Rate Of Platelet Activation Research Articles

Simulation of the effect of hemolysis on thrombosis in blood-contacting medical devices

Heart failure, broadly characterized by the gradual decline of the ability of the heart to maintain adequate blood flow throughout the body's vascular network of veins and arteries, is one of the leading causes of death worldwide. Mechanical Circulatory Support is one of the few available alternative interventions for late-stage heart failure with reduced ejection fraction. A ventricular assist device is surgically implanted and connected to the left and or right heart ventricles to provide additional bloodflow, off-loading the work required by the heart to maintain circulation. Modern mechanical circulatory support devices generate non-physiological flow conditions that can lead to the damage and rupture of blood cells (hemolysis), and the formation of blood clots (thrombosis), which pose severe health risks to the patient. It is essential to improve prediction tools for blood damage to reduce the risk of hemolysis and thrombosis. A simulation-based approach examines the interaction between hemolysis and thrombosis. Incompressible finite-volume computational fluid dynamics simulations are executed on an open-hub axial flow ventricular assist device. A continuum model of thrombosis and the intrinsic coagulation process is extended to include the effect of hemolysis. The model accounts for the effect of activation of platelets by shear stress, paracrine signaling, adhesion, and hemoglobin and ADP released during hemolysis. The effect of hemolysis with thrombosis is modelled by accounting for the hyper-adhesivity of von-Willebrand Factor on extracellular hemoglobin, and the increased rate of platelet activation induced by ADP release. Thrombosis is assessed at varying inflow rates and rotor speeds, and cases are executed where thrombosis is affected by ADP release and Hb-induced hyper-adhesivity. It is found that there is a non-negligible effect from hemolysis on thrombosis across a range of rotor speeds, and that hyperadhesivity plays a dominant role in thrombus formation in the presence of hemolysis.

Preparation Method and Quality Evaluation of Novel Frozen Human Platelets

To optimize the technical parameters related to the preparation of novel frozen human platelets and formulate corresponding protocol for its preparation. Novel frozen human platelets were prepared with O-type bagged platelet-rich plasma (PRP), the key technical parameters (DMSO addition, incubation time, centrifugation conditions, etc.) of the preparation process were optimized, and the quality of the frozen platelets was evaluated by routine blood tests, apoptosis rate, platelet activation rate and surface protein expression level. In the preparation protocol of novel frozen human platelets, the operation of centrifugation to remove supernatant was adjusted to before the procedure of platelets freezing, and the effect of centrifugation on platelets was minimal when the centrifugation condition was 800×g for 8 min. In addition, platelets incubated with DMSO for 30 min before centrifugation exhibited better quality after freezing and thawing. The indexes of novel frozen human platelets prepared with this protocol remained stable after long-term cryopreservation. The preparation technique of novel frozen human platelets was established and the protocol was formulated. It was also confirmed that the quality of frozen platelets could be improved by incubating platelets with DMSO for 30 min and then centrifuging them at 800×g for 8 min in the preparation of novel frozen human platelets.

Preparation of aminated poly (L‐lactic acid) film coated with multilayers of mussel‐inspired heparin‐like carboxymethylcellulose sodium and chitosan for anticoagulant materials

AbstractIn this study, dopamine hydrochloride was grafted onto the sulfated carboxymethylcellulose sodium with different sulfated degree (DA‐g‐SCMCS) as heparin‐like polysaccharides. By studying the preliminary structure and coagulation time of DA‐g‐SCMCS, the DA‐g‐SCMC2 possessing good anticoagulant properties were selected to be assembled layer by layer on the surface of aminated poly (L‐lactic acid) (APLLA) film with chitosan (CS). The characterization properties including morphologies and wettability of DA‐g‐SCMC2/CS multilayers modified APLLA films were studied by scanning electron microscope and water contact angle instrument. Anticoagulation tests revealed that the (DA‐g‐SCMC2/CS)15 multilayers modified APLLA film experienced a decrease of 37.5% and 73.68% in the adsorption of bovine serum albumin (BSA) and fibrinogen (FBG), respectively. And the platelet adhesion ability was reduced by 92.42% compared with the APLLA film. The degree of platelet activation (PF4 and TAT) and hemolysis rates of modified APLLA films also decreased significantly. Besides, the modified APLLA films promoted the growth of L929 cells and exhibited remarkable anti‐inflammatory and antibacterial abilities. To summarize, the APLLA films modified with heparin‐like multifunctional DA‐g‐SCMC2/CS multilayers exhibit outstanding biocompatibility, cytocompatibility, anti‐inflammatory, and antibacterial characteristics, showcasing their potential as anticoagulation materials.

