Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors.

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3-5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

Dissecting thrombus-directed chemotaxis and random movement in neutrophil near-thrombus motion in flow chambers.

BACKGROUND: Thromboinflammation is caused by mutual activation of platelets and neutrophils. The site of thromboinflammation is determined by chemoattracting agents release by endothelium, immune cells, and platelets. Impaired neutrophil chemotaxis contributes to the pathogenesis of Shwachman-Diamond syndrome (SDS). In this hereditary disorder, neutrophils are known to have aberrant chemoattractant-induced F-actin properties. Here, we aim to determine whether neutrophil chemotaxis could be analyzed using our previously developed ex vivo assay of the neutrophils crawling among the growing thrombi. METHODS: Adult and pediatric healthy donors, alongside with pediatric patients with SDS, were recruited for the study. Thrombus formation and granulocyte movement in hirudinated whole blood were visualized by fluorescent microscopy in fibrillar collagen-coated parallel-plate flow chambers. Alternatively, fibrinogen, fibronectin, vWF, or single tumor cells immobilized on coverslips were used. A computational model of chemokine distribution in flow chamber with a virtual neutrophil moving in it was used to analyze the observed data. RESULTS: The movement of healthy donor neutrophils predominantly occurred in the direction and vicinity of thrombi grown on collagen or around tumor cells. For SDS patients or on coatings other than collagen, the movement was characterized by randomness and significantly reduced velocities. Increase in wall shear rates to 300-500 1/s led to an increase in the proportion of rolling neutrophils. A stochastic algorithm simulating leucocyte chemotaxis movement in the calculated chemoattractant field could reproduce the experimental trajectories of moving neutrophils for 72% of cells. CONCLUSIONS: In samples from healthy donors, but not SDS patients, neutrophils move in the direction of large, chemoattractant-releasing platelet thrombi growing on collagen.

Redox regulation of platelet function and thrombosis.

Platelets are well-known players in several cardiovascular diseases such as atherosclerosis and venous thrombosis. There is increasing evidence demonstrating that reactive oxygen species (ROS) are generated within activated platelets. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of ROS generation in platelets. Ligand binding to platelet receptor glycoprotein (GP) VI stimulates intracellular ROS generation consisting of a spleen tyrosine kinase-independent production involving NOX activation and a following spleen tyrosine kinase-dependent generation. In addition to GPVI, stimulation of platelet thrombin receptors (protease-activated receptors [PARs]) can also trigger NOX-derived ROS production. Our recent study found that mitochondria-derived ROS production can be induced by engagement of thrombin receptors but not by GPVI, indicating that mitochondria are another source of PAR-dependent ROS generation apart from NOX. However, mitochondria are not involved in GPVI-dependent ROS generation. Once generated, the intracellular ROS are also involved in modulating platelet function and thrombus formation; therefore, the site-specific targeting of ROS production or clearance of excess ROS within platelets is a potential intervention and treatment option for thrombotic events. In this review, we will summarize the signaling pathways involving regulation of platelet ROS production and their role in platelet function and thrombosis, with a focus on GPVI- and PAR-dependent platelet responses.

Kinetics and regulation of coagulation factor X activation by intrinsic tenase on phospholipid membranes.

BACKGROUND: Factor X activation by the phospholipid-bound intrinsic tenase complex is a critical membrane-dependent reaction of blood coagulation. Its regulation mechanisms are unclear, and a number of questions regarding diffusional limitation, pathways of assembly and substrate delivery remain open. METHODS: We develop and analyze here a detailed mechanism-driven computer model of intrinsic tenase on phospholipid surfaces. Three-dimensional reaction-diffusion-advection and stochastic simulations were used where appropriate. RESULTS: Dynamics of the system was predominantly non-stationary under physiological conditions. In order to describe experimental data, we had to assume both membrane-dependent and solution-dependent delivery of the substrate. The former pathway dominated at low cofactor concentration, while the latter became important at low phospholipid concentration. Factor VIIIa-factor X complex formation was the major pathway of the complex assembly, and the model predicted high affinity for their lipid-dependent interaction. Although the model predicted formation of the diffusion-limited layer of substrate for some conditions, the effects of this limitation on the fXa production were small. Flow accelerated fXa production in a flow reactor model by bringing in fIXa and fVIIIa rather than fX. CONCLUSIONS: This analysis suggests a concept of intrinsic tenase that is non-stationary, employs several pathways of substrate delivery depending on the conditions, and is not particularly limited by diffusion of the substrate.

