Aggregation In Platelet-rich Plasma Research Articles

Does Yogurt Enriched with Platelet-Activating Factor Inhibitors from Olive Oil By-Products Affect Gut Microbiota and Faecal Metabolites in Healthy Overweight Subjects? (A randomized, parallel, three arm trial.).

The effect of the daily consumption of a low-fat yogurt (150 g) enriched with Platelet-Activating Factor receptor (PAF-R) antagonists, or the plain one, on gut microbiota and faecal metabolites was investigated in healthy overweight subjects. A randomized, three-arm, double-blind, placebo-controlled, parallel-group study was performed that lasted 8 weeks. Blood and stools were collected and analyzed before and after the intervention. Our findings revealed that the intake of the enriched yogurt resulted in a significant increase in the levels of Bifidobacterium spp., Clostridium perfringens group and Firmicutes-to-Bacteroidetes (F/B) ratio. On the other hand, a significant increase in the levels of Lactobacillus and C. perfringens group was detected after the intake of the plain yogurt. The increase in the levels of C. perfringens group was inversely associated with the plasma catabolic enzyme of PAF, namely LpPLA2 (lipoprotein-associated phospholipase A2), a cardiovascular risk marker that has been linked with inflammation and atherosclerosis. Moreover, in the enriched with PAF-R antagonists yogurt group, the increased levels of C. perfringens group were also associated with lower PAF action assessed as ex vivo human platelet-rich plasma (PRP) aggregation. Additionally, a higher % increase in molar ratio of Branched Short Chain Fatty Acids (BSCFAs) was detected for both yogurt groups after the 8 week-intervention compared to control. The consumption of the enriched yogurt also resulted in a significant drop in faecal caproic levels and a trend for lower ratio of butyrate to total volatile fatty acids (VFAs) compared to baseline levels. Yogurt consumption seems to favorably affect gut microbiota while its enrichment with PAF-R antagonists from olive oil by-products, may provide further benefits in healthy overweight subjects. ClinicalTrials.gov (NCT02259205).

Apolipoprotein A-IV polymorphisms Q360H and T347S attenuate its endogenous inhibition of thrombosis

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbβ3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s−1) and low (300s−1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module

AbstractActivation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

TISSUE ENGINEERING MATRIX BASED ON POLYURETHANE: IN VITRO RESEARCH

HighlightsThe article focuses on a new polyurethane-based material that has been developed and tested in vitro. This highly porous material with satisfactory physical and mechanical, hemocompatibility and matrix properties, obtained by using an electrospinning method, is suitable for the fabrication of cardiovascular products. AbstractAim. To manufacture a polyurethane-based tissue engineered matrix and study its physical and mechanical characteristics, hemocompatibility and matrix properties in comparison with decellularized xenopericardium and sheep carotid artery.Methods. Matrices based on polyurethane were produced by electrospinning. The surface structure was studied by scanning electron microscopy, the physical and mechanical characteristics were studied using a Zwick/Roell Universal testing machine, hemocompatibility was studied according to ISO 10993-4-2020, and the matrix properties of the material were studied in a cell experiment with Ea.hy 926.Results. The structure of the 12% polyurethane matrix was represented by a fibrous network with interpenetrating pores. The physical and mechanical characteristics of polyurethane matrices corresponded to the parameters of the carotid artery of sheep more than xenopericardium. Polyurethane had optimal hemocompatibility: hemolysis of erythrocytes did not exceed 0.52%, platelet aggregation corresponded to the aggregation of platelet-rich plasma – 80%. Platelet adhesion to the surface of the polyurethane matrix is statistically significantly lower than adhesion to the xenopericardium (p = 0.0041). Cell adhesion, viability and metabolic activity of Ea.hy 926 cultured on the surface of polyurethane matrices were higher relative to xenopericardium: cell density was 236.3 [198.5; 264.6] cells/mm2 (p = 0.458), viability 19.0 [16.0; 25.0] % (p = 0.0145).Conclusion. Physical and mechanical characteristics, hemocompatibility and matrix properties of polyurethane confirmed its suitability for potential use for the needs of cardiovascular surgery.