Inhibitory Effect of Heparin/Polyarginine/Hyaluronic Acid Complex on Coronary Artery Restenosis by Loading Vascular Endothelial Growth Factor

This research was aimed to investigate the inhibitory effect of heparin/polyarginine/hyaluronic acid (HPHA) complex loaded with vascular endothelial growth factor (VEGF) on coronary artery restenosis (CAR). Dopamine was grafted to obtain dopamine-hyaluronic acid (DPA) composite material, and heparin/polyarginine (HP) nanoparticles (NPs) loaded with VEGF were obtained through electrostatic binding (ESB). The HP NPs loaded with VEGF mixed with hyaluronic acid (HA) were mixed in a 10:1 volume ratio to obtain a composite solution, namely HPHA complex. Next, the sodium metaperiodate was added to form a hydrogel. The samples were characterized using a zeta potential (ZP) analyzer, transmission electron microscopy (TEM), Fourier transform infrared spectrometer (FTIR), and water contact angle (WCA). Enzyme-linked immunosorbent assay (ELISA) and toluidine blue O (TBO) staining were employed to assess the immobilization and release of VEGF and heparin. Rhodamine staining was adopted to evaluate the adhesion of platelets, fibrinogen, macrophages, and umbilical artery smooth muscle cells (UASMCs). Furthermore, CCK-8 assay was performed to evaluate the adhesion and proliferation of endothelial cells (ECs). The results revealed that compared to 0 mg/L, the particle size (PS), polydispersity index (PDI), and θ angle decreased, and the absolute value of ZP increased at 100, 200, 300, and 400 mg/L. The cumulative release (CR) of VEGF at 200 mg/L was higher to that at 0 mg/L, while that of heparin was lower (P <0.05). The platelet surface coverage rate (PSCR), relative platelet activation rate (RPAR), and fibrinogen coverage rate (FCR) at 10 and 20 days were lower and exhibited great difference to those at 0 mg/L (P <0.05). Compared to groups II and III, group I presented a markedly higher number of macrophages and UASMCs on the first and third days of culture (P <0.05). 3 days after culture, adhesion and proliferation of ECs in groups U, I, and K increased greatly (P <0.05). In conclusion, the HPHA complex prepared from HP NPs loaded with VEGF and HA in this research exhibited good stability, uniformity, hydrophilicity, and blood compatibility. It effectively blocked the adhesion of macrophages and UASMCs and promoted the proliferation of ECs, thus conforming to the mechanism of inhibiting CAR after coronary stent intervention.

BIO3: Advancement of a Novel Polymeric Transcatheter Aortic Valve as an Alternative to Tissue-based Valves

