Association of A1AT With Poor Functional Outcome in Patients With Acute Ischemic Stroke

Jou nal o he Ame ican Hea Associa ion J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 1 ORIGINAL RESEARCH Associa ion o A1AT Wi h Poo Func ional Ou come in Pa ien s Wi h Acu e Ischemic S oke PaulaGa cía- Rod íguez , MSc; Ma cel Lamana- Vall e dú , BSc; Daisy R. Guamán- Pilco , MSc; Anna Penalba, MLT; Lau a Rami o , PhD; Alba Sima s , PhD; Júlia Fau a, PhD; Alejand o Bus aman e , MD, PhD; Noelia Díaz- T oyano , BSc; Joan Mon ane , MD, PhD BACKGROUND: This s udy aimed o in es iga e whe he A1AT (α- 1 an i ypsin) bloods eam le els, measu ed acu ely a e is- chemic s oke, can p edic he ou come o pa ien s wi h s oke. METHODS AND RESULTS: Two coho s o pa ien s wi h s oke we e s udied independen ly and e ospec i ely: a pilo coho o 59 pa ien s and a la ge eplica i e s udy wi h 527 pa ien s. Blood samples we e d awn a hospi al admission (<6 hou s a e s oke onse ) be o e any ea men was gi en. A1AT le els we e analyzed. Pa ien s we e ollowed a e he e en , and unc ional ou comes we e e alua ed a in- hospi al (discha ge) and mid e m ( hi d mon h) ollow- up acco ding o he modi ied Rankin Scale (conside ing modi ied Rankin Scale sco e >2 a poo ou come). Associa ion s udies be ween A1AT le els and unc ional ou comes we e conduc ed. We also e alua ed he added alue o A1AT as a p ognos ic bioma ke o e he clinical model (sex, age, p emo bid modi ied Rankin Scale and Na ional Ins i u es o Heal h S oke Scale sco es) wi h he likelihood a io es . In bo h s udies, he le els o A1AT we e highe in pa ien s who had wo se ou comes, we e olde , and had highe Na ional Ins i u es o Heal h S oke Scale sco es a admission. In he pilo s udy, highe le els o A1AT we e also associa ed wi h dea h a discha ge and a 3 mon hs a e s oke (P=0.035 and P=0.023, espec i ely). The addi ion o A1AT o he clinical model did no show enough e idence o inc easing he i o he model o he da a in ei he coho . CONCLUSIONS: Based on ou da a, we canno claim ha A1AT is an independen bioma ke o ischemic s oke. Ne e heless, A1AT is po en ially in ol ed in s oke ou comes and migh be explo ed as a po en ial he apeu ic a ge . Key Wo ds: A1AT ■ bioma ke ■ ou come ■ s oke S oke is conside ed he second leading cause o bo h disabili y and dea h wo ldwide. Almos 50% o s oke su i o s p esen disabili y ollowing he e en .1 P e en ing dea h and disabili y caused by s oke is a majo esea ch aim, and apid diagnosis and ea men can amelio a e he consequences p o- oked by ischemia. Iden i ying pa ien s who a e mo e likely o ha e poo ou comes migh be impo an o guiding p ope he apy managemen .2 F om he neu ologis ’s pe spec i e, p edic ing clin- ical and unc ional ou comes a e acu e ischemic s oke is challenging. S oke ou come is known o be s ongly in luenced by he baseline demog aphic and clinical cha ac e is ics o pa ien s and he cause and loca ion o he s oke, among many o he ac- o s.3 Howe e , clinical complica ions ha occu la e du ing he cou se o he disease, including hemo - hagic ans o ma ions, ce eb al edema, in ec ions, o Co espondence o: Joan Mon ane , MD, PhD, Neu o ascula Resea ch G oup, IBiS/Hospi al Uni e si a io Vi gen Maca ena/CSIC/Uni e si y o Se ille, A enue Manuel Siu o s/n, 41013 Se ille, Spain. Email: jmon ane [email protected] This a icle was sen o Michelle H. Leppe , MD, MBA, Associa e Edi o , o e iew by expe e e ees, edi o ial decision, and inal disposi ion. Supplemen al Ma e ial is a ailable a h ps:// www. ahajo u nals. o g/ doi/ suppl/ 10. 1161/ JAHA. 124. 036727 Fo Sou ces o Funding and Disclosu es, see page 9. © 2025 The Au ho (s). Published on behal o he Ame ican Hea Associa ion, Inc., by Wiley. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed and is no used o comme cial pu poses. JAHA is a ailable a : www.ahajou nals.o g/jou nal/jaha Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 2 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come ecu en e en s, migh also be key ac o s ha a ec s oke mo bidi y.4–6 In his scena io, he iden i ica ion o blood bioma ke s ha aid in p edic ing pa ien s wi h s oke ou comes migh help clinicians selec hose pa- ien s who should be admi ed o speci ic s oke ca e uni s o ongoing clinical ials o ensu e ideal pa ien ea men and imp o e pa ien ou comes.7 To da e, he e has been an inc ease in he li e a u e assessing he associa ion o blood bioma ke s wi h pa ien ou - comes a e s oke, bu he clinical alue o hese bio- ma ke s in guiding decision- making s a egies emains unce ain.8 A1AT (α- 1 an i ypsin) is he mos abundan se ine p o einase inhibi o in human blood. The s anda d plasma concen a ion o A1AT ypically anges om 80 o 220 mg/dL.9 I s main unc ion is o inhibi neu ophil elas ase, bu i also has an i- in lamma o y and an ia- pop o ic p ope ies and is in ol ed in p o eos asis.10,11 Addi ionally, se e al s udies ha e sugges ed ha A1AT migh play a ole in he s abili y o a e ial walls, be- cause he e is an associa ion be ween A1AT le els and he o ma ion and up u e o in ac anial and ao ic an- eu ysms.12–14 Se e e A1AT de iciency has been linked o he de elopmen o emphysema, b onchiec asis, and li e disease.15 In con as , A1AT has been shown o inc ease apidly in he ci cula ion du ing acu e sys emic in lamma o y esponses, including s oke. In his ega d, highe le els o A1AT in he bloods eam a e also associa ed wi h an ele a ed isk o de eloping s oke.16 Howe e , he associa ion be ween A1AT and s oke ou come and i s possible ole as a bioma ke o s oke p ognosis emain unknown.2 This s udy aimed o de e mine whe he ci cula ing A1AT le els measu ed acu ely a e ischemic s oke (he ea e e e ed o a s oke) could p edic pa ien s wi h s oke ou comes in he subacu e phase (a dis- cha ge) and in he mid e m (3 mon hs). METHODS Da a A ailabili y The da a ha suppo he indings o his s udy a e a ailable om he co esponding au ho upon easonable eques . S udy Design and Popula ion In his s udy, 2 analyses we e pe o med: a i s pilo s udy and a subsequen eplica ion s udy. In bo h co- ho s, samples om he Blood Bioma ke s o he Ea ly Diagnosis o S oke: The S oke- CHIP S udy coho we e analyzed. The S oke- CHIP s udy was an obse a- ional, e ospec i e, and mul icen e s udy pe o med a he eme gency depa men s o 6 hospi als in Ca alonia om Augus 2012 o No embe 2013.17 The inclusion c i e ia o his s udy included he ollowing pa ame e s: suspec ed s oke a he i s medical assessmen wi h pe sis en symp oms when a i ing a he eme gency oom, age >18 yea s, <6 hou s om symp om onse o blood sample collec ion, blood collec ion p eceding h omboly ic ea men wi h PA ( issue- ype plasmino- gen ac i a o ) (i eligible), and signed in o med consen . The p esen s udy has adhe ed o he S eng hening he Repo ing o Obse a ional S udies in Epidemiology (STROBE) guidelines.18 Fo he pilo s udy, pa ien s wi h acu e ischemic s oke admi ed o he eme gency depa men a Hospi al Uni e si a i Vall d’Heb on (Ba celona, Spain) wi hin he i s 6 hou s a e symp om onse we e p o- spec i ely s udied. A o al o 81 pa ien s wi h ischemic s oke we e ec ui ed. Th ee o hese pa ien s wi h ischemic s oke ecei ed PA a a s anda d 0.9 mg/kg dose (10% bolus, 90% con inuous in usion o 1 hou ), 21 pa ien s we e subjec ed o mechanical h ombec- omy, and 2 pa ien s unde wen bo h epe usion s a - egies. In he eplica ion s udy, pa ien s’ samples we e om di e en cen e s. Samples om 863 pa ien s wi h ischemic s oke we e s udied; 312 pa ien s wi h s oke ecei ed PA a a s anda d 0.9 mg/kg dose (10% bolus, 90% con inuous in usion o 1 hou ), 52 pa ien s we e subjec ed o mechanical h ombec omy, and 30 pa- ien s unde wen bo h epe usion s a egies. Excluding RESEARCH PERSPECTIVE Wha Is New? • Ou s udy e eals ha A1AT (α- 1 an i ypsin) has a ole in s oke p ognosis. Speci ically, highe le els o A1AT in he acu e phase seem o be associa ed wi h poo ou comes a e ischemic s oke. Wha A e he Clinical Implica ions? • The p esen s udy ein o ces he impo ance o u he in es iga ing he ole o A1AT in he acu e phase o s oke o guiding decision- making p ocesses on he managemen and ea men o pa ien s wi h ischemic s oke. Nons anda d Abb e ia ions and Ac onyms A1AT α- 1 an i ypsin mRS modi ied Rankin Scale NIHSS Na ional Ins i u es o Heal h S oke Scale PA issue plasminogen ac i a o Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 3 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come ea ed pa ien s, he numbe o pa ien s in he pilo and eplica ion s udy we e 59 and 527, espec i ely. Clinical P o ocol The s udy was app o ed by he Hospi al Uni e si a i