Flavonol and A-type procyanidin-rich extracts of Prunus spinosa L. flower exhibit anticoagulant activity through direct thrombin inhibition, but do not affect platelet aggregation in vitro

Background: Blackthorn flower (Prunus spinosa L.) is a traditional herbal remedy recommended for treating cardiovascular diseases (CVDs).Aim: This in vitro study investigates the effects of flavonol and A-type procyanidin-rich blackthorn flower extracts on the hemostatic system, including the blood plasma coagulation cascade and platelet aggregation.Methods: Six distinct extracts, characterized through various techniques, including LC-MS/MS, were assessed at in vivo-relevant levels (1–50 μg/mL) for their antithrombotic activity. The thrombin, prothrombin, and activated partial thromboplastin times were measured. Additionally, the thrombin enzymatic activity was tested using the chromogenic substrate S-2238 and fibrinogen as the physiological substrate of the enzyme. To gain insights into the mechanism of action, the interactions between the primary extracts’ constituents, their potential metabolites, and thrombin were examined in silico. The computational analyses were complemented by in vitro experiments and circular dichroism spectroscopy. The platelet aggregation in human platelet-rich plasma was assessed after ADP or collagen stimulation. Furthermore, the extracts’ biocompatibility was tested on human peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs).Results: The extracts slightly prolonged the prothrombin and thrombin times and effectively inhibited the thrombin’s enzymatic activity, reducing its amidolytic and proteolytic functions at 50 μg/mL by 91.2% and 74.8%, respectively. In silico molecular docking demonstrated a strong binding affinity of the examined polyphenols and their metabolites to thrombin. Most analytes bound exclusively within the enzyme active site; however, afzelin, kaempferitrin, and procyanidin A2 revealed the affinity to additional binding sites, including exosite I. The structure-activity relationship of flavonols as thrombin inhibitors was studied in vitro. Circular dichroism spectroscopy confirmed that the interactions between thrombin and the compounds (even at 1 μg/mL) induce alterations within the α-helices’ secondary structure, resulting in noticeable changes in the enzyme’s CD spectrum. On the other hand, the extracts did not influence platelet aggregation. Eventually, their cellular biocompatibility with PBMCs and RBCs was confirmed.Conclusion: The extracts directly inhibit thrombin, a critical serine protease in hemostasis and a prime anticoagulant drug target, and do not exhibit antiplatelet effects. This study enhances the knowledge of the biological activity of blackthorn flowers and supports their traditional use in CVDs.

In vitro comparison of platelet aggregation between zalunfiban and selatogrel in healthy donors

Abstract Background Two subcutaneously injected antiplatelet drugs, zalunfiban and selatogrel, are under development for prehospital administration for STEMI and (N)STEMI, respectively. Zalunfiban is a glycoprotein IIb/IIIa inhibitor that produces high-grade inhibition of platelet aggregation induced by ADP or a thrombin receptor activating peptide (TRAP) within 15 minutes. Selatogrel is a P2Y12 inhibitor that produces high-grade inhibition of platelet aggregation initiated by ADP within 15 minutes. We have studied the relative potencies of the drugs at levels achievable in vivo in inhibiting platelet aggregation. Since trisodium citrate (TSC) has been reported to enhance the antiplatelet effect of zalunfiban and inhibit the antiplatelet effect off selatogrel, aggregation studies were performed with both blood anticoagulated with TSC and the non-chelating anticoagulant PPACK. Whereas both ADP and thrombin are present at the site of vascular injury within seconds, we tested the effects of both ADP and TRAP. Purpose To compare directly the effects of zalunfiban and selatogrel on ADP- and TRAP-induced platelet aggregation. Methods Light transmission platelet aggregometry was performed within 4 hours of blood collection (BioData PAP-8E). Whole blood from healthy donors not taking aspirin (N=10) was anticoagulated with 3.2% TSC and 100 μM PPACK. Platelet-rich plasma (PRP) was prepared and adjusted to a platelet count of 250,000/µl. Three clinically relevant incremental concentrations of zalunfiban and selatogrel were added to PRP and platelet aggregation was initiated with 20 μM ADP or 20 μM TRAP. Tracings were recorded for at least 10 minutes after agonist addition. Platelet aggregation was quantified based on the instrument’s measurement of the primary slope (PrSl) of aggregation, which is a measure of the change in light transmission per unit time sustained over at least a 15 second period. Results In TSC-anticoagulated PRP, zalunfiban was more effective in inhibiting the PrSl of both ADP- and TRAP-induced platelet aggregation at all drug concentrations [Figure 1, panel A and B]. In PPACK-anticoagulated PRP, selatogrel showed greater inhibition of ADP-induced platelet aggregation at the lowest concentration tested, similar inhibition at the intermediate concentration, and slightly less inhibition at the highest doses. In sharp contrast, with TRAP-induced aggregation, zalunfiban demonstrated dose-related inhibition, reaching greater than 80% inhibition at the highest dose, whereas selatogrel produced less than 20% inhibition at all three doses. Conclusion Selatogrel is a potent inhibitor of ADP-induced platelet aggregation, but has low impact on TRAP-induced platelet aggregation, whereas zalunfiban is a potent inhibitor of both ADP and TRAP-induced platelet aggregation. The difference between the drugs needs to be considered in light of the known generation of thrombin at the site of vascular injury within seconds.Comparison of Platelet Inhibition