Chemical Adjustment of Fibrinolysis.

Fibrinolysis is the process of the fibrin-platelet clot dissolution initiated after bleeding has been stopped. It is regulated by a cascade of proteolytic enzymes with plasmin at its core. In pathological cases, the balance of normal clot formation and dissolution is replaced by a too rapid lysis, leading to bleeding, or an insufficient one, leading to an increased thrombotic risk. The only approved therapy for emergency thrombus lysis in ischemic stroke is recombinant tissue plasminogen activator, though streptokinase or urokinase-type plasminogen activators could be used for other conditions. Low molecular weight compounds are of great interest for long-term correction of fibrinolysis dysfunctions. Their areas of application might go beyond the hematology field because the regulation of fibrinolysis could be important in many conditions, such as fibrosis. They enhance or weaken fibrinolysis without significant effects on other components of hemostasis. Here we will describe and discuss the main classes of these substances and their mechanisms of action. We will also explore avenues of research for the development of new drugs, with a focus on the use of computational models in this field.

Force balance ratio is a robust predictor of arterial thrombus stability.

Thrombus formation on a damaged vessel wall can lead to the formation of a stable occlusive/subocclusive clot or unstable embolizing thrombus. Both outcomes can cause significant health damage. The mechanisms that regulate maximum thrombus size, its stability, and embolization in both micro- and macrocirculation are poorly understood. To investigate the impact of flow and intrathrombus forces on the stability of homogeneous and heterogeneous platelet thrombi in a wide range of thrombus geometries, critical interplatelet forces, vessel diameters, and hydrodynamic conditions, we took advantage of the recently developed in silico models. To perform analysis of thrombus stability/embolization in arterioles, we used our previously developed particle-based 2D model with a single-platelet resolution. Its results and predictions were further extended to a 3D case and the large spatial scales of arteries using novel particle-based and continuum 3D models. We found a robust quantitative parameter, termed force balance ratio, which quantifies the balance between destabilizing hydrodynamic and stabilizing interplatelet forces. This parameter predicts whether a homogeneous thrombus (or the shell of a heterogeneous thrombus) with a particular value of critical interplatelet forces will embolize under given hydrodynamic conditions. Our simulations also predict that, for a given magnitude of critical interplatelet forces, the longer thrombi are more stable than the shorter ones. Furthermore, the aggregates formed on top of the severe stenosis are more stable than thrombi formed at moderate stenosis. Taken together, our results give new insights into the interplay between critical interplatelet forces, local hydrodynamics, and overall thrombus stability against the flow.

H2O2-Responsive Polymeric Micelles of Biodegradable Aliphatic Poly(carbonate)s as Promising Therapeutic Agents for Inflammatory Diseases.

Excessive amounts of reactive oxygen species (ROS) cause various biological damages and are involved in many diseases, such as cancer, inflammatory and thrombotic complications, and neurodegenerative diseases. Thus, ROS-responsive polymers with inherent ROS scavenging activity and biodegradability are extremely needed for the efficient treatment of ROS-related diseases. Here, this work fabricates the amphiphilic diblock copolymer PEG-b-PBC via ring-opening polymerization (ROP) of phenylboronic acid ester conjugated cyclic carbonate monomer. The copolymer easily forms micelles (BCM) and scavenges ROS rapidly. BCM not only releases the delivered drug but degrades to produce the small molecules p-hydroxybenzyl alcohol (HBA) with anti-inflammatory capability in the presence of H2O2. BCM can reduce the oxidative stress of human umbilical vein endothelial cells (HUVEC) and the levels of inflammatory factors secreted by macrophages, showing antioxidative and anti-inflammatory activity. Finally, BCM exerts a significant capability to reduce the complications of inflammation and thrombosis in vivo. The biodegradable aliphatic poly(carbonate)s have the potential to be used for drug delivery systems (DDS) for diseases induced by reactive oxygen species.

Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

OBJECTIVES: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. METHODS: For activation, 2.5 µM CaCl2 , 5 µM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. RESULTS: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. CONCLUSIONS: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

Perfluorocarbon Nanoparticles Incorporating Ginkgolide B: Artificial O2 Carriers with Antioxidant Activity and Antithrombotic Effect.

