the macromolecular complex involved in calcium release and indirect effects on the expression of myosin heavy chain isoforms.

The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

1972 ◽  
Vol 28 (01) ◽  
pp. 031-048 ◽  
Author(s):  
W. H. E Roschlau ◽  
R Gage
Keyword(s):  
Platelet Aggregation ◽  
Aspergillus Oryzae ◽  
Degradation Products ◽  
Blood Platelet ◽  
Human Platelets ◽  
Fibrinolytic Enzyme ◽  
Inhibitory Effects ◽  
Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Differential Ability of Agonists to Express Distinct Pools of Fibrinogen (Gpllb/llla) Receptors which Can Mediate the Aggregation of Human Platelets

1989 ◽  
Vol 62 (03) ◽  
pp. 955-961 ◽  
Author(s):  
Ian S Watts ◽  
Rebecca J Keery ◽  
Philip Lumley
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Surface Membrane ◽  
Blood Platelet ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Platelet Surface ◽  
Human Platelet Aggregation ◽  
Differential Ability ◽  
Blood Platelet Aggregation

SummaryWe have investigated the effect of two procedures that modify human platelet surface membrane glycoprotein (Gp) IIb and IIIa complexes upon whole blood platelet aggregation to a range of agonists. (A) Irreversible disruption of complexes by temporary (30 min) Ca2+-deprivation with EGTA at 37° C. (B) Binding of a monoclonal antibody M148 to the complex. EGTA exposure abolished aggregation to ADP, adrenaline and PAF. In contrast, full aggregation curves to collagen and U-46619 could still be established. EGTA exposure reduced M148 binding to platelets by 80%. Excess M148 abolished aggregation to ADP, PAF, collagen and U-46619. However, upon removal of unbound antibody from platelets full aggregation curves to collagen and U-46619 but not to ADP and PAF could be re-established. Thus human platelet aggregation to ADP, PAF and adrenaline appears absolutely dependent upon surface membrane GpIIb/IIIa complexes. In contrast, collagen and U-46619 cause expression of an additional distinct pool of Gp complexes inaccessible to EGTA and M148 in unstimulated platelets which is intimately involved in aggregation to these agonists.

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