The Anti-Aggregative Potential of Resolvin E1 on Human Platelets.

Resolvin E1 is a metabolite of eicosapentaenoic acid (EPA) which is one of the omega-3 polyunsaturated fatty acids (omega-3 PUFAs). The antiplatelet properties of omega-3 PUFAs are well known, but the effect of resolvin E1 on platelets via the collagen receptors is extremely poorly reported. We investigated the effect of resolvin E1 on collagen-induced platelet aggregation, activation, and reactivity, and also platelet membrane fluidity. The ultimate and statistically significant results showed that resolvin E1 may inhibit platelet reactivity due to the reduction of collagen-induced platelet aggregation in platelet-rich plasma and isolated platelets, but not in whole blood. Also, resolvin E1 significantly reduced P-selectin exposure on collagen-stimulated platelets. Moreover, we demonstrated that resolvin E1 can maintain platelet membrane structure (without increasing membrane fluidity). The association between platelet reactivity and membrane fluidity, including resolvin E1 and collagen receptors requires further research. However, the goal of this study was to shed light on the molecular mechanisms behind the anti-aggregative effects of resolvin E1 on platelets, which are still not fully clarified. We also indicate an innovative research direction focused on further analysis and then use of omega-3 PUFAs metabolites as antiplatelet compounds for future applications in the treatment and prevention of cardiovascular diseases.

Ex vivo STUDY OF THE ACTION OF INTEGRIN RECEPTORS ANTAGONIST FROM ECHIS MULTISQUAMATIS SNAKE VENOM ON PLATELETS OF PREGNANT WOMEN WITH COMPICATIONS DURING GESTATION

Aim. In our work, we studied platelet aggregation in blood plasma of pregnant women and estimated the possibility of ex vivo normalization of aggregation rate using a polypeptide from the Echis multisquamatus snake venom. Previous reports demonstrated that it directly interacts with glycoprotein IIb/IIIa receptors on the surface of platelets, preventing their adhesion, thereby affecting the degree of aggregation. Methods. chromatography followed by size-exclusion chromatography on Superdex 75 using the FPLC system (ӒKTA, GE Healthcare, USA). Analysis of molecular weight of protein components was performed using SDS-PAGE. The concentration of protein was measured using spectrophotometer Optizen POP (Korea) at 280 nm. The ability of obtained protein to inhibit platelet aggregation was measured directly by aggregometry. Blood samples of women with placental disfunction during pregnancy (n = 28) were kindly provided by “Perinatal Center of Kyiv”. This study was approved by the Ethics Commission of the Shupyk National Medical Academy of Postgraduate Education and the Ethics Commission of the Kyiv Perinatal Center (# 3 from 05/05/2020). Aggregation of platelet-rich plasma (PRP) induced by ADP was investigated using aggregometry on the AP 2110 (Solar, Belarus). We compared the rate of platelet aggregation in the presence vs absence of platelet aggregation inhibitor. Results. Two-step chromatography protocol allowed us to obtain the polypeptide from the venom of Echis multisquamatus that possessed the anti-aggregatory action. SDS-PAGE analysis confirmed the homogeneity of obtained polypeptide with apparent molecular weight 14 kDa that corresponds to the platelet aggregation inhibitor reported earlier. Initial studies of ADPinduced platelet aggregation allowed selecting active concentration for the effective inhibitory action as 0.02 mg/ml. Conclusions. Platelet aggregation inhibitor from Echis multisquamatis snake venom of can be assumed as the effective agent that reduce the rate of platelet aggregation. We demonstrated it efficacy in platelet rich plasma of pregnant women that had placenta dysfunction. The use of direct antagonist of platelet integrin receptors was assumed as the prospective approach for suppressing of platelet reactivity in particular during complicated pregnancy.

