RESUMEN

BACKGROUND/AIMS: The Kunitz Protease Inhibitor (KPI) domain of protease nexin 2 (PN2) potently inhibits coagulation factor XIa. Recombinant KPI has been shown to inhibit thrombosis in mouse models, but its clearance from the murine circulation remains uncharacterized. The present study explored the pharmacokinetic and pharmacodynamic effects of fusing KPI to human serum albumin (HSA) in fusion protein KPIHSA. METHODS: Hexahistidine-tagged KPI (63 amino acids) and KPIHSA (656 amino acids) were expressed in Pichia pastoris yeast and purified by nickel-chelate chromatography. Clearance profiles in mice were determined, as well as the effects of KPI or KPIHSA administration on FeCl3-induced vena cava thrombus size or carotid artery time to occlusion, respectively. RESULTS: Fusion to HSA increased the mean terminal half-life of KPI by 8-fold and eliminated its interaction with the low density lipoprotein receptor-related protein. KPI and KPIHSA similarly reduced thrombus size and occlusion in both venous and arterial thrombosis models when administered at the time of injury, but only KPI was effective when administered one hour before injury. CONCLUSIONS: Albumin fusion deflects KPI from rapid in vivo clearance without impairing its antithrombotic properties and widens its potential therapeutic window.

Asunto(s)

Precursor de Proteína beta-Amiloide/genética , Albúmina Sérica Humana/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Área Bajo la Curva , Factores de Coagulación Sanguínea/antagonistas & inhibidores , Factores de Coagulación Sanguínea/metabolismo , Cloruros/toxicidad , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Compuestos Férricos/toxicidad , Semivida , Histidina/genética , Humanos , Radioisótopos de Yodo/química , Ratones , Oligopéptidos/genética , Dominios Proteicos/genética , Curva ROC , Receptores de LDL/química , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/uso terapéutico , Albúmina Sérica Humana/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Trombosis/inducido químicamente , Trombosis/prevención & control

RESUMEN

INTRODUCTION: Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid ß-protein precursor (AßPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AßPP are abundant in brain. Regions of AßPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AßPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. METHODS: To determine the extent of KPI function of AßPP in regulating cerebral thrombosis we generated a recombinant reactive center KPIR13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AßPP knock out and AßPP/KPIR13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. RESULTS: Recombinant mutant KPIR13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AßPP/KPIR13I mutant mice were similarly deficient as AßPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. CONCLUSIONS: We demonstrate that the anti-thrombotic function of AßPP primarily resides in the KPI activity of the protein.

Asunto(s)

Precursor de Proteína beta-Amiloide/metabolismo , Coagulación Sanguínea , Trombosis Intracraneal/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animales , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/genética , Trombosis de las Arterias Carótidas/metabolismo , Hemorragia Cerebral/sangre , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Técnicas de Sustitución del Gen , Humanos , Trombosis Intracraneal/sangre , Trombosis Intracraneal/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Platelets are the first peripheral source of amyloid precursor protein (APP). They possess the proteolytic machinery to produce Aß and fragments similar to those produced in neurons, and thus offer an ex-vivo model to study APP processing and changes associated with Alzheimer's disease (AD). Platelet process APP mostly through the α-secretase pathway to release soluble APP (sAPP). They produce small amounts of Aß, predominantly Aß40 over Aß42. sAPP and Aß are stored in α-granules and are released upon platelet activation by thrombin and collagen, and agents inducing platelet degranulation. A small proportion of full-length APP is present at the platelet surface and this increases by 3-fold upon platelet activation. Immunoblotting of platelet lysates detects APP as isoforms of 130 kDa and 106-110 kDa. The ratio of these of APP isoforms is significantly lower in patients with AD and mild cognitive impairment (MCI) than in healthy controls. This ratio follows a decrease that parallels cognitive decline and can predict conversion from MCI to AD. Alterations in the levels of α-secretase ADAM10 and in the enzymatic activities of α- and ß-secretase observed in platelets of patients with AD are consistent with increased processing through the amyloidogenic pathway. ß-APP cleaving enzyme activity is increased by 24% in platelet membranes of patients with MCI and by 17% in those with AD. Reports of changes in platelet APP expression with MCI and AD have been promising so far and merit further investigation as the search for blood biomarkers in AD, in particular at the prodromal stage, remains a priority and a challenge.