Background: Transcatheter aortic valve replacement (TAVR) is a minimally invasive alternative to surgical valve replacement, originally targeted to elderly patients with aortic stenosis (AS), now increasingly utilized in younger lower risk AS patients. With growth of TAVR the shortcomings of current tissue-based valves are becoming prevalent, namely limited durability and thromboembolic risk. To address these, a polymeric TAV named PolyV-2 (27 mm, PolyNova Cardiovascular Inc, Stony Brook, NY) was designed, optimized, and further tested. Methods: The design of the PolyV-2 (Figure A) was achieved with a battery of in silico simulations including the use of finite element analysis (FEA, Abaqus 2020, Dassault Systems, Providence, RI) to optimize the radial force of the stent frame and minimize leaflet stresses during the cardiac cycle. The PolyV-2 employs a sutureless design via coating the stent with polymer, bypassing problematic hand suturing manufacture of tissue valves. The PolyV-2 utilizes a thermoplastic elastomer polymeric material which was tested in vitro with accelerated wear durability testing according to ISO 5840-3 (2015) with periodic performance testing, evaluated against tissue-based surgical and TAV devices for platelet activation rate (PAR) in a pulsatile flow loop, and evaluated for calcification risk with similar wear testing in a pro-calcific medium. To assess the deposition of calcifications in the valves, both µCT and ICP spectroscopy were used. Results: The optimization process of the PolyV-2 leaflets (Figure B) was able to greatly lower the peak and mean stresses throughout over the cardiac cycle by employing a variable thickness across the entire leaflet. With success of this in silico optimization process, the leaflets were then tested in vitro (Figure C) in accelerated wear testing, performing 1Billion cycles w/o failure – correlating to 25 years of efficacy in vivo, exceeding the 250 million minimum required by the FDA. The material also demonstrated favorable hemocompatiblity as to PAR (Figure D) with activation significantly lower than a commercial tissue TAVR device, more comparable to a surgical device. The pro-calcific wear testing demonstrated no deposition of calcific salts within the polymer leaflet (µCT image, Figure E) compared to the tissue valve and minimal calcium detected with ICP spectroscopy. Conclusion: To mitigate the persistent complications of tissue-based TAV devices, the polymeric PolyV-2 valve was optimized through a battery of in silico and in vitro trials to offer superior hemodynamics, performance, and durability. Proper design and material considerations are essential to ensure successful translation, demonstration of enhanced bio/hemocompatibility and advance of the PolyV-2 through in vivo animal trials.

The rate of platelet activation determines thrombus size and structure at arterial shear

BackgroundThe response of platelets to activating stimuli and pharmaceutical agents varies greatly within the normal population. Current platelet function tests are used to measure end-point levels of platelet activation without taking the speed at which platelets activate into account, potentially missing vital metrics to characterize platelet reactivity. ObjectivesTo identify variability, to agonists and among individuals, in platelet activation kinetics and assess the impact of this on thrombus formation. MethodsWe have developed a bespoke real-time flow cytometry assay and analysis package to measure the rate of platelet activation over time using 2 parameters of platelet activation, fibrinogen binding and P-selectin exposure. ResultsThe rate of platelet activation varied considerably within the normal population but did not correlate with maximal platelet activation, demonstrating that platelet activation rate is a separate and novel metric to describe platelet reactivity. The relative rate of platelet response between agonists was strongly correlated, suggesting that a central control mechanism regulates the rate of platelet response to all agonists. ConclusionFor the first time, we have shown that platelet response rate corresponds to thrombus size and structure, wherein faster responders form larger, more densely packed thrombi at arterial, but crucially not venous, shear. We have demonstrated that the rate of platelet activation is an important metric in stratifying individual platelet responses and will provide a novel focus for the design and development of antiplatelet therapy, targeting high-shear thrombosis without exacerbating bleeding at low shear.

Experimental Investigation of Microfluidic Device for Platelet Activation.