Effects of uridine and nucleotides on hemostasis parameters

Several purinergic receptors have been identified on platelets which are involved in hemostatic and thrombotic processes. The aim of the present study was to investigate the effects of uridine and its nucleotides on platelet aggregation and hemostasis in platelet-rich plasma (PRP) and whole blood. The effects of uridine, UMP, UDP, and UTP at different final concentrations (1 to 1000 µM) on platelet aggregation were studied using an aggregometer. In PRP samples, platelet aggregation was induced by ADP, collagen and epinephrine 3min after addition of uridine, UMP, UDP, UTP and saline (as a control). All thromboelastogram experiments were performed at 1000 µM final concentrations of uridine and its nucleotides in whole blood. UDP and UTP were also tested in thromboelastogram with PRP. Our results showed that UDP, and especially UTP, inhibited ADP- and collagen-induced aggregation in a concentration-dependent manner. In whole blood thromboelastogram experiments, UDP stimulated clot formation while UTP suppressed clot formation. When thromboelastogram experiments were repeated with PRP, UTP's inhibitory effect on platelets was confirmed, while UDP's stimulated clot forming effect disappeared. Collectively, our data showed that UTP inhibited platelet aggregation in a concentration-dependent manner and suppressed clot formation. On the other hand, UDP exhibited distinct effects on whole blood or PRP in thromboelastogram. These data suggest that the difference on effects of UTP and UDP might have arisen from the different receptors that they stimulate and warrant further investigation with regard to their in vivo actions on platelet aggregation and hemostasis.

Modulation of platelet functions by European toad (Bufo Bufo) skin secretions components

Background: A growing number of reports indicate that amphibian skin secretions may have a remarkable medical importance; however, the effects of the components of some dermal secretions on blood platelets and hemostasis are inadequately recognized. Since our previous studies demonstrated that the general Bufo bufo skin secretions induced platelet aggregation in platelet-rich plasma, this work was designed to study the effects of the components of some fractions on platelet functions to comprehend its possible mechanism of action as platelet modulators. Methods: Chromatographic separation of B. bufo general skin secretions was carried out using size exclusion chromatography. Rabbit platelets were purified by column chromatography on Sepharose 4B. Various aspects of platelet function such as activation, aggregation, and adhesion were evaluated. Results: One fraction, out of 7, dose-dependently induced aggregation of isolated platelets and was used in further experiments. The studied fraction was shown to induce platelet adhesion onto fibrinogen-coated surface. Furthermore, the results demonstrated the effects of the fraction on some processes that involved in platelets activation: The fraction components facilitated (Ca2+) i mobilization and attenuated platelets Akt phosphorylation, but had no effect on platelet serotonin secretion. Membrane integrity was determined using lactate dehydrogenase (LDH) assay. No increased LDH release was recorded that means no platelet damage, which could lead to misinterpretation of the data, occurred. Conclusion: The results suggest that components of the B. bufo skin secretions may be a promising source of natural compounds which can modulate platelet functions.

Heparin and heparin proteoglycan-mimetics activate platelets via PEAR1 and PI3Kβ

BackgroundPlatelet endothelial aggregation receptor 1 (PEAR1) is a single-transmembrane orphan receptor primarily expressed on platelets and endothelial cells. Genetic variants of PEAR1 have repeatedly and independently been identified to be associated with cardiovascular diseases, including coronary artery disease. ObjectivesWe have identified sulfated fucoidans and their mimetics as ligands for PEAR1 and proposed that its endogenous ligand is a sulfated proteoglycan. The aim of this study was to test this hypothesis. MethodsA heparin proteoglycan-mimetic (HPGM) was created by linking unfractionated heparin (UFH) to albumin. The ability of the HPGM, UFH and selectively desulfated heparins to stimulate platelet aggregation and protein phosphorylation was investigated. Nanobodies against the 12th to 13th epidermal growth factor-like repeat of PEAR1 and phosphoinositide 3-kinase (PI3K) isoform-selective inhibitors were tested for the inhibition of platelet activation. ResultsWe show that HPGM, heparin conjugated to an albumin protein core, stimulates aggregation and phosphorylation of PEAR1 in washed platelets. Platelet aggregation was abolished by an anti-PEAR1 nanobody, Nb138. UFH stimulated platelet aggregation in washed platelets, but desulfated UFH did not. Furthermore, HPGM, but not UFH, stimulated maximal aggregation in platelet-rich plasma. However, both HPGM and UFH increased integrin αIIbβ3 activation in whole blood. By using PI3K isoform-selective inhibitors, we show that PEAR1 activates PI3Kβ, leading to Akt phosphorylation. ConclusionOur findings reveal that PEAR1 is a receptor for heparin and HPGM and that PI3Kβ is a key signaling molecule downstream of PEAR1 in platelets. These findings may have important implications for our understanding of the role of PEAR1 in cardiovascular disease.

