Phosphorylation of protein S by platelet kinases enhances its activated protein C cofactor activity

Abstract

Protein S (PS) is a multifunctional plasma protein of the hemostatic and inflammatory pathways, although mechanisms for its regulation are poorly understood. Since certain plasma proteins are regulated through extracellular phosphorylation, we investigated whether the anticoagulant activity of PS is regulated through phosphorylation by platelet-secreted kinases. PS was phosphorylated on exposure to activated platelets or their releasates, as judged by immunoblotting for phospho-amino acids and PS. PS phosphorylation was reduced by specific inhibitors of casein kinase 1 (CK1) and casein kinase 2 (CK2) (10 μM D4476, 100 μM CK2-inhibitory peptide YNLKSKSSEDIDESS). Involvement of CKs in PS phosphorylation was confirmed using purified CK1/CK2. Phosphorylation of PS by purified CK1 did not affect its activated protein C (APC) cofactor activity in activated partial thromboplastin time assays in PS-depleted plasma. However, phosphorylation of PS by CK2 or by CK1/CK2 increased PS cofactor activity ∼1.5-fold (158.7±4.8%, P<0.01) or ∼2-fold (191.5±6.4%, P<0.0001), respectively. The APC cofactor activity of PS in PS-depleted plasma exposed to platelet-secreted kinases was enhanced, while CK2 but not CK1 inhibitors reduced APC cofactor activity. Mass spectrometry revealed a phosphorylated CK2 site at Thr37 within the N-terminal Gla-domain. Thus, platelet-mediated extracellular phosphorylation of PS is a potential mechanism by which its activity is regulated.

The Human Platelet as an Innate Immune Cell: Interactions Between Activated Platelets and the Complement System

“…Protein kinase activities in the form of protein kinase A (PKA) (93), several isoforms of protein kinase C (PKC) (115), and casein kinases 1 and 2 (CK1 and CK2) (98–100, 102, 103, 107–115) have all been identified in the releasate from activated platelets. They have all been shown to mediate extracellular phosphorylation of specific plasma proteins, in particular proteins within the cascade systems, provided that ATP and Ca 2+ are present in sufficient amounts.…”

“…Furthermore, it has been suggested that anticoagulation is regulated by platelet-mediated phosphorylation: Coagulation factor Va becomes more readily inactivated by activated protein C after phosphorylation by platelet casein kinase (128). More recently, platelet-secreted casein kinase(s) have been shown to phosphorylate protein S, thereby enhancing its activated protein C cofactor activity (103). In that publication, the authors postulated that extracellular platelet-mediated phosphorylation of protein S is a previously unrecognized mechanism for regulating its anticoagulant activity, and that role of phosphorylation most likely applies to all the substrates discussed here.…”

In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion

“…Previous studies have identified in vitro phosphorylation of extracellular plasma proteins such as fibrinogen, complement protein C3, vitronectin, factor V and protein S by kinases, including casein kinase 1, casein kinase 2, protein kinase C and protein kinase A . However, these kinases are predominately localized in the cytoplasm and/or nucleus and not likely to encounter extracellular proteins.…”

Influence of hyperinsulinemic – hypoglycemic clamp on induced platelet aggregation, activity of physiological anticoagulants and von Willebrand factor in patients with type I diabetes

“…Свободный протеин S имеет два механизма антикоагулянтного влияния. С од-ной стороны, активация тромбоцитов сопровождается выделением ими киназ, которые сразу же усиливают фосфорилирование свободного протеина S и тем самым повышают в 1,5-2,0 раза его кофакторную активность к протеину С [32]. Фосфорилированный протеин S об-ладает высоким сродством к отрицательно заряженным фосфолипидам и тем самым улучшает контакт активи-рованного протеина С с мембраной за счет образова-ния комплекса с ним.…”