Comparison of effects of anti-thrombin aptamers HD1 and HD22 on aggregation of human platelets, thrombin generation, fibrin formation, and thrombus formation under flow conditions

HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of HD1 and HD22 on thrombin-, ristocetin-, and collagen-induced human platelet aggregation, on thrombin generation and fibrin formation in human plasma, as well as on thrombus formation in human whole blood under flow conditions were assessed. The dissociation constants for HD1 and HD22 complexes with thrombin in simulated plasma ionic buffer were also evaluated. HD1 was more potent than HD22 in terms of inhibiting thrombin-induced platelet aggregation in platelet-rich plasma (PRP; 0.05-3 mu M) and in washed platelets (WPs; 0.005-3 mu M): approximately 8.31% (+/- 6.99% SD) and 89.53% (+/- 11.38% SD) for HD1 (0.5 mu M) and HD22 (0.5 mu M), respectively. Neither HD1 nor HD22 (3 mu M) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 mu M) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 mu M: 1677.53 +/- 55.77 (nM +/- min) vs. control 2271.71 +/- 423.66 (nM +/- min)], inhibited fibrin formation (lag time at 3 mu M: 33.70 min +/- 8.01 min vs. control 7.91 min +/- 0.91 min) and reduced thrombus formation under flow conditions [AUC(30) at 3 mu M: 758.30 +/- 344.23 (kPa +/- min) vs. control 1553.84 +/- 118.03 (kPa +/- min)]. HD22 (3 mu M) also delayed thrombin generation but increased the thrombin peak. HD22 (3 mu M) shortened the lag time of fibrin generation (5.40 min +/- 0.26 min vs. control 7.58 min +/- 1.14 min) but did not modify thrombus formation (3, 15 mu M). K-d values for the HD1 complex with thrombin was higher (257.8 +/- 15.0 nM) than the K-d for HD22 (97.6 +/- 2.2 nM). In conclusion, HD1 but not HD22 represents a potent anti-thrombotic agent, confirming the major role of exosite I in the action of thrombin. HD22 aptamer blocking exosite II displays weaker anti-platelet and anti-coagulant activity, with surprising activating effects on thrombin and fibrin generation most likely induced by HD22-induced allosteric changes in thrombin dynamic structure.</p

Comparison of Effects of Anti-thrombin Aptamers HD1 and HD22 on Aggregation of Human Platelets, Thrombin Generation, Fibrin Formation, and Thrombus Formation Under Flow Conditions

HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of HD1 and HD22 on thrombin-, ristocetin-, and collagen-induced human platelet aggregation, on thrombin generation and fibrin formation in human plasma, as well as on thrombus formation in human whole blood under flow conditions were assessed. The dissociation constants for HD1 and HD22 complexes with thrombin in simulated plasma ionic buffer were also evaluated. HD1 was more potent than HD22 in terms of inhibiting thrombin-induced platelet aggregation in platelet-rich plasma (PRP; 0.05–3 μM) and in washed platelets (WPs; 0.005–3 μM): approximately 8.31% (±6.99% SD) and 89.53% (±11.38% SD) for HD1 (0.5 μM) and HD22 (0.5 μM), respectively. Neither HD1 nor HD22 (3 μM) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 μM) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 μM: 1677.53 ± 55.77 (nM⋅min) vs. control 2271.71 ± 423.66 (nM⋅min)], inhibited fibrin formation (lag time at 3 μM: 33.70 min ± 8.01 min vs. control 7.91 min ± 0.91 min) and reduced thrombus formation under flow conditions [AUC30 at 3 μM: 758.30 ± 344.23 (kPa⋅min) vs. control 1553.84 ± 118.03 (kPa⋅min)]. HD22 (3 μM) also delayed thrombin generation but increased the thrombin peak. HD22 (3 μM) shortened the lag time of fibrin generation (5.40 min ± 0.26 min vs. control 7.58 min ± 1.14 min) but did not modify thrombus formation (3, 15 μM). Kd values for the HD1 complex with thrombin was higher (257.8 ± 15.0 nM) than the Kd for HD22 (97.6 ± 2.2 nM). In conclusion, HD1 but not HD22 represents a potent anti-thrombotic agent, confirming the major role of exosite I in the action of thrombin. HD22 aptamer blocking exosite II displays weaker anti-platelet and anti-coagulant activity, with surprising activating effects on thrombin and fibrin generation most likely induced by HD22-induced allosteric changes in thrombin dynamic structure

Modified biovectors for the tuneable activation of anti-platelet carbon monoxide release

This communication describes the anti-platelet effects of a new class of cis-rhenium(II)- dicarbonyl-vitamin B12 complexes (B12-ReCORMs) with tuneable CO releasing properties

Surface modifications of superparamagnetic iron oxide nanoparticles (SPION) to improve their properties as a contrast agent in MRI technique

