Supplemental Fibrinogen Restores Platelet Inhibitor-Induced Reduction in Thrombus Formation without Altering Platelet Function: An In Vitro Study

2020 ◽  
Vol 120 (11) ◽  
pp. 1548-1556
Author(s):  
Thomas Bärnthaler ◽  
Elisabeth Mahla ◽  
Gabor G. Toth ◽  
Rufina Schuligoi ◽  
Florian Prüller ◽  
...  
Keyword(s):  
Antiplatelet Therapy ◽  
Platelet Activation ◽  
Platelet Function ◽  
Dual Antiplatelet Therapy ◽  
Thrombus Formation ◽  
In Vitro Study ◽  
Coronary Intervention ◽  
Calcium Flux ◽  
Major Surgery

Abstract Background For patients treated with dual antiplatelet therapy, standardized drug-specific 3-to-7 day cessation is recommended prior to major surgery to reach sufficient platelet function recovery. Here we investigated the hypothesis that supplemental fibrinogen might mitigate the inhibitory effects of antiplatelet therapy. Methods and Results To this end blood from healthy donors was treated in vitro with platelet inhibitors, and in vitro thrombus formation and platelet activation were assessed. Ticagrelor, acetylsalicylic acid, the combination of both, and tirofiban all markedly attenuated the formation of adherent thrombi, when whole blood was perfused through collagen-coated microchannels at physiological shear rates. Addition of fibrinogen restored in vitro thrombus formation in the presence of antiplatelet drugs and heparin. However, platelet activation, as investigated in assays of P-selectin expression and calcium flux, was not altered by fibrinogen supplementation. Most importantly, fibrinogen was able to restore in vitro thrombogenesis in patients on maintenance dual antiplatelet therapy after percutaneous coronary intervention. Conclusion Thus, our in vitro data support the notion that supplementation of fibrinogen influences the perioperative hemostasis in patients undergoing surgery during antiplatelet therapy by promoting thrombogenesis without significantly interfering with platelet activation.

scholarly journals Analysis of Platelet Function from Thromboelastography Examination in Patients with Single and Multiple Antiplatelet Therapy after Percutaneous Coronary Intervention in Dr. Saiful Anwar Malang

2020 ◽  
Vol 7 (1) ◽  
pp. 19-28
Author(s):  
Sasmojo Widito ◽  
Dadang Hendrawan ◽  
Dedy Irawan
Keyword(s):  
Percutaneous Coronary Intervention ◽  
Antiplatelet Therapy ◽  
Platelet Function ◽  
Dual Antiplatelet Therapy ◽  
Coronary Intervention ◽  
Primary Therapy ◽  
Research Subjects ◽  
Platelet Function Test ◽  
Percutaneous Coronary ◽  
Significant Difference

Each year, approximately 3 million people with coronary heart disease worldwide undergo percutaneous coronary intervention (PCI). Dual antiplatelet therapy (DAPT) with aspirin and P2Y12 inhibitors became the primary therapy for 6-12 months after PCI. DAPT can be continued > 12 months at a high risk of thrombosis. About 9-10% of patients with dual antiplatelet therapy still experience ischemia. The platelet function examination by thromboelastography (TEG). This research is an analytic observational study using a cross-sectional method. This study was conducted in Saiful Anwar General Hospital. Patients were divided into two groups: (1) on-single antiplatelet therapy; (2) on-dual antiplatelet therapy. The outcome measured result of the platelet function test was divided into standard, low platelet function, and platelet hypercoagulability. An analysis of the differences between single or multiple antiplatelet administration and the platelet function results was performed. There were 52 research subjects, each group of single and multiple antiplatelet therapies as many as 26 people, most of the subjects were male (82.6%) with a mean age of 57. The results of this study showed that there was no significant difference in the results of platelet function examinations between single and multiple antiplatelet therapies after 12 months of dual antiplatelet therapy.

scholarly journals The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

2021 ◽  
Author(s):  
Leila Revollo ◽  
Glenn Merrill-Skoloff ◽  
Karen De Ceunynck ◽  
James R. Dilks ◽  
Mattia Bordoli ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Secretory Pathway ◽  
Thrombus Formation ◽  
Extracellular Proteins ◽  
Normal Platelet

AbstractTyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have dramatic changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2(T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

scholarly journals The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

Blood ◽  
2021 ◽  
Author(s):  
Leila Denise Revollo ◽  
Glenn Merrill-Skoloff ◽  
Karen De Ceunynck ◽  
James R Dilks ◽  
Shihui Guo ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Secretory Pathway ◽  
Thrombus Formation ◽  
Extracellular Proteins ◽  
Normal Platelet

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and a-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls, but normal bleeding times. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

Abstract 571: A Novel High-Throughput Whole Blood Immuno-Counting Assay is Useful in the Assessment of Platelet Responses to Antiplatelet Therapy

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Marie Lordkipanidze ◽  
Natalia Dovlatova ◽  
Mohammad Algahtani ◽  
Martina H Lundberg ◽  
Timothy D Warner ◽  
...  
Keyword(s):  
Platelet Count ◽  
Antiplatelet Therapy ◽  
Platelet Function ◽  
Whole Blood ◽  
Dual Antiplatelet Therapy ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Treated Sample ◽  
Function Testing

Background: The current gold standard in platelet function testing, light transmission aggregometry, is time- and labor-intensive, and uses platelet-rich plasma which makes it sub-optimal for high throughput testing. In order to reduce blood manipulation prior to platelet function testing and to study multiple platelet activation pathways simultaneously, we have developed a 96-well plate-based assay carried out in whole blood, where aggregation is measured as a decrease in the number of fluorescently-labeled single platelets by flow cytometry. Aim: To investigate whether a 96-well plate-based whole blood assay can be used to assess platelet function. Methods: Platelet function in response to 5 concentrations of lyophilized arachidonic acid (AA), ADP, collagen, epinephrine, TRAP, U46619, and ristocetin, was assessed in healthy volunteers (n=20) to establish normal ranges. The effect of antiplatelet drugs was assessed in vitro by incubation with aspirin (100 μM), cangrelor (1 μM) or both (n=20), and in patients on dual antiplatelet therapy (n=20). After addition of 40 μl of whole blood per well, the plate was shaken for 5 min at 1000 rpm at 37°C; a fixative solution (Platelet Solutions, Nottingham) was applied to stop platelet aggregation and allow analysis in a central laboratory. Fixed whole blood samples (stable for up to 9 days) were labeled with FITC-conjugated CD42a and assessed by flow cytometry. Aggregation was calculated as (Platelet count in vehicle-treated sample - Platelet count in agonist-stimulated sample) / Platelet count in vehicle-treated sample x 100. Results: Dose-response curves were readily assessable for all agonists and intra-individual variability was minimal in healthy volunteers (CV<10%). In vitro addition of aspirin alone resulted in inhibition of AA- and collagen-induced aggregation, whereas cangrelor induced a shift in dose-response to most agonists in addition to profound inhibition of ADP responses. In patients on dual antiplatelet therapy, the pattern of response was consistent with the results obtained with in vitro agents. Conclusions: A 96-well plate-based whole blood assay with a minimal blood volume requirement (<2 ml) could be used to provide a global portrait of platelet responses to antiplatelet agents.

P1931Effect of de-escalated switching dual antiplatelet therapy after acute myocardial infarction in patients undergoing percutaneous coronary intervention: real-world data from a nationwide cohort study

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
C.-Y Hsu ◽  
J.-S Yeh ◽  
C.-Y Huang
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Percutaneous Coronary Intervention ◽  
Antiplatelet Therapy ◽  
Platelet Function ◽  
Dual Antiplatelet Therapy ◽  
Coronary Intervention ◽  
Risk Of Death ◽  
Percutaneous Coronary ◽  
Function Testing

