Design, manufacturing and testing of a green non-isocyanate polyurethane prosthetic heart valve.

peer reviewedThe sole effective treatment for most patients with heart valve disease is valve replacement by implantation of mechanical or biological prostheses. However, mechanical valves represent high risk of thromboembolism, and biological prostheses are prone to early degeneration. In this work, we aim to determine the potential of novel environmentally-friendly non-isocyanate polyurethanes (NIPUs) for manufacturing synthetic prosthetic heart valves. Polyhydroxyurethane (PHU) NIPUs are synthesized via an isocyanate-free route, tested in vitro, and used to produce aortic valves. PHU elastomers reinforced with a polyester mesh show mechanical properties similar to native valve leaflets. These NIPUs do not cause hemolysis. Interestingly, both platelet adhesion and contact activation-induced coagulation are strongly reduced on NIPU surfaces, indicating low thrombogenicity. Fibroblasts and endothelial cells maintain normal growth and shape after indirect contact with NIPUs. Fluid-structure interaction (FSI) allows modeling of the ideal valve design, with minimal shear stress on the leaflets. Injection-molded valves are tested in a pulse duplicator and show ISO-compliant hydrodynamic performance, comparable to clinically-used bioprostheses. Poly(tetrahydrofuran) (PTHF)-NIPU patches do not show any evidence of calcification over a period of 8 weeks. NIPUs are promising sustainable biomaterials for the manufacturing of improved prosthetic valves with low thrombogenicity

Nutrition Phytochemicals Affecting Platelet Signaling and Responsiveness:Implications for Thrombosis and Hemostasis

Cardiovascular disease, in particular due to arterial thrombosis, is a leading cause of mortality and morbidity, with crucial roles of platelets in thrombus formation. For multiple plant-derived phytochemicals found in common dietary components, claims have been made regarding cardiovascular health and antiplatelet activities. Here we present a systematic overview of the published effects of common phytochemicals, applied in vitro or in nutritional intervention studies, on agonist-induced platelet activation properties and platelet signaling pathways. Comparing the phytochemical effects per structural class, we included general phenols: curcuminoids (e.g., curcumin), lignans (honokiol, silybin), phenolic acids (caffeic and chlorogenic acid), derivatives of these (shikimic acid), and stilbenoids (isorhapontigenin, resveratrol). Furthermore, we evaluated the flavonoid polyphenols, including anthocyanidins (delphinidin, malvidin), flavan-3-ols (catechins), flavanones (hesperidin), flavones (apigenin, nobiletin), flavonols (kaempferol, myricetin, quercetin), and isoflavones (daidzein, genistein); and terpenoids including carotenes and limonene; and finally miscellaneous compounds like betalains, indoles, organosulfides (diallyl trisulfide), and phytosterols. We furthermore discuss the implications for selected phytochemicals to interfere in thrombosis and hemostasis, indicating their possible clinical relevance. Lastly, we provide guidance on which compounds are of interest for further platelet-related research

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects

Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients

Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca2+ elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients

Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics

Abstract Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules

Ultra-high throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein coupled receptor activation

Antiplatelet drugs targeting G-protein coupled receptors (GPCR), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high throughput (UHT) method based on intracellular [Ca2+]i rises to differentiate GPVI and GPCR effects on platelets. In 96-, 384- or 1536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i rise when stimulated with the GPVI agonist collagen-related peptide (CRP), or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five early and late parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results provide proof-of-principle of distinct receptor-type dependent Ca2+ signaling curves platelets, which allow identification of new inhibitors in a UHT way

Protein C or Protein S deficiency associates with paradoxically impaired platelet-dependent thrombus and fibrin formation under flow

BACKGROUND: Low plasma levels of protein C or protein S are associated with venous thromboembolism rather than myocardial infarction. The high coagulant activity in patients with thrombophilia with a (familial) defect in protein C or S is explained by defective protein C activation, involving thrombomodulin and protein S. This causes increased plasmatic thrombin generation. OBJECTIVE: Assess the role of platelets in the thrombus‐ and fibrin‐forming potential in patients with familial protein C or protein S deficiency under high‐shear flow conditions. PATIENTS/METHODS: Whole blood from 23 patients and 15 control subjects was perfused over six glycoprotein VI–dependent microspot surfaces. By real‐time multicolor microscopic imaging, kinetics of platelet thrombus and fibrin formation were characterized in 49 parameters. RESULTS AND CONCLUSION: Whole‐blood flow perfusion over collagen, collagen‐like peptide, and fibrin surfaces with low or high GPVI dependency indicated an unexpected impairment of platelet activation, thrombus phenotype, and fibrin formation but unchanged platelet adhesion, observed in patients with protein C deficiency and to a lesser extent protein S deficiency, when compared to controls. The defect extended from diminished phosphatidylserine exposure and thrombus contraction to delayed and suppressed fibrin formation. The mechanism was thrombomodulin independent, and may involve negative platelet priming by plasma components. [Image: see text

Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26–34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1