Ischemic stroke primarily leads to insufficient oxygen delivery in ischemic area. Prompt reperfusion treatment for restoration of oxygen is clinically suggested but mediates more surging reactive oxygen species (ROS) generation and oxidative damage, known as ischemia-reperfusion injury (IRI). Therefore, the regulation of oxygen content is a critical point to prevent cerebral ischemia induced pathological responses and simultaneously alleviate IRI triggered by the sudden oxygen restoration. In this work, we constructed a perfluorocarbon (PFC)-based artificial oxygen nanocarrier (PFTBA-L@GB), using an ultrasound-assisted emulsification method, alleviates the intracerebral hypoxic state in ischemia stage and IRI after reperfusion. The high oxygen solubility of PFC allows high oxygen efficacy. Furthermore, PFC has the adhesion affinity to platelets and prevents the overactivation of platelet. The encapsulated payload, ginkgolide B (GB) exerts its anti-thrombosis by antagonism on platelet activating factor and antioxidant effect by upregulation of antioxidant molecular pathway. The versatility of the present strategy provides a practical approach to build a simple, safe, and relatively effective oxygen delivery agent to alleviate hypoxia, promote intracerebral oxygenation, anti-inflammatory, reduce intracerebral oxidative stress damage and thrombosis and caused by stroke.

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

BACKGROUND: Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. METHODS: Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. RESULTS: We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. CONCLUSIONS: Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. IMPACT: Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

The Ways of the Virus: Interactions of Platelets and Red Blood Cells with SARS-CoV-2, and Their Potential Pathophysiological Significance in COVID-19.

The hematological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important in COVID-19 pathophysiology. However, the interactions of SARS-CoV-2 with platelets and red blood cells are still poorly understood. There are conflicting data regarding the mechanisms and significance of these interactions. The aim of this review is to put together available data and discuss hypotheses, the known and suspected effects of the virus on these blood cells, their pathophysiological and diagnostic significance, and the potential role of platelets and red blood cells in the virus's transport, propagation, and clearance by the immune system. We pay particular attention to the mutual activation of platelets, the immune system, the endothelium, and blood coagulation and how this changes with the evolution of SARS-CoV-2. There is now convincing evidence that platelets, along with platelet and erythroid precursors (but not mature erythrocytes), are frequently infected by SARS-CoV-2 and functionally changed. The mechanisms of infection of these cells and their role are not yet entirely clear. Still, the changes in platelets and red blood cells in COVID-19 are significantly associated with disease severity and are likely to have prognostic and pathophysiological significance in the development of thrombotic and pulmonary complications.

Platelets provide robustness of spatial blood coagulation to the variation of initial conditions.

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 105/µL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

Phenylboronic Ester-Bridged Chitosan/Myricetin Nanomicelle for Penetrating the Endothelial Barrier and Regulating Macrophage Polarization and Inflammation against Ischemic Diseases.

The brain and liver are more susceptible to ischemia and reperfusion (IR) injury (IRI), which triggers the reactive oxygen species (ROS) burst and inflammatory cascade and results in severe neuronal damage or hepatic injury. Moreover, the damaged endothelial barrier contributes to proinflammatory activity and limits the delivery of therapeutic agents such as some macromolecules and nanomedicine despite the integrity being disrupted after IRI. Herein, we constructed a phenylboronic-decorated chitosan-based nanoplatform to deliver myricetin, a multifunctional polyphenol molecule for the treatment of cerebral and hepatic ischemia. The chitosan-based nanostructures are widely studied cationic carriers for endothelium penetration such as the blood-brain barrier (BBB) and sinusoidal endothelial barrier (SEB). The phenylboronic ester was chosen as the ROS-responsive bridging segment for conjugation and selective release of myricetin molecules, which meanwhile scavenged the overexpressed ROS in the inflammatory environment. The released myricetin molecules fulfill a variety of roles including antioxidation through multiple phenolic hydroxyl groups, inhibition of the inflammatory cascade by regulation of the macrophage polarization from M1 to M2, and endothelial injury repairment. Taken together, our present study provides valuable insight into the development of efficient antioxidant and anti-inflammatory platforms for potential application against ischemic disease.

Bioinspired Platelet-Anchored Electrospun Meshes for Tight Inflammation Manipulation and Chronic Diabetic Wound Healing.