Purification and characterization of platelet aggregation inhibitor from the venom of Bitis arietans

Disintegrins are the antagonists of integrin receptors that can be found mostly in snakes’ venom. They can inhibit platelet aggregation, thus preventing the formation of blood clots. By blocking the integrin receptors of cancer cells, disintegrins can inhibit proliferation and metastasis. Thus, the search for new sources of disintegrins and development of methods of their purification is an important task of modern biotechnolo­gy. This work was dedicated to the purification and characterization of inhibiting polypeptides from Bitis arietans­ venom. Crude venom of B. arietans was fractionated using ion-exchange chromatography on Q Sepharose followed by size-exclusion chromatography on Superdex 75 using FPLC method. Analysis of molecular weight of protein components was performed using SDS-PAGE and MALDI-TOF analysis on Voyager-DE. Aggregation of platelet-rich plasma (PRP) in the presence of platelet aggregation inhibitor was investigated using aggregometry on the AR2110. MTT test was used for measuring HeLa cells proliferation and survival in vitro. Two-step chromatography allowed us to obtain fraction that contained polypeptides possessing the dose-dependent inhibitory action on adenosine diphosphate (ADP)-induced platelet aggregation in PRP. SDS-PAGE showed that obtained fraction contained two polypeptides with molecular weight 9.0 and 13.67 kDa according­ to MALDI-TOF analysis. Purified polypeptides inhibited ADP-induced platelet aggregation with IC50 0.09 mg/ml. However, 0.005 mg/ml of fraction suppressed viability of HeLa cells according to MTT test on 20%. Discovered biological effects of fractions allowed us to conclude the possible use of these polypeptides as anti-aggregatory or anti-proliferative agents. Keywords: antithrombotic action, disintegrins, glycoprotein IIb/IIIa, platelets, snake venom

P17.10: Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation. This work was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development [Project No. PJ015607 (HK and KO)], Rural Development Administration, Republic of Korea. This research was partly supported by Hallym University Research Fund (HL).

Platelet aggregation under the conditions of vortex flow in vitro in patients with chronic heart failure

Aim. To compare the effect of increased concentrations of aggregation inducers (five-fold addition) under standard conditions and under the conditions of vortex flow in vitro on platelet aggregation in patients with chronic heart failure (CHF).Materials and methods. The study included 28 patients. The activity of platelet aggregation in platelet-rich plasma (PRP) was evaluated according to light transmission curves (%) and the average size of aggregates (relative units (rel. units)). The aggregation inducer was added once at 10 seconds of the study (standard procedure) and five times at 10 seconds, 1, 2, 3, and 4 minutes of the study with a constant stirring rate of 800 rpm. The same parameters were evaluated under the conditions of vortex flow, which was created by changing the stirring rate of the PRP from 800 rpm to 0 rpm and again to 800 rpm by pressing the centrifugation button on the analyzer.Results. In the course of the study, the size of the aggregates increased in patients with CHF only under the conditions of vortex flow. When a collagen aggregation inducer was added both at the concentration of 2 mmol / l and 10 mmol / l, platelet aggregation parameters increased under the conditions of vortex flow. During the study of epinephrine-induced platelet aggregation in patients with CHF, an increase in the aggregation parameters was revealed, both at five-fold addition of the inducer and under the conditions of vortex flow compared with the standard method.Conclusion. The proposed methodological approaches to creating the conditions for vortex flow in vitro and to five-fold addition of epinephrine showed an increase in the size of the aggregates and the degree of platelet aggregation. Collagen-induced aggregation under the conditions of vortex flow revealed 7 (25%) patients with high residual platelet reactivity (HRPR), and epinephrine-induced aggregation detected 15 (54%) patients with HRPR.

Risks in Induction of Platelet Aggregation and Enhanced Blood Clot Formation in Platelet Lysate Therapy: A Pilot Study.

