β-adrenergic receptor (βAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells.
Heparin and its low-molecular-weight heparin (LMWH) derivatives are widely used clinical anticoagulants. These drugs are critical for the practice of medicine in applications including kidney dialysis, cardiopulmonary bypass, and in the management of venous thromboembolism. Currently, these drugs are derived from livestock, primarily porcine intestine. The worldwide dependence on a single animal species has made the supply chain for this critical drug quite fragile, leading to the search for other sources of these drugs, including bovine tissues such as bovine intestine or lung. A number of laboratories are currently examining the similarities and differences between heparins prepared from porcine and bovine tissues. The current study is designed to compare LMWH prepared from bovine heparins through chemical β-elimination, a process currently used to prepare the LMWH, enoxaparin, from porcine heparin. Using top-down, bottom-up, compositional analysis and bioassays, LMWHs, derived from bovine lung and intestine, are shown to closely resemble enoxaparin.
Unfractionated heparin is the first anticoagulant drug and has been successfully used clinically for over 80 years. Heparin and its analogues are used during surgery and dialysis and are often used to coat indwelling catheters and other devices where the vascular system is exposed. Most of the heparins used clinically are derived from porcine intestinal mucosa. However, heparins have also been manufactured from tissues of other mammalian species such as cows and sheep. Recently there have been attempts to generate bioengineered heparin in order to overcome contamination and antigenicity problems. Currently there are some concerns about the shortage of the porcine heparins as they are widely used in the manufacturing of the low-molecular-weight heparins. Moreover, due to cultural and religious reasons in some countries, alternative sources of heparins are needed. The Food and Drug Administration and other regulatory agencies have considered alternative sourcing of heparin for potential substitution of porcine heparin and are currently reviewing this matter. Numerous studies are ongoing to understand the structure-activity relationships of these various heparins. In this article, heparins from different animal sources were studied to determine the extent of biosimilarity between them. For these investigations, 10 batches each of bovine mucosal heparin (BMH), ovine mucosal heparin (OMH), and porcine mucosal heparin (PMH) were studied. These studies have demonstrated that OMH and PMH have comparable anticoagulant and antiproteases activities. However, BMH exhibited somewhat a lower potency compared to OMH and PMH in functional assays.
Introduction: Bovine mucosal heparins (BMH) are currently being developed for re-introduction for both medical and surgical indications. BMH active pharmaceutical ingredient (API) exhibits a somewhat weaker USP potency when compared to PMHs. We hypothesized that when dosages are normalized based on the USP reference heparin, BMH will exhibit comparable in vitro and in vivo effects to those produced by PMH. Therefore, studies were developed to compare the APIs of bovine and porcine heparin.Materials and Methods: API versions of PMH were obtained from Celsus Laboratories (Franklin, OH) and Medefil (Glen Ellen, IL). API versions of BMH were obtained from Kin Master (Passo Fundo, Brazil). Each of these heparins was assayed for their molecular weight profile, AT affinity, USP potency, and anticoagulant/antiprotease profiles using standard laboratory methods. In vitro protamine neutralization studies were carried out. Antithrombotic and hemorrhagic effects were measured in rats and pharmacodynamic profiles were assessed in primates.Results: Size exclusion chromatography demonstrated that the mean molecular weight of BMH was ~15% higher than that of PMH (BMH: 20.1 ± 0.8 kDa and PMH: 17.5 ± 0.7 kDa). BMH exhibited an anti-Xa potency of 130 U/mg while PMH had an anti-Xa potency of 185 U/mg. In the anticoagulant and antiprotease assays, the BMH exhibited lower functionality which was proportional to USP potency. When the BMH was compared with PMH at potency adjusted concentrations, it showed identical concentration-response curves in the aPTT and anti-protease assays. However, in the protamine neutralization studies, BMH required slightly higher amounts of protamine in contrast to PMH. BMH and PMH administered to rats at equivalent anti-Xa unit dosages resulted in comparable antithrombotic activity and prolongation of bleeding time. Similar pharmacodynamic profiles were observed in primates when BMH and PMH were dosed on an anti-Xa U/kg basis.Conclusion: BMH, when used at comparable anti-Xa unit levels, is comparable to PMH, however, it requires proportionally higher amount of protamine due to the increased mass for adjusting to higher potency. Additional studies on the structural characterization, interactions with PF4 and in vivo neutralization studies are ongoing.
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