Tight manipulation of the initial leukocytes infiltration and macrophages plasticity toward the M2 phenotype remain a challenge for diabetic wound healing. Inspired by the platelet function and platelet-macrophage interaction, a platelet-anchored polylactic acid-b-polyethylene glycol-b-polylactic acid (PLA-PEG-PLA) electrospun dressing is developed for inflammatory modulation and diabetic wounds healing acceleration. PLA-PEG-PLA electrospun meshes encapsulated with thymosin ß4 (Tß4) and CaCl2 is fabricated with electrospinning, followed by immersion of electrospun mesh in platelet-rich plasma to firmly anchor the platelets. It is demonstrated that the anchored platelets on electrospun mesh can enhance the initial macrophage recruitment and control the Tß4 release from electrospun meshes to facilitate the macrophages polarization to the M2 phenotype. The inflammatory regulation promotes the expression of vascular endothelial growth factor and the migration of vascular endothelial cells for angiogenesis, resulting in accelerated diabetic wounds healing. Therefore, this work paved a new way to design platelet-inspired electrospun meshes for inflammation manipulation and diabetic wound healing.

Integral assays of hemostasis in hospitalized patients with COVID-19 on admission and during heparin thromboprophylaxis.

BACKGROUND: Blood coagulation abnormalities play a major role in COVID-19 pathophysiology. However, the specific details of hypercoagulation and anticoagulation treatment require investigation. The aim of this study was to investigate the status of the coagulation system by means of integral and local clotting assays in COVID-19 patients on admission to the hospital and in hospitalized COVID-19 patients receiving heparin thromboprophylaxis. METHODS: Thrombodynamics (TD), thromboelastography (TEG), and standard clotting assays were performed in 153 COVID-19 patients observed in a hospital setting. All patients receiving treatment, except extracorporeal membrane oxygenation (ECMO) patients (n = 108), were administered therapeutic doses of low molecular weight heparin (LMWH) depending on body weight. The ECMO patients (n = 15) were administered unfractionated heparin (UFH). RESULTS: On admission, the patients (n = 30) had extreme hypercoagulation by all integral assays: TD showed hypercoagulation in ~75% of patients, while TEG showed hypercoagulation in ~50% of patients. The patients receiving treatment showed a significant heparin response based on TD; 77% of measurements were in the hypocoagulation range, 15% were normal, and 8% remained in hypercoagulation. TEG showed less of a response to heparin: 24% of measurements were in the hypocoagulation range, 59% were normal and 17% remained in hypercoagulation. While hypocoagulation is likely due to heparin treatment, remaining in significant hypercoagulation may indicate insufficient anticoagulation for some patients, which is in agreement with our clinical findings. There were 3 study patients with registered thrombosis episodes, and all were outside the target range for TD parameters typical for effective thromboprophylaxis (1 patient was in weak hypocoagulation, atypical for the LMWH dose used, and 2 patients remained in the hypercoagulation range despite therapeutic LMWH doses). CONCLUSION: Patients with COVID-19 have severe hypercoagulation, which persists in some patients receiving anticoagulation treatment, while significant hypocoagulation is observed in others. The data suggest critical issues of hemostasis balance in these patients and indicate the potential importance of integral assays in its control.

Accelerated death of megakaryocytes from Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder caused by WAS gene mutations resulting in haematopoietic/immune cell defects. Recent studies report accelerated death of WAS platelets and lymphocytes. Data on megakaryocyte (MK) maturation, viability and their possible role in thrombocytopenia development in WAS are limited. In this study we evaluate the MK viability and morphology in untreated, romiplostim-treated WAS patients compared with normal controls. The study included 32 WAS patients and 17 healthy donors. MKs were captured from bone marrow aspirates by surface-immobilized anti-GPIIb-IIIa antibody. Viability (by phosphatidylserine [PS] externalization), distribution by maturation stages and size of MK were determined by light microscopy. MK distribution by maturation stages in patients differed from controls. 40 ± 22% of WAS MKs versus 23 ± 11% of normal MKs were at maturation stage 3 (p = 0.02), whereas 24 ± 20% in WAS and 39 ± 14% in controls had megakaryoblast morphology (p = 0.05). Romiplostim treatment changed the MK maturation stages distribution close to normal. PS-positive (PS+) MK in WAS was significantly higher (21 ± 21%) than in healthy controls (2 ± 4%, p < 0.01). WAS patients with more damaging truncating mutations and higher disease score had higher PS+ MK fraction (Spearman r = 0.6, p < 0.003). We conclude that WAS MKs have increased cell death tendency and changes in maturation pattern. Both could contribute to thrombocytopenia in WAS patients.

New Hybrid Tetrahydropyrrolo[3,2,1-ij]quinolin-1-ylidene-2-thioxothiazolidin-4-ones as New Inhibitors of Factor Xa and Factor XIa: Design, Synthesis, and In Silico and Experimental Evaluation.