Abstract Background Recently, both unguided (platelet function testing independent) and guided (platelet function testing dependent) DAPT de-escalation strategies have been investigated in different clinical studies but the data is still limited and conflicting. The aim of this study was to examine the effect of switching dual antiplatelet therapy (DAPT) on the major vascular risk after acute myocardial infarction (AMI) in patients undergoing percutaneous coronary intervention (PCI) by using Taiwan National Health Insurance Research Database. Methods In total, 1,903 and 4,059 patients defined as switched to aspirin and clopidogrel (switched DAPT) and continuation of aspirin and ticagrelor (unswitched DAPT) cohort, respectively who had received PCI during AMI hospitalization, on aspirin and ticagrelor initially and without occurring adverse events at 3 months were evaluated between 2013 and 2015. An inverse probability treatment of weighted approach was adopted to balance the baseline differences between two groups and Cox proportional hazard regression and competing risk regression were used to evaluated the effect of switching DAPT on death, AMI readmission, major bleeding and non-major clinically relevant bleeding. Results The incidence rates (per 100 person-year) of death and AMI readmission were 3.97 (95% confidence interval [CI] = 3.19–4.84) and 3.84 (95% CI = 3.09–4.73) in switched cohort and 1.83 (95% CI = 1.47–2.24) and 2.23 (95% CI = 1.82–2.68) in unswitched cohort, respectively. After adjustment for patients' clinical variables, switched cohort had higher risk of death (adjusted hazard ratio = 2.18, 95% CI = 1.62–2.93, P<0.001), and AMI readmission (adjusted sub-distribution ratio = 1.72, 95% CI = 1.27–2.34, P<0.001) compared to these in unswitched cohort; however, there was no difference in the risk of bleeding. Subgroup analysis showed a similar findings in many specific groups, except the patients who were younger age and had lower comorbidity score. Conclusion Switching DAPT might increase the risk of death and AMI readmission among patients with AMI undergoing PCI.

scholarly journals Vorapaxar

2013 ◽  
pp. 88-95
Author(s):  
Gianluca Airoldi ◽  
Mauro Campanini
Keyword(s):  
Antiplatelet Therapy ◽  
Platelet Activation ◽  
Dual Antiplatelet Therapy ◽  
Absolute Risk ◽  
Standard Of Care ◽  
Adenosine Diphosphate ◽  
Coronary Intervention ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Thrombin Inhibition

Antiplatelet drugs are the cornerstone of treatment for patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention. Clopidogrel and aspirin improve long-term vascular clinical outcomes in these patients and have become a standard of care. However, many patients still experience ischemic/thrombotic events, and it appears that insufficient response to both aspirin and clopidogrel contribute to this failure. Newer P2Y12 receptor blocker therapy resulted in only an approximately 2% reduction in absolute risk compared with clopidogrel. This indicates that residual ischemic events are mediated by other pathways that are unblocked by current dual antiplatelet therapy. Thrombin is the most potent platelet agonist (over 1000 times more than adenosine diphosphate on a molar basis). Thrombin-mediated platelet activation depends on proteaseactivated receptor (PAR) binding. PAR-1 is the main receptor for thrombin on human platelets; PAR-4 may contribute to platelet activation at much higher concentrations of thrombin. Inhibition of the PAR-1 may provide additional benefits over the standard dual antiplatelet therapy in attenuating ischemic event in patients with ACS. Vorapaxar is a new highly selective oral PAR-1 antagonist that inhibits thrombin-induced platelet activation. We review the pharmacokinetic, pharmacodynamic and clinical profile of vorapaxar. Although preliminary data indicated that vorapaxar may have the potential to improve ischemic outcomes without significantly increasing bleeding, more recent larger clinical trials seem to be less optimistic about both its effectiveness and safety. At this time, the role of vorapaxar in the settings of atherothrombotic disorders is not clear. Although it may be associated with less bleeding than P2Y12 receptor blockers, its antithrombotic effectiveness and side effects are major concerns.

scholarly journals Opposing Roles of GSK3α and GSK3β Phosphorylation in Platelet Function and Thrombosis

2021 ◽  
Vol 22 (19) ◽  
pp. 10656
Author(s):  
Samantha F. Moore ◽  
Ejaife O. Agbani ◽  
Andreas Wersäll ◽  
Alastair W. Poole ◽  
Chris M. Williams ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Glycogen Synthase ◽  
Thrombus Formation ◽  
Resistant Mouse ◽  
Function Expression ◽  
Substrate Phosphorylation ◽  
Granule Secretion

