RESUMEN
High-density materials, such as titanium, used for spinal stabilization, introduces several critical issues in proton therapy (PT). Artefacts affect both contouring and dose calculation. Subsequently, artefacts need to be corrected which is a time-consuming process. Besides, titanium causes proton interactions that are unaccounted for in dose calculation. The result is a suboptimal treatment plan, and indeed decreased local controls have been reported for these patients. Carbon fiber reinforced polyetheretherketone (CFR-PEEK) implant material, which is of low density, potentially solves these issues. For this study, we designed a unique phantom to compare the effects of titanium and CFR-PEEK implants in PT. The phantom contains four interchangeable spinal inserts representing a native spine, and three different spinal stabilizations consisting of titanium only, CFR-PEEK only, and a combination of titanium and CFR-PEEK. All phantom scenarios received the standard treatment workup. Two planning approaches were investigated: a single field plan and a multi-field optimized plan with spinal cord sparing. For both plans we analyzed the following aspects: total volume of artefacts on CT images, time required for artefact correction, effect of planning CT correction on dose calculation, plan robustness to range and set up uncertainties, and finally the discrepancy between the calculated dose and the delivered dose with Gafchromic® film. The CFR-PEEK implant had a 90% reduction of artefacts on CT images and subsequently severely reduced the time for artefact correction with respect to the titanium-only implant. Furthermore, the CFR-PEEK as opposed to titanium did not influence the robustness of the plan. Finally, the titanium implants led to hardware-related discrepancies between the planned and the measured dose while the CFR-PEEK implant showed good agreement. As opposed to titanium, CFR-PEEK has none to minor effects on PT. The use of CFR-PEEK is expected to optimize treatment and possibly improve outcomes for patients that require spinal stabilization.
Asunto(s)
Fibra de Carbono/química , Cetonas/química , Fantasmas de Imagen , Polietilenglicoles/química , Prótesis e Implantes , Terapia de Protones/métodos , Neoplasias de la Columna Vertebral/radioterapia , Titanio/química , Benzofenonas , Humanos , Polímeros , Planificación de la Radioterapia Asistida por ComputadorRESUMEN
Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equimolar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.
Asunto(s)
Factor VIIIa/antagonistas & inhibidores , Factor V/farmacología , Integrina alfaXbeta2/farmacología , Proteína C/farmacología , Proteína S/antagonistas & inhibidores , Anticoagulantes/antagonistas & inhibidores , Anticoagulantes/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Factor VIIIa/metabolismo , Humanos , Integrina alfaXbeta2/metabolismo , Cinética , Membranas Artificiales , Fosfolípidos/metabolismo , Unión Proteica , Proteína C/metabolismo , Proteína S/efectos de los fármacos , Proteína S/metabolismo , Proteína S/farmacologíaRESUMEN
We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.
Asunto(s)
Complemento C4b/metabolismo , Proteínas Inactivadoras de Complemento , Factor Va/antagonistas & inhibidores , Factor Va/metabolismo , Glicoproteínas , Proteína C/fisiología , Receptores de Complemento/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , Arginina/genética , Sitios de Unión de Anticuerpos/genética , Unión Competitiva/genética , Unión Competitiva/inmunología , Línea Celular , Cricetinae , Activación Enzimática , Factor Va/genética , Glutamina/genética , Humanos , Riñón , Unión Proteica/genética , Proteína C/antagonistas & inhibidores , Proteína C/metabolismo , Proteína S/fisiología , Receptores de Complemento/genética , Receptores de Complemento/inmunología , Receptores de Complemento/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Eliminación de SecuenciaRESUMEN
An important mechanism of regulation of blood coagulation is the anticoagulant protein C pathway. In this pathway, the anticoagulant activity of activated protein C is increased by its cofactor protein S. The cofactor activity of protein S can be regulated by binding to complement regulatory C4b-binding protein (C4BP). The sites of interaction of protein S and C4BP are discussed.
Asunto(s)
Anticoagulantes/sangre , Complemento C4b/metabolismo , Integrina alfaXbeta2/sangre , Proteína S/metabolismo , Animales , Sitios de Unión , Coagulación Sanguínea/fisiología , Interacciones Farmacológicas , Humanos , Proteína C/metabolismo , Proteína S/antagonistas & inhibidoresRESUMEN
Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S.
Asunto(s)
Complemento C4b/fisiología , Proteínas Inactivadoras de Complemento , Glicoproteínas , Proteína S/fisiología , Receptores de Complemento/fisiología , Secuencia de Consenso , Humanos , Repeticiones de Microsatélite , Proteínas Recombinantes de FusiónRESUMEN
C4b-binding protein (C4BP) regulates the complement system and the anticoagulant activity of protein S. Protein S can bind to C4BP, resulting in a decreased cofactor activity of protein S for anticoagulant activated protein C. C4BP contains several identical a-chains and a single 3-chain. Each chain contains Short Consensus Repeats (SCRs). By making chimeras of 13-chain SCRs fused to tissue-type plasminogen activator (tPA chimeras), we found that 13-chain SCR-2 contributed to the interaction of 13-chain SCR-1 with protein S (van de Poel RHL, Meijers JCM, Bouma BN. J Biol Chem 274:15144-15150, 1999). Chimeras containing C4BP a-chains with SCR-1, SCR-l +2 or SCR-l +2+3 replaced by their 13-chain counterpart had affinities for protein S similar to C4BP (Hardig Y, Dahlb¿ck B. J Biol Chem 271:20861-20867, 1996). This was not in agreement with the finding that Beta-chain SCR-2 contributed to the interaction and could be explained by the possibility that alpha-chain SCR-2 in the alpha-chain chimeras contributed comparable with Beta-chain SCR-2 in the tPA chimeras. To investigate this we constructed a tPA chimera containing Beta-chain SCR-1 and alpha-chain SCR-2 (Beta1alpha2). Binding studies showed that Beta1alpha2 had a lower affinity compared with SCR-1 +2, indicating that alpha-chain SCR-2 did not contribute to the interaction. The difference with the alpha-chain chimeras may be explained by the fact that the alpha-chain chimeras were linked by their C-terminal cysteines, resulting in multiple binding sites in a single molecule. Thereby, the effect of a lower affinity of each alpha-chain chimera may have been masked. The studies performed here help to clarify the apparent inconsistencies in two previous reports about the contribution of the SCR-2 domain in C4BP to protein S binding. In conclusion, Beta-chain SCR-2 specifically contributes to the interaction of SCR-1 with protein S.