Platelet concentrates (PCs) are widely used in regenerative medicine; as it is produced from freeze–thawing PC, platelet lysate (PL) has a longer shelf life. The thrombotic risk of PL therapy needs to be explored since PL and PC contain cytokines that contribute to platelet aggregation and thrombus formation. Whole blood samples of 20 healthy subjects were collected; PL was produced from PCs with expired shelf life through freeze–thawing. The direct mixing of PL with platelet-rich plasma (PRP) or whole blood was performed. In addition, rotational thromboelastometry (ROTEM) was used to investigate whether PL enhanced coagulation in vitro; the effects of fibrinogen depletion and anticoagulants were evaluated to prevent hypercoagulation. The results showed that PL induced platelet aggregation in both PRP and whole blood. In ROTEM assays, PL was shown to cause a significantly lower clotting onset time (COT) and clot formation time (CFT), and a significantly greater α angle and maximum clot firmness (MCF). Compared with the controls, which were 1:1 mixtures of normal saline and whole blood, fibrinogen depletion of PL showed no significant difference in CFT, α angle and MCF. Moreover, heparin- and rivaroxaban-added PL groups demonstrated no clot formation in ROTEM assays. Platelet lysate-induced hypercoagulability was demonstrated in vitro in the present study, which could be prevented by fibrinogen depletion or the addition of an anticoagulant.

ACTION OF VENOM OF VIPERA SNAKE OF UKRAINE ON BLOOD COAGULATION in vitro

Aim. In this study we focused on the search of fibrinogen-targeted proteases in the venom of Vipera renardi, Vipera nikolskii and Vipera berus. Venom of Vipera berus was also fractionated on Q-sepharose and action of separated fractions on human blood plasma, platelets and red cells was studied. Methods. Analysis of protein mixtures was performed using SDS-PAGE. Аction on the blood coagulation system was analyzed using the APTT assay. Identification of protein components with fibrinolytic activity was performed using enzyme-electrophoresis with fibrinogen as the substrate. Fractionation of V. berus venom was performed on Q-sepharose using FPLC system Acta Prime. Action of separated fractions on ADP-induced platelet aggregation in platelet rich blood plasma was analyzed using Aggregometer AP 2110. Hemolytic action of fractions was estimated using fresh human red cells. Amount of released hemoglobin was estimated by spectrophotometry on Optizen POP. Results. All studied venoms had different protein compositions with major protein fractions in the range from 25 kDa to 130 kDa. Both V. berus and V. nikolskii venoms taken in 1:200 dilutions reduced the time of clotting in APTT test from 25 to 13 s. In contrast, V. renardi venom in the same dilution prolonged the clotting time from 25 s to 180 s that we assumed as the result of fibrinogen-specific protease presence. According to enzyme-electrophoresis data all studied venoms contained fibrinogen-specific proteases with the apparent molecular weights for V. berus, V. nikolskii – 25-55 kDa. and V. renardi – 55-75 kDa. Fractionation of crude venom of V. berus allowed obtaining several fractions eluted at different concentrations of NaCl: 0.1; 0.2; 0.3; 0.5 М. Non-binded fraction was also collected. Conclusions. Thus, the components of Vipera venoms living in Ukraine can be used for basic biochemical research. At the same time, care should be taken in the case of envenomation, as the presence of fibrinogenolytic enzymes in the venom can lead to hemorrhage. Further characterization of fibrinogen-specific protease from V. berus venom is a promising task for biotechnology.

An optimized agonist peptide of protease-activated receptor 4 and its use in a validated platelet-aggregation assay

Protease-activated receptor 4 (PAR4) is a promising drug target to improve the efficacy/safety window of antiplatelet agents. The native peptide GYPGQV and the more potent peptide AYPGKF, are PAR4-specific activators. However, these PAR4 agonist peptides (APs) elicit an agonist response, for example, platelet aggregation, at concentrations of 50 to 1000 µM in platelet-function assays, thereby limiting their utility to monitor the pharmacodynamic effects of PAR4 antagonists over a wide concentration range. Improved pharmacodynamic assays are needed for clinical development of PAR4 antagonists. We attempted to identify potent PAR4 APs to aid development of robust assays for optimization of PAR4 antagonists. Using an AYPG-based biased phage display peptide library approach followed by chemical peptide optimization, A-Phe(4-F)-PGWLVKNG was identified. This peptide demonstrated an EC50 value of 3.4 µM in a platelet-aggregation assay, which is 16-fold more potent than AYPGKF. Using this new PAR4 AP, a platelet-rich plasma-aggregation assay using light-transmission aggregometry was developed and validated in a series of precision and reproducibility tests. PAR4 antagonist responses to PAR4 AP A-Phe(4-F)-PGWLVKNG (12.5 µM to 100 µM) were subsequently evaluated in this assay in vitro and ex vivo in a human study using BMS-986120, a PAR4 antagonist that entered clinical studies.