Wound healing is accelerated when Platelet Rich Plasma is activated and growth factors are released. In this study, it was aimed to stimulate platelets without using chemical stimulants. Two types of mechanical platelet activation methods have been proposed in this study. The first one is a microfluidic chip developed with the shear-induced platelet activation approach. The second one is a piezo-based ultrasound-assisted device which provides platelet activation by stimulating with an ultrasonic wave (0.55 and 1.1 MHz). Three different microfluidic chip designs were worked out to determine the optimal shear stress characteristics; 8-nodes (2789 µs, 288 shear pulses, and 98.3 dyne/cm2), 40-nodes (2765 µs, 1440 shear pulses, and 95.5 dyne/cm2) and pillar-shaped (1030 µs, 1656 shear pulses, and 48.1 dyne/cm2). The highest platelet activation rate (72.7%) was obtained from the chips with 8-nodes. In the ultrasound-assisted device, 32.4% activation rate was obtained from ultrasound waves with 0.55 MHz frequency and 10 Vp-p amplitude. These activation rates, determined by CD62P (P-Selectin) expression, are significantly higher than spontaneous activation of intact platelets (8.5%). In addition, the gradual increase in activation of stimulated platelets with incubation at room temperature showed that activation continued after stimulation. The results showed that these microfluidic devices can be used for platelet activation to enhance the effect of PRP treatment and might reduce adverse immune reactions that may happened due to the use of exogenous activator substances. Fast-response, low-cost, easy-to-use and controllable biomedical device have been developed for PRP applications.

Kinetx: A Combined Flow Cytometry Assay and Analysis Software Framework to Quantitatively Measure and Categorize Platelet Activation in Real-time.

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests measure endpoint responses after a defined time period and not the rate of platelet activation. However, the rate at which platelets convert extracellular stimuli into a functional response is an essential factor in determining how efficiently they can respond to injury, bind to a forming thrombus, and signal to recruit other platelets. This paper describes a flow cytometry-based platelet function assay that enables simultaneous data acquisition and sample stimulation and utilizes newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux. Kinetix was developed in R so that users can alter parameters such as degree of smoothing, identification of outlying data points, or time scales. To aid users unfamiliar with the R environment, Kinetix analysis of data can be performed by a single command. Together, this allows real-time platelet activation metrics, such as rate, acceleration, time to peak-rate, time to peak-calcium, and qualitative shape changes, to be accurately and reproducibly measured and categorized. Kinetic measurements of platelet activation give a unique insight into platelets' behavior during the first stages of activation and may provide a method of predicting the recruitment of platelets into a forming thrombus.

Is “one size fits all” anti-aggregation really effective? Variability in the response to P2Y12 receptor inhibitors in obese patients

Abstract Background Different “ex vivo” studies have shown both a greater platelet activation and higher rates of resistance to clopidogrel in obese patients. Although there is less evidence, less prasugrel activity has also been observed in these patients. Our aim was to study the variability of the response to clopidogrel, ticagrelor and prasugrel in obese patients, defined as a body mass index ≥30. Methods Prospective, multicenter, observational, pharmacodynamic study, conducted in a Spanish population of patients with an acute coronary syndrome (ACS) treated with percutaneous coronary intervention (PCI) and double anti-aggregation with acetylsalicylic acid and a P2Y12 receptor inhibitor. Platelet function tests were performed the morning after the ICP and 30 days after it, including: 1) VerifyNow P2Y12 assay; 2) multiple electrode aggreometry (Multiplate); and 3) VASP analysis. Results Of the total patients included (988), 300 were obese (30.3%). The obese group was younger (62.8±12 years vs 64.9±12), had a higher incidence of arterial hypertension (76.3% vs. 56.7%), diabetes mellitus (35% vs. 27.5%); and lower incidence of chronic kidney disease (7.7% vs. 17%). There were no differences in the acute phase (day 1 after PCI) in the pharmacodynamic response to any of the P2Y12 inhibitors used. After 30 days, greater platelet aggregation (decreased response) was documented in obese patients treated with prasugrel according to VASP tests (PRI in non-obese 23.9±13% vs. 30.4±14.7% in obese, p 0.035) and MEA (area under the aggregation units curve in non-obese 251.7±104.1 vs 320±166.7 in obese, p 0.007) and a numerical trend with VerifyNow. A trend in the same direction was also observed in patients treated with clopidogrel that did not reach statistical significance with all the platelet function tests used. No differences were observed in the ticagrelor group. Conclusion Obese patients with an ACS treated with PCI have a worse response to thienopyridines than non-obese patients in the maintenance phase of antiaggregant treatment, while the response to ticagrelor is not affected by obesity. Completing the clinical follow-up proposed by the registry is necessary to know if these differences have an implication in cardiovascular events. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Fondo de Investigaciones Sanitarias (FIS)

Accelerating availability of clinically-relevant parameter estimates from thromboelastogram point-of-care device.