W toku badań zsyntezowano multifunkcjonalne superparamagnetyczne nanocząstki tlenku żelaza SPION (ang. Superparamagnetic Iron Oxide Nanoparticles). Prace rozpoczęto od przygotowania kationowej pochodnej chitozanu, którą wykorzystano do pokrycia SPION. Następnie, powierzchnia biokompatybilnego układu została aktywowana przez dołączenie do niej grup tosylowych. Pozwoliło to na funkcjonalizację powierzchni nanocząstek przez dołączenie przeciwciał przeciw selektynie P i VCAM-1 oraz przeciwciał II-rzędowych IgG znakowanych fluorescencyjnie Texas Red-X. W ten sposób otrzymano nośnik leku, który w sposób celowany może być dostarczany do miejsca stanu zapalnego tętnicy. Badania in vitro pozwoliły sprawdzić skuteczność kierowanego dostarczenia SPION sfunkcjonalizowanych przeciwciałami do miejsca tętnicy objętego wczesnym stanem miażdżycowym.The aim of the research was the synthesis of multifunctional superparamagnetic iron oxide nanoparticles (SPION). The first step was preparation of a cationic chitosan derivative which was used as a surface coating of the SPION. Next, the surface of the biocompatible system was activated by attaching tosyl groups. That stage led to functionalization of the SPION’s surface by Anti P-selectin, Anti VCAM-1 antibodies and IgG secondary antibodies labeled with fluorescent dye Texas Red-X. That way a drug carrier was obtain which could be actively delivered to an inflammation area in an artery. The in vitro studies allowed to check effectiveness of the targeted delivery of SPION functionalized by antibodies to an early atherosclerosis area in an artery

Modified biovectors for the tuneable activation of anti-platelet carbon monoxide release

This communication describes the anti-platelet effects of a new class of cis-rhenium(II)-dicarbonyl-vitamin B12 complexes (B12-ReCORMs) with tuneable CO releasing properties

Novel nanostructural contrast for magnetic resonance imaging of endothelial inflammation : targeting SPIONs to vascular endothelium

This study aimed to develop superparamagnetic iron oxide nanoparticles (SPIONs) targeted to the areas of vascular endothelium changed in the initial inflammation process, a first step of numerous cardiovascular diseases. Iron oxide nanoparticles coated with a cationic derivative of chitosan (CCh) and having attached monoclonal antibodies (anti VCAM-1 and anti P-selectin) were successfully prepared. Owing to electrostatic stabilization, they form a stable colloidal dispersion in aqueous media. The superparamagnetic properties of the resulting SPION-CCh-anti-VCAM-1 maghemite nanoparticles were proved by magnetometric and Mössbauer measurements. In vitro studies confirmed the specific interaction of anti-VCAM-1 antibodies bound to the surface of SPIONs with endothelial cells of aorta of db/db mice, known to display endothelial inflammation associated with diabetes. The nanoparticles obtained were also visualized using MRI in the aortic arch of ApoE/LDLR-/- mice displaying endothelial inflammation associated with atherosclerosis

Nitric oxide deficiency and endothelial–mesenchymal transition of pulmonary endothelium in the progression of 4T1 metastatic breast cancer in mice

Abstract Background Mesenchymal transformation of pulmonary endothelial cells contributes to the formation of a metastatic microenvironment, but it is not known whether this precedes or follows early metastasis formation. In the present work, we characterize the development of nitric oxide (NO) deficiency and markers of endothelial–mesenchymal transition (EndMT) in the lung in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in mice. Methods NO production, endothelial nitric oxide synthase (eNOS) phosphorylation status, markers of EndMT in the lung, pulmonary endothelium permeability, and platelet activation/reactivity were analyzed in relation to the progression of 4T1 breast cancer metastasis to the lung, as well as to lung tissue remodeling, 1–5 weeks after 4T1 cancer cell inoculation in Balb/c mice. Results Phosphorylation of eNOS and NO production in the lungs of 4T1 breast cancer-bearing mice was compromised prior to the development of pulmonary metastasis, and was associated with overexpression of Snail transcription factor in the pulmonary endothelium. These changes developed prior to the mesenchymal phenotypic switch in the lungs evidenced by a decrease in vascular endothelial-cadherin (VE-CAD) and CD31 expression, and the increase in pulmonary endothelial permeability, phenomena which coincided with early pulmonary metastasis. Increased activation of platelets was also detected prior to the early phase of metastasis and persisted to the late phase of metastasis, as evidenced by the higher percentage of unstimulated platelets binding fibrinogen without changes in von Willebrand factor and fibrinogen binding in response to ADP stimulation. Conclusions Decreased eNOS activity and phosphorylation resulting in a low NO production state featuring pulmonary endothelial dysfunction was an early event in breast cancer pulmonary metastasis, preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT) evidenced by a decrease in VE-CAD and CD31 expression. The latter coincided with development of the first metastatic nodules in the lungs. These findings suggest that early endothelial dysfunction featured by NO deficiency rather than EndMT, might represent a primary regulatory target to prevent early pulmonary metastasis