Multiparameter Evaluation of the Platelet-Inhibitory Effects of Tyrosine Kinase Inhibitors Used for Cancer Treatment.

Current antiplatelet drugs for the treatment of arterial thrombosis often coincide with increased bleeding risk. Several tyrosine kinase inhibitors (TKIs) for cancer treatment inhibit platelet function, with minor reported bleeding symptoms. The aim of this study was to compare the antiplatelet properties of eight TKIs to explore their possible repurposing as antiplatelet drugs. Samples of whole blood, platelet-rich plasma (PRP), or isolated platelets from healthy donors were treated with TKI or the vehicle. Measurements of platelet aggregation, activation, intracellular calcium mobilization, and whole-blood thrombus formation under flow were performed. Dasatinib and sunitinib dose-dependently reduced collagen-induced aggregation in PRP and washed platelets; pazopanib, cabozantinib, and vatalanib inhibited this response in washed platelets only; and fostamatinib, axitinib, and lapatinib showed no/limited effects. Fostamatinib reduced thrombus formation by approximately 50% on collagen and other substrates. Pazopanib, sunitinib, dasatinib, axitinib, and vatalanib mildly reduced thrombus formation on collagen by 10–50%. Intracellular calcium responses in isolated platelets were inhibited by dasatinib (>90%), fostamatinib (57%), sunitinib (77%), and pazopanib (82%). Upon glycoprotein-VI receptor stimulation, fostamatinib, cabozantinib, and vatalanib decreased highly activated platelet populations by approximately 15%, while increasing resting populations by 39%. In conclusion, the TKIs with the highest affinities for platelet-expressed molecular targets most strongly inhibited platelet functions. Dasatinib, fostamatinib, sunitinib, and pazopanib interfered in early collagen receptor-induced molecular-signaling compared with cabozantinib and vatalanib. Fostamatinib, sunitinib, pazopanib, and vatalanib may be promising for future evaluation as antiplatelet drugs.

Preferential Inhibition of Fibrin Mediated Thrombosis in Rodent Models By JNJ-9375, a Novel exosite1 Thrombin Inhibitor