Modeling approaches offer a novel way to detect and predict coagulopathy in trauma patients. A dynamic model, built and tested on thromboelastogram (TEG) data, was used to generate a virtual library of over 160,000 simulated RapidTEGs. The patient-specific parameters are the initial platelet count, platelet activation rate, thrombus growth rate, and lysis rate (P(0), k1, k2, and k3, respectively). Patient data from both STAAMP (n = 182 patients) and PAMPer (n = 111 patients) clinical trials were collected. A total of 873 RapidTEGs were analyzed. One hundred sixteen TEGs indicated maximum amplitude (MA) below normal and 466 TEGs indicated lysis percent above normal. Each patient's TEG response was compared against the virtual library of TEGs to determine library trajectories having the least sum-of-squared error versus the patient TEG up to each specified evaluation time ∈ (3, 4, 5, 7.5, 10, 15, 20 minutes). Using 10 nearest-neighbor trajectories, a logistic regression was performed to predict if the patient TEG indicated MA below normal (<50 mm), lysis percent 30 minutes after MA (LY30) greater than 3%, and/or blood transfusion need using the parameters from the dynamic model. The algorithm predicts abnormal MA values using the initial 3 minutes of RapidTEG data with a median area under the curve of 0.95, and improves with more data to 0.98 by 10 minutes. Prediction of future platelet and packed red blood cell transfusion based on parameters at 4 and 5 minutes, respectively, provides equivalent predictions to the traditional TEG parameters in significantly less time. Dynamic model parameters could not predict abnormal LY30 or future fresh-frozen plasma transfusion. This analysis could be incorporated into TEG software and workflow to quickly estimate if the MA would be below or above threshold value within the initial minutes following a TEG, along with an estimate of what blood products to have on hand. Therapeutic/Care Management: Level IV.

Measurement of Platelet Function in an Experimental Stroke Model With Aspirin and Clopidogrel Treatment.

Dual antiplatelet treatment (DAPT) increases the risk of tPA-associated hemorrhagic transformation (HT) in ischemic stroke. To investigate the effects of DAPT in rodents, reliable indicators of platelet function utilizing a minimally invasive procedure are required. We here established a fluorescence-based assay to monitor DAPT efficiency in a mouse model of ischemic stroke with HT. Male C57/BL6 mice were fed with aspirin and clopidogrel (ASA+CPG). Venous blood was collected, stimulated with thrombin, labeled with anti-CD41-FITC and anti-CD62P-PE, and analyzed by flow cytometry. Subsequently, animals were subjected to experimental stroke and tail bleeding tests. HT was quantified using NIH ImageJ software. In ASA+CPG mice, the platelet activation marker CD62P was reduced by 40.6 ± 4.2% (p < 0.0001) compared to controls. In vitro platelet function correlated inversely with tail bleeding tests (r = −0.8, p = 0.0033, n = 12). Twenty-four hours after drug withdrawal, platelet activation rates in ASA+CPG mice were still reduced by 20.2 ± 4.1% (p = 0.0026) compared to controls, while tail bleeding volumes were increased by 4.0 ± 1.4 μl (p = 0.004). Conventional tests using light transmission aggregometry require large amounts of blood and thus cannot be used in experimental stroke studies. In contrast, flow cytometry is a highly sensitive method that utilizes small volumes and can easily be incorporated into the experimental stroke workflow. Our test can be used to monitor the inhibitory effects of DAPT in mice. Reduced platelet activation is indicative of an increased risk for tPA-associated cerebral hemorrhage following experimental stroke. The test can be applied to individual animals and implemented flexibly prior and subsequent to experimental stroke.

