Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca2؉ sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K ؉ depolarization. The Ca 2؉ -sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In ␣-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5-O-(␥-thiotriphosphate), and histamine plus guanyl-5-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca 2؉ sensitization of smooth muscle contraction.

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca2+ sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr695 of the MLCP regulatory subunit (MYPT1) and at Thr38 of the MLCP inhibitor protein CPI‐17 results in inhibition of MLCP activity. We have previously demonstrated that CPI‐17 Thr38 phosphorylation plays an important role in G‐protein‐mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site‐ and phospho‐specific antibodies, phosphorylation of MYPT1 Thr695 and CPI‐17 Thr38 was examined along with MYPT1 Thr850, which is a non‐inhibitory Rho‐kinase site. We found that both CPI‐17 Thr38 and MYPT1 Thr850 were phosphorylated in response to agonists or GTPγS concurrently with contraction and myosin phosphorylation in α‐toxin‐permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr695 did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho‐kinase inhibitor Y‐27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr850 and CPI‐17 Thr38, respectively, in intact VD while MYPT1 Thr695 phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr695 is independent of stimulation of G‐proteins, Rho‐kinase or PKC. In the phasic PV, phosphorylation of CPI‐17 Thr38 may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI‐17 concentration, probably utilizes other Ca2+ sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI‐17.

Using permeabilized, arterial smooth muscle strips where membrane-associated pathways remain intact but intracellular Ca 2؉ stores are depleted, we investigated mechanism(s) for the Ca 2؉ desensitization of contractile force by cGMP. The nonhydrolyzable analog 8-bromo-cGMP, when applied to these strips with submaximal Ca 2؉ levels clamped, dramatically and reversibly reduced the steady state levels of phosphorylation at 20-kDa myosin light chain and contractile force, with a nanomolar concentration required to obtain 50% reduction. Supramaximal concentrations of 8-bromocGMP (10 M), however, did not change the steady state relationship between phosphorylation and force. When light chain phosphatase activity was blocked at pCa 6.7, 10 M 8-bromo-cGMP did not affect the rates of rise of light chain phosphorylation and contractile force. When light chain kinase activity was blocked, 10 M 8-bromocGMP significantly accelerated light chain dephosphorylation and force relaxation from the maximal contraction steady state. The light chain phosphorylation time course of a pCa 6.0-induced contraction in the presence of 8-bromo-cGMP exhibited kinetics that are predictable from a mathematical model in which only light chain phosphatase activity is increased. The results of this study strongly suggest that cGMP indirectly activates light chain phosphatase, the first proposed mechanism for cGMP-induced Ca 2؉ desensitization in vasodilatation.The primary mechanisms that regulate smooth muscle contraction and relaxation are, respectively, phosphorylation of the regulatory 20-kDa myosin light chain (MLC 20 ) 1 at Ser-19 by MLC 20 kinase (MLCK) and its dephosphorylation by MLC 20 phosphatase (MLCP) (1, 2). Typically, intracellular Ca 2ϩ levels modulate the MLCK to MLCP activity ratio and ultimately the degree of contractile force because MLCK activity depends on the amount of the Ca 2ϩ -calmodulin complex, which itself hinges on cytosolic Ca 2ϩ levels. In many cases, however, the sensitivity of force to Ca 2ϩ can be changed by physiological modulation of the Ca 2ϩ dependence of MLC 20 phosphorylation (3, 4) or by other mechanisms such as thin filament disinhibition (5, 6).The nucleotide cyclic GMP has emerged as a potent, physiological second messenger involved in both vasodilator action and failure of vasoconstrictor activity. The neighboring endothelium, endogenous circulating hormones, as well as clinically administered nitrovasodilators all function to widen the lumen of vessels by stimulating guanylyl cyclase in vascular smooth muscle to produce cGMP (7). To date, cGMP has been implicated in lowering intracellular Ca 2ϩ (8, 9) and in decreasing sensitivity of the contractile force to Ca 2ϩ (10,11). A myriad of Ca 2ϩ lowering mechanisms have since been reportedly shown (see Ref. 9 for references): increased Ca 2ϩ sequestration, increased Ca 2ϩ efflux, decreased Ca 2ϩ influx through decreased Ca 2ϩ channel activity and through hyperpolarization via increased K ϩ channel activity, and decreased Ca 2ϩ release through antagonism of ...

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

Various smooth muscles have unique contractile characteristics, such as the degree of Ca2+ sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)‐induced Ca2+ sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI‐17, and the thin filament regulator h‐calponin. Immunohistochemical and Western blot analyses indicated that CPI‐17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI‐17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI‐17 concentration of the phasic vas deferens. In contrast to CPI‐17 expression patterns, phasic muscles contained more MLCP myosin‐targeting subunit than tonic tissues. Calponin expression was not statistically different. Addition of phorbol ester to α‐toxin‐permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI‐17 and myosin light chain (MLC) at submaximal [Ca2+]i. These responses were several‐fold greater in femoral artery as compared to vas deferens. We conclude that the expression ratio of CPI‐17 to MLCP correlates with the Ca2+ sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI‐17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI‐17 and high MLCP content. This implicates CPI‐17 inhibition of MLCP as an important component in modulating vascular muscle tone.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein‐coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Abstract-Ca2ϩ ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca 2ϩ with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca 2ϩ transient, we compared Ca 2ϩ signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during ␣ 1 agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38Ϯ0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca 2ϩ rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca 2ϩ release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca 2ϩ -dependent protein kinase C. This was followed by a slow Ca 2ϩ -independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29Ϯ0.09 at rest to the peak of 0.68Ϯ0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca 2ϩ sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca 2ϩ rise induces a rapid inhibition of MLC phosphatase coincident with the Ca 2ϩ -induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content. (Circ Res. 2007;100:121-129.)