We have previously reported an improved therapeutic index (TI) for JNJ-9375 vs apixaban in rat and cynomolgus monkey models of venous thrombosis and bleeding. We have also reported that JNJ-9375 preferentially inhibited the fibrin component of the thrombus without significant impact on the platelet component under low and high shear conditions in a human translational model of thrombosis (Badimon chamber model). We hypothesized that the novel mode of action of JNJ-9375 which by selectively inhibiting exosite 1 substrates from binding to thrombin (such as fibrinogen and PAR-1) may allow for other thrombin interactions at the active site and exosite 2 including platelet activation by PAR-4 and GP1b.In the present studies, we have compared the antithrombotic effects of JNJ-9375, the active site thrombin inhibitor dabigatran and the factor Xa inhibitor apixaban in well characterized and widely used rat AV-shunt and carotid ferric chloride models of venous and arterial thrombosis, respectively. JNJ-9375, dabigatran and apixaban all maximally inhibited thrombus formation in a dose dependent manner in the rat AV-shunt model of venous thrombosis. Significant inhibition of thrombus weights of 50%, 44% and 57% were observed at 0.3 mg/kg, i.v., 0.1 mg/kg, i.v. and 1 mg/kg, i.v. with JNJ-9375, dabigatran and apixaban respectively. Dose dependent prolongation in coagulation parameters were also observed ex-vivo with JNJ-9375 (TT, ECT, aPTT and PT), dabigatran (TT, ECT, aPTT) and apixaban (aPTT and PT). In addition, there were dose dependent increases in drug exposure.In the rat ferric chloride model of arterial thrombosis, JNJ-9375, dabigatran and apixaban administration also resulted in dose dependent inhibition of thrombus formation as assessed by the time to occlusion (TTO) of the carotid artery and area under the curve (AUC) for carotid blood flow. However, the dose required to inhibit arterial thrombosis Vs venous thrombosis varied substantially for the different agents. With JNJ-9375 we only observed a significant inhibition of arterial thrombosis (TTO and AUC) at a dose of 10 mg/kg, i.v. (Vs 0.3 mg/kg in the AV-shunt model) which represents a 33-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). In contrast, we observed a significant inhibition of arterial thrombosis with dabigatran (AUC) and apixaban (TTO and AUC) at doses of 0.3 mg/kg, i.v. and 3 mg/kg, i.v. (Vs 0.1 mg/kg and 1 mg/kg in the AV-shunt model) respectively. These doses of dabigatran and apixaban represent a 3-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). We have also documented dose dependent prolongations in coagulation parameters and drug exposure with all three agents in the these studies. In addition, we performed ex-vivo platelet aggregation in platelet rich plasma with various platelet agonists (ADP, collagen, AA, rat and human thrombin) in those animals dosed with JNJ-9375. As anticipated and consistent with its mechanism of action, JNJ-9375 only inhibited platelet aggregation to thrombin stimulation but not with other platelet agonists confirming the specificity of JNJ-9375. We observed about 50%, >95% and >95% inhibition of thrombin-induced platelet aggregation at 1, 3 and 10 mg/kg, i.v. with JNJ-9375 respectively. However, we only observed inhibition of arterial thrombosis at 10 mg/kg.In summary, we have demonstrated dose dependent inhibition of thrombosis with JNJ-9375, dabigatran and apixaban in venous and arterial model of thrombosis in rats. However, we have observed a larger difference in the dose required for inhibition of arterial Vs venous thrombosis with JNJ-9375 (33-fold) compared to a more modest 3-fold shift observed for dabigatran and apixaban. These findings with JNJ-9375 demonstrate an anticoagulant profile that preferentially inhibits fibrin mediated clot formation with a more modest impact on platelet driven thrombosis in the present models. This differential profile may account in part for the improved TI over apixaban we had reported earlier in animal models. This profile suggests JNJ-9375 which is currently in early clinical development may also be more amenable to dosing in combination with antiplatelet therapy for indications such as acute coronary syndromes and needs to be tested experimentally. DisclosuresChintala:Janssen Research & Development: Employment. Li:Janssen R&D: Employment. Huang Devine:Janssen Research & Development: Employment. Bunce:Janssen R&D: Employment.

Electrical impedance vs. light transmission aggregometry: Testing platelet reactivity to antiplatelet drugs using the MICELI POC impedance aggregometer as compared to a commercial predecessor

BackgroundPatients' responses to antiplatelet therapy significantly vary, with individuals showing high residual platelet reactivity associated with thrombosis. To personalize thrombosis management, platelet function testing has been suggested as a promising tool able to monitor the antithrombotic effect of antiplatelet agents in real-time. We have prototyped the MICELI, a miniature and easy-to-use electrical impedance aggregometer (EIA), measuring platelet aggregation in whole blood. Here, we tested the capability of the MICELI aggregometer to quantify platelet reactivity on antiplatelet agents, as compared with conventional light-transmission aggregometry (LTA). MethodsPlatelet aggregation in ACD-anticoagulated whole blood and platelet-rich plasma of healthy donors (n = 30) was evaluated. The effect of clopidogrel, ticagrelor, cangrelor, cilostazol, and tirofiban on ADP-induced aggregation was tested, while aspirin was evaluated with arachidonic acid and collagen. Platelet aggregation was recorded using the MICELI or BioData PAP-8E (Bio/Data Corp.) aggregometers. ResultsThe MICELI aggregometer detected an adequate and comparable dose-dependent decrease of platelet aggregation in response to increments of drugs' concentrations, as compared to LTA (the inter-device R2 = 0.79–0.93). Platelet aggregation in platelet-rich plasma recorded by LTA showed higher sensitivity to antiplatelet agents, but it couldn't distinguish between different drug doses as indicated by saturation of the aggregatory response. ConclusionPlatelet aggregation in whole blood as recorded by EIA represents a better model system for evaluation of platelet reactivity as compared with platelet aggregation in platelet-rich plasma as recorded by LTA, since EIA takes into consideration the modulatory effect of other blood cells on platelet hemostatic function and pharmacodynamics of antiplatelet drugs in vivo. As such, the MICELI impedance aggregometer could be potentially employed for the point-of-care monitoring of platelet function in patients on-treatment for personalized tailoring of their antiplatelet regimen.