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets

AbstractG protein–coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6−/− mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6−/− mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6−/− platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5′-diphosphate stimulation in GRK6−/− platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6−/− platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation.

GRK6 Regulates the Hemostatic Response to Injury through Its Rate-Limiting Effects on GPCR Signaling in Platelets

Inappropriate platelet activation remains a major cause of cardiovascular and cerebrovascular diseases. Most agonists activate platelets through G protein-coupled receptors (GPCRs). However, questions remain about mechanisms that provide negative feedback towards activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in WT controls during the early stages of thrombus formation, with a rapid increase of platelet accumulation at site of injury. Platelet activation in the absence of GRK6 is enhanced, but in an agonist-selective manner. Responses to PAR4 agonist peptide or ADP stimulation in GRK6-/- platelets are increased compared to WT control littermates, while the response to TxA2 is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that in human platelets, platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase of the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Collectively, these observations, for the first time, show that 1) GRK6 regulates the hemostatic response to injury by thrombin and ADP, 2) it mediates platelet activation by reducing PAR1/4- and P2Y12-dependent signaling, and 3) GRK6 limits the rate of platelet activation during early stage of thrombus growth and helps prevent inappropriate platelet activation. Disclosures No relevant conflicts of interest to declare.

Scutellarin Suppresses Platelet Aggregation and Stalls Lesional Progression in Mouse With Induced Endometriosis.

Platelets play an important role in the development of endometriosis. Scutellarin is a flavonoid isolated from a medicinal herb traditionally used as a potent antiplatelet agent. In this study, we sought to evaluate its potential therapeutic effect, if any, in mice with induced endometriosis. Endometriosis was induced in 27 female Balb/c mice by intraperitoneal injection of uterine fragments. Two weeks after the induction, the 27 mice were randomly divided in equal sizes into 3 groups: untreated, which received only vehicle, and low-dose and high-dose groups, which received low- and high dose of scutellarin treatment. Hotplate test was administrated to all mice before endometriosis induction, and before and after the scutellarin treatment. Two weeks after the treatment, a blood sample was drawn before sacrifice and all lesions were harvested. The peripheral platelet activation rate and total lesion weight were assessed, and immunohistochemistry and histochemistry analyses were performed to evaluate the extent of proliferation, angiogenesis, fibroblast-to-myofibroblast transdifferentiation (FMT), and fibrosis in lesions. Compared with untreated mice, mice in both low-dose and high-dose groups had significantly reduced lesion weight and improved hyperalgesia. Scutellarin also reduced the peripheral-activated platelets rate and resulted in significantly reduced platelet aggregation, cellular proliferation, angiogenesis, the extent of FMT, and the extent of fibrosis in lesions. Thus, we conclude that scutellarin is efficacious in treating endometriosis in vivo by suppressing platelet aggregation, inhibiting proliferation, angiogenesis, and fibrogenesis, resulting in reduced lesion size and improved pain behavior. As such, scutellarin may be a potentially promising therapeutics for the treatment of endometriosis.

Enhanced Platelet Sensitivity to IGF-1 in Patients with Type 2 Diabetes Mellitus.

Diabetes mellitus is characterized by increased platelet activation which is determined by many factors including changes in the expression of membrane proteins. The aim of this study was to investigate the sensitivity of human platelets to the insulin-like growth factor (IGF) system in patients with poorly controlled type 2 diabetes mellitus (DM2). Ligand binding was analyzed using 125I-labelled IGF-1 and insulin, and relative expression of insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) was evaluated by Western blotting. Platelet aggregation in the presence of IGF-1 was studied by the plate aggregometry assay. We found that platelets from DM2 patients exhibited significantly higher IGF-1 binding and upregulation of IGF-1R expression in comparison with healthy individuals. Both insulin binding and IR expression were lower in the DM2 group, but the differences with the healthy control were statistically insignificant. The potentiating effect of IGF-1 on the thrombin-induced activation of platelets was detected in both groups but was significantly more pronounced in the DM2 patients. The initial rate of platelet activation in the presence of IGF-1 positively correlated with the concentration of glycated hemoglobin. Platelets isolated from DM2 patients displayed elevated expression of the IGF-1R subunits, which might have contributed to the higher sensitivity of these cells to IGF-1 in thrombin-initiated aggregation by increasing the rate of platelet activation. However, further experiments are needed to investigate the role of IGF-1 in thrombotic complications that usually accompany diabetes.