Despite extensive research in the field of thrombotic diseases, the prevention of blood clots remains an important area of study. Therefore, the development of new anticoagulant drugs with better therapeutic profiles and fewer side effects to combat thrombus formation is still needed. Herein, we report the synthesis and evaluation of novel pyrroloquinolinedione-based rhodanine derivatives, which were chosen from 24 developed derivatives by docking as potential molecules to inhibit the clotting factors Xa and XIa. For the synthesis of new hybrid derivatives of pyrrolo[3,2,1-ij]quinoline-2-one, we used a convenient structural modification of the tetrahydroquinoline fragment by varying the substituents in positions 2, 4, and 6. In addition, the design of target molecules was achieved by alkylating the amino group of the rhodanine fragment with propargyl bromide or by replacing the rhodanine fragment with 2-thioxoimidazolidin-4-one. The in vitro testing showed that eight derivatives are capable of inhibiting both coagulation factors, two compounds are selective inhibitors of factor Xa, and two compounds are selective inhibitors of factor XIa. Overall, these data indicate the potential anticoagulant activity of these molecules through the inhibition of the coagulation factors Xa and XIa.

Novel SLFN14 mutation associated with macrothrombocytopenia in a patient with severe haemorrhagic syndrome.

BACKGROUND: Platelet-type bleeding disorder 20 (BDPLT20), as known as SLFN14-related thrombocytopenia, is a rare inherited thrombocytopenia (IT). Previously, only 5 heterozygous missense mutations in the SLFN14 gene have been reported. METHODS: A comprehensive clinical and laboratory examination of a 17-year-old female patient with macrothrombocytopenia and severe mucocutaneous bleeding was performed. Examination was carried out using standardized questionnaires to assess bleeding, high-throughput sequencing (Next Generation Sequencing), optical and fluorescence microscopy, flow cytometry with activation and analysis of intracellular calcium signaling of platelets, light transmission aggregometry and thrombus growth in the flow chamber. RESULTS: Analysis of the patient's genotype revealed a previously undescribed c.655 A > G (p.K219E) variant in the hotspot of the SLFN14 gene. Immunofluorescence and brightfield examination of platelets in the smear showed heterogeneity in cells size, including giant forms over 10 µm (normal size 1-5) in diameter, with vacuolization and diffuse distribution of ß1-tubulin and CD63. Activated platelets showed impaired contraction and shedding/internalization of GPIb. GP IIb/IIIa clustering was increased at rest and attenuated upon activation. Intracellular signalling study revealed impaired calcium mobilization upon TRAP 35.97 nM (reference range 180 ± 44) and CRP-XL 10.08 nM (56 ± 30) stimulation. Aggregation with ADP, collagen, TRAP, arachidonic acid and epinephrine was impaired in light transmission aggregometry; agglutination with ristocetin persisted. In the flow chamber with a shear rate of 400 s-1 platelet adhesion to collagen and clot growth were impaired. CONCLUSION: The revealed disorders of phenotype, cytoskeleton and intracellular signaling explain the nature of SLFN14 platelet dysfunction and the patient's severe hemorrhagic syndrome.

Bioactive fibrous scaffolds with programmable release of polypeptides regulate inflammation and extracellular matrix remodeling.

Inflammation manipulation and extracellular matrix (ECM) remodeling for healthy tissue regeneration are critical requirements for tissue engineering scaffolds. To this end, the bioactive polycaprolactone (PCL)-based scaffolds are fabricated to release aprotinin and thymosin ß4 (Tß4) in a programmable manner. The core part of the fiber is composed of hyaluronic acid and Tß4, and the shell is PCL, which is further coated with heparin/gelatin/aprotinin to enhance biocompatibility. The in vitro assay demonstrates that the controlled release of aprotinin prevents initial excessive inflammation. The subsequent release of Tß4 after 3 days induces the transition of macrophages from M1 into M2 polarization. The manipulation of inflammatory response further controls the expression of transforming growth factor-ß and fibroblast activation, which oversee the quantity and quality of ECM remodeling. In addition, the gradual degradation of the scaffold allows cells to proliferate within the platform. In vivo implant evaluation convinces that PCL-based scaffolds possess the high capability to control the inflammatory response and restore the ECM to normal conditions. Hence, our work paves a new way to develop tissue engineering scaffolds for inflammation manipulation and ECM remodeling with peptide-mediated reactions.