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3β. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3β(Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/β phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/β reduced thrombin-mediated platelet aggregation, integrin αIIbβ3 activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3β phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3β resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/β KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3β KI. In conclusion, our data indicate that GSK3α and GSK3β have differential roles in regulating platelet function.

scholarly journals Preparations from Selected Cucurbit Vegetables as Antiplatelet Agents – An in Vitro Study

2021 ◽  
Author(s):  
Agata Rolnik ◽  
Bartosz Skalski ◽  
Anna Stochmal ◽  
Beata Olas
Keyword(s):  
Platelet Activation ◽  
Cucurbita Pepo ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
In Vitro Study ◽  
Blood Platelet ◽  
Arachidonic Acid Metabolism ◽  
Platelet Activity ◽  
Activated Platelets

Abstract Increased blood platelet activation plays an important role in cardiovascular diseases (CVDs). Recent experiments indicate that certain fruits and vegetables, including onion, garlic, and beetroot, have anti-platelet potential and therefore may reduce the likelihood of CVDs. While vegetables from the Cucuritaceae family are known to exerting beneficial antioxidant and anti-inflammatory effects, their effects on blood platelet activation are poorly understood. Therefore, the aim of the present study was to determine the effect on platelet adhesion of preparations from selected cucurbits: pumpkin (Cucirbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds). It also evaluates the activity of these preparations on enzymatic lipid peroxidation in thrombin-activated washed blood platelets by TBARS assay. The study also determines the anti-platelet and anticoagulant properties of these five cucurbit preparations in whole blood by flow cytometry and with the total thrombus-formation analysis system (T-TAS) and evaluates the cytotoxicity of the tested preparations against platelets based on LDH activity. The results indicate that the yellow Cucurbita pepo var. patisoniana preparation demonstrated stronger anti-platelet properties than the other tested preparations, reducing the adhesion of thrombin-activated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. None of the preparations was found to cause platelet lysis. Our findings provide new information on the anti-platelet activity of the tested cucurbit preparations and their potential for treating CVDs associated with platelet hyperactivity.

Abstract 52: IkB Kinase beta is a Key Modulator of Platelet Function, and the Remodeling of CARMA1-Bcl10-MALT1 Complex Formation

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Zubair A Karim
Keyword(s):  
Complex Formation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Clot Retraction ◽  
Functional Responses ◽  
Knockout Mouse Model ◽  
Iκb Kinase

Secretion plays an important role in platelet function, and hence the secretory machinery offers a unique target to modulate thrombogenesis. We previously established that IκB kinase (IKK)-β is phosphorylated upon platelet activation, regulates their SNARE machinery, and involves CBM (CARMA1, Bcl10, and MALT1) complex formation. However, the detailed role of IKKβ in platelet function, the mechanism by which it regulates CBM complex formation and downstream effector activation remain elusive. Using a knockout mouse model system ( IKK β flox/flox -PF4Cre), we first showed that IKKβ plays a vital role in thrombus formation, and that it does so, in part, by regulating platelet functional responses, including dense and alpha granule release, αIIbβ3 activation, and PS exposure. Furthermore, we observed defects in compound fusion, platelet spreading, actin remodeling, and clot retraction. To this end, under clot retraction conditions in the knockout platelets, 7S complex formation was found to be inhibited. In terms of its signaling, using knockout platelets, we observed that IKKβ is required for the recruitment of Bcl10/MALT1 to CARMA1 upon platelet activation, i.e., CBM complex formation, and that this was due to the defective phosphorylation of Bcl10 and the IKKγ polyubiquitination. In conclusion, our data shows that IKKβ is a key regulator of platelet activation, in vitro and in vivo , and the remodeling of the CBM complex. Furthermore, our findings indicate that inducible clustering of signaling mediators and the formation of higher-order multi-protein complexes (i.e., the CBM complex) is a dynamic process, that supports nonlinear signaling networks and is important for platelet activation.

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