Potential different impact of inhibition of thrombin function and thrombin generation rate for the growth of thrombi formed at site of endothelial injury under blood flow condition

IntroductionThrombin inhibitor and anti-Xa are now widely used in clinical practice. However, the difference between thrombin inhibitor and anti-Xa in prevention of thrombosis is still to be elucidated. Materials and methodsComputer simulator implementing the function of platelet, coagulation, fibrinolysis and blood flow was developed. The function of thrombin is defined as to activated platelet at the rate of 0.01 s−1 and to produce fibrin at the rate of 0.1 s−1 in control. The effect of thrombin inhibitor was settled to reduce the rate of platelet activation and fibrin generation changed from 10 to 100% as compared to the control. The local thrombin generation rate on activated platelet was settled as 1.0 s−1 as a control. The effect of anti-Xa was settled to reduce to thrombin generation rate on activated platelet from 10% to 100% as compared to the control. The sizes of thrombi formed at site of endothelial injury in the presence and absence of thrombin inhibitor and anti-Xa were compared. Results and conclusionsThe size of thrombi formed by 30-s perfusion of blood at site of endothelial injury reduced both in the presence of thrombin inhibitor and anti-Xa. There was significant positive relationship between thrombin inhibitor effect and the size of formed thrombi with R value of 0.96. (p < 0.0001) However, the sizes of thrombi were not influence by anti-Xa until it decreased 30% or less as compared to control. There was no significant relationship between anti-Xa effect and the size of formed thrombi. (R = 0.39, p = 0.09) Our results suggest the different dose-dependent effects of thrombin inhibitor and anti-Xa on thrombus formation at least in specific conditions. Computer simulation may help to predict quantitative antithrombotic effects of various antithrombotic agents.

Mean platelet volume as a diagnostic marker in children with acute hepatitis A

Objective The aim of this study was to assess the level of mean platelet volume (MPV) in children with acute hepatitis A as compared with the control group. Background MPV is a marker that indicates platelet size, function, and the rate of platelet production and activation. Patients and methods Our study was conducted on 100 Egyptian children, aged from 2 to 14 years (58 male and 42 female) previously diagnosed as having hepatitis A as a patient group and 100 apparently healthy age-matched and sex-matched children as a control group. All of them were subjected to full history, physical examination, C-reactive protein, erythrocyte sedimentation rate, complete blood count, MPV levels, liver function tests (alanine aminotransferase and aspartate aminotransferase), serum bilirubin level (total and direct), prothrombin time, and platelet count. The diagnosis of hepatitis A virus infection was based on antihepatitis A virus immunoglobulin M positivity. Results MPV in the study group was significantly lower than the control group (P 0.05), but the MPV level was directly proportionate to the platelet counts (P Conclusion This study demonstrated that MPV is a negative acute-phase reactant for children with acute hepatitis A.

Further Evidence for Hypercoagulability in Women With Ovarian Endometriomas.

