The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity

Background: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis. Methods: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time. Results: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time. Conclusion: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound

Macrophage Polarization is Deregulated in Haemophilia

Macrophages make important contributions to inflammation and wound healing. We show here that macrophage polarization is deregulated in haemophilia in response to macrophage colony-stimulating factor (M-CSF) and partially in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). As a result, haemophilia macrophages exhibit a specific impairment of M-CSF-mediated functions involved in wound healing such as clot invasion and phagocytosis. Haemophilia monocytes express reduced amounts of the receptors for M-CSF and GM-CSF, which correlates with a failure to express tumour necrosis factor α (TNFα) and CD163 in M-CSF-treated haemophilia macrophages and reduced expression of TNFα and CD206 after treatment with GM-CSF. Protein expression in response to M-CSF was regained with respect to CD163 and CD206 after embedding haemophilia monocytes in clotted plasma suggesting that a functioning coagulation system has positive effects on macrophage M2 polarization. Mimicking the functional deficits of haemophilia macrophages in normal macrophages was possible by adding leptin, which we found to be elevated in the blood of haemophilia patients, to a monocyte cell line. The increase of leptin occurred in conjunction with C-reactive protein in a body mass index-controlled cohort suggesting that haemophilia patients harbour chronic low-grade inflammation. Together, our data indicate that impaired clotting in haemophilia patients leads to increased inflammation and a deregulation in macrophage differentiation, which may explain the commonly observed deficits in wound healing and tissue regeneration

Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells

The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules

Concizumab restores thrombin generation potential in patients with haemophilia: Pharmacokinetic/pharmacodynamic modelling results of concizumab phase 1/1b data

Introduction: Concizumab enhances thrombin generation (TG) potential in haemophilia patients by inhibiting tissue factor pathway inhibitor (TFPI). In EXPLORER3 (phase 1b), a dose‐dependent pharmacokinetic/pharmacodynamic (PK/PD) relationship was confirmed between concizumab dose, free TFPI and TG potential. Aim: Determine the association between concizumab exposure, PD markers (free TFPI; peak TG) and bleeding episodes to establish the minimum concizumab concentration for achieving sufficient efficacy. Methods: Free TFPI predictions were generated using an estimated concizumab‐free TFPI exposure‐response (Emax) model based on concizumab phase 1/1b data for which simultaneously collected concizumab and free TFPI samples were available. Concizumab concentration at the time of a bleed was predicted using a PK model, based on available data for concizumab doses >50 μg/kg to ≤9 mg/kg. Peak TG vs concizumab concentration analyses and an Emax model were constructed based on EXPLORER3 observations. Results: The Emax model showed a tight PK/PD relationship between concizumab exposure and free TFPI; free TFPI decreased with increasing concizumab concentration. A strong correlation between concizumab concentration and peak TG was observed; concizumab >100 ng/mL re‐established TG potential to within the normal reference range. Estimated EC50 values for the identified concizumab‐free TFPI and concizumab‐TG potential models were very similar, supporting free TFPI as an important biomarker. A correlation between bleeding episode frequency and concizumab concentration was indicated; patients with a concizumab concentration >100 ng/mL experienced less frequent bleeding. The PK model predicted that once‐daily dosing would minimize within‐patient concizumab PK variability.Novo Nordis

Erysense, a Lab-on-a-Chip-Based Point-of-Care Device to Evaluate Red Blood Cell Flow Properties With Multiple Clinical Applications

In many medical disciplines, red blood cells are discovered to be biomarkers since they “experience” various conditions in basically all organs of the body. Classical examples are diabetes and hypercholesterolemia. However, recently the red blood cell distribution width (RDW), is often referred to, as an unspecific parameter/marker (e.g., for cardiac events or in oncological studies). The measurement of RDW requires venous blood samples to perform the complete blood cell count (CBC). Here, we introduce Erysense, a lab-on-a-chip-based point-of-care device, to evaluate red blood cell flow properties. The capillary chip technology in combination with algorithms based on artificial neural networks allows the detection of very subtle changes in the red blood cell morphology. This flow-based method closely resembles in vivo conditions and blood sample volumes in the sub-microliter range are sufficient. We provide clinical examples for potential applications of Erysense as a diagnostic tool [here: neuroacanthocytosis syndromes (NAS)] and as cellular quality control for red blood cells [here: hemodiafiltration (HDF) and erythrocyte concentrate (EC) storage]. Due to the wide range of the applicable flow velocities (0.1–10 mm/s) different mechanical properties of the red blood cells can be addressed with Erysense providing the opportunity for differential diagnosis/judgments. Due to these versatile properties, we anticipate the value of Erysense for further diagnostic, prognostic, and theragnostic applications including but not limited to diabetes, iron deficiency, COVID-19, rheumatism, various red blood cell disorders and anemia, as well as inflammation-based diseases including sepsis

Hemophilia treatment in 2021: choosing the”optimal” treatment using an integrative, patient-oriented approach to shared decision-making between patients and clinicians

The mainstay of hemophilia treatment is to prevent bleeding through regular long-term prophylaxis and to control acute breakthrough bleeds. Various treatment options are currently available for prophylaxis, and treatment decision-making is a challenging and multifaceted process of identifying the most appropriate option for each patient. A multidisciplinary expert panel convened to develop a practical, patient-oriented algorithm to facilitate shared treatment decision-making between clinicians and patients. Key variables were identified, and an algorithm proposed based on five variables: bleeding phenotype, musculoskeletal status, treatment adherence, venous access, and lifestyle. A complementary, patient-focused preference tool was also hypothesized, with the aim of exploring individual patients' priorities, preferences, and goals. It is hoped that the proposed algorithm and the hypothesized patient preference tool will assist in selecting a treatment for each patient that is as efficient as possible in preventing bleeds while also accounting for the patient's expectations and prioritiesFunded by a grant from Novo Nordis

Factor VIII: Long-established role in haemophilia A and emerging evidence beyond haemostasis

Abstract Factor VIII protein (FVIII) as a coagulation replacement factor has for decades been used as the standard of care for management of people with haemophilia A. It is effective for treatment of bleeding events, as prophylaxis to prevent bleeding events and preserve joint function, and to support surgery in people with haemophilia A. Despite long experience in treating haemophilia A, we are only beginning to understand the functions of FVIII beyond its established role as a coenzyme to factor IXa to expedite thrombin generation through the intrinsic pathway of coagulation. Here, we review the current role of FVIII coagulant (FVIII:C) in haemophilia A management and emerging evidence for the role of FVIII across multiple systems, including the cardiovascular system, angiogenesis and maintenance of bone health. For instance, supraphysiological FVIII levels are a risk factor for venous thromboembolism. von Willebrand factor (VWF), which forms a non-covalent complex with circulating FVIII, is an established marker and regulator of angiogenesis. In a mouse model of haemophilia, treatment with FVIII decreased expression of receptor activator of nuclear factor kappa-Β ligand (RANKL), a marker for bone turnover. Longitudinal follow-up data in people with haemophilia A are needed to confirm and extend these observations

PTFEP-Al2O3 hybrid nanowires reducing thrombosis and biofouling

Thrombosis and bacterial infection are major problems in cardiovascular implants. Here we demonstrated that a superhydrophobic surface composed of poly(bis(2,2,2-trifluoroethoxy)phosphazene) (PTFEP)-Al2O3 hybrid nanowires (NWs) is effective to reduce both platelet adhesion/activation and bacterial adherence/colonization. The proposed approach allows surface modification of cardiovascular implants which have 3D complex geometries. © 2019 The Royal Society of Chemistry