Our previous studies have shown that platelets play a crucial role in the development of endometriosis, and women with endometriosis appear to be in a state of hypercoagulability. However, a recent study could only replicate part of our previous finding, casting doubts on this notion. We further investigated this question through a cross-sectional study by measuring additional coagulation factors in women with and without endometriosis. To this end, we conducted a cross-sectional study of 100 women with laparoscopically and pathologically diagnosed ovarian endometriomas (OMA) and another 100 women without endometriosis. The platelet count; platelet activation rate; maximum platelet aggregation rate; plasma levels of D-dimer, fibrinogen, fibrin degradation products (FDPs), plasma soluble P-selectin (sP-sel), and prothrombin fragment 1+2 (F1+2); prothrombin time; thrombin time (TT); and activated partial thromboplastin time were measured before surgery and 3 months after surgery, and their clinical data were recorded. These measurements were also performed in control patients. We found that, compared with controls, women with OMA had a significantly higher platelet activation rate and platelet aggregation rate, elevated plasma D-dimer, fibrinogen, FDPs, sP-sel, and F1+2 levels as well as shortened TT. Remarkably, TT was prolonged, and all the other coagulation measurements, except plasma fibrinogen level, were significantly reduced 3 months after surgical removal of endometriotic lesions. Thus, our study provides another piece of evidence that endometriosis is a hypercoagulable disease, and anticoagulation therapy may hold promises in treating endometriosis.

Freeze-Dried Human Platelet-Rich Plasma Retains Activation and Growth Factor Expression after an Eight-Week Preservation Period.

Study DesignControlled laboratory study.PurposeThis study aimed to evaluate the efficacy of platelet-rich plasma (PRP) stored at room temperature (RT), frozen, or after freeze-drying.Overview of LiteraturePRP enriches tissue repair and regeneration, and is a novel treatment option for musculoskeletal pathologies. However, whether biological activity is preserved during PRP storage remains uncertain.MethodsPRP was prepared from blood of 12 healthy human volunteers (200 mL/person) and stored using three methods: PRP was stored at RT with shaking, PRP was frozen and stored at −80℃, or PRP was freeze-dried and stored at RT. Platelet counts and growth factor content were examined immediately after preparation, as well as 2, 4, and 8 weeks after storage. Platelet activation rate was quantified by flow cytometry.ResultsPlatelet counts were impossible to determine in many RT samples after 2 weeks, but they remained at constant levels in frozen and freeze-dried samples, even after 8 weeks of storage. Flow cytometry showed approximately 80% activation of the platelets regardless of storage conditions. Almost no growth factors were detected in the RT samples after 8 weeks, while low but significant expression was detected in the frozen and freeze-dried PRP. Over time, the mean relative concentrations of various growth factors decreased significantly or disappeared in the RT group. In the frozen group, levels were maintained for 4 weeks, but decreased significantly by 8 weeks (p <0.05). The freeze-dried group maintained baseline levels of growth factors for the entire 8-week duration.ConclusionsFreeze-drying enables PRP storage while maintaining bioactivity and efficacy for extended periods.

ASSESSMENT OF PLATELET CONCENTRATE PREPARED FROM FRESH AND OVERNIGHT HELD WHOLE BLOOD

Separation of platelet rich plasma (PRP) within 8 hours after holding whole blood at ambient temperature is the current practice in the preparation of platelet concentrate (PC). However, the ability to hold whole blood for up to 24 hours prior to PC preparation would allow every unit of whole blood to be used regardless of the distance from the collection site to the processing centre. Therefore the aim of this study is to assess and compare the platelet quality and activation of the platelet concentrate prepared from fresh whole blood (within 8 hours from collection) and overnight hold of whole blood at room temperature. In this study, 23 units of PC were prepared freshly from whole blood (i.e., within 8 hours of collection) and another 23 units were prepared after 24 hours of storage at 20–24 ºC. The following parameters of each unit of PC were assessed: pH, total white blood cell (TWBC) count, platelet count, presence of swirling, and platelet activation. When the parameters were compared between groups, no significant difference in platelet activation rate was found on sampling days 1, 3, and 5. pH and TWBC count for both groups were within the quality requirements of the National Blood Centre (&gt; 75% units tested fall within the standard), but not all of the samples complied with the standard requirement for platelet count. All units of PC prepared after 24 hours showed the presence of swirling, whereas one unit of PC in the fresh group did not show swirling activity after 3 days of storage. Delaying whole blood processing for up to 24 hours does not significantly affect certain in vitro quality or activation parameters as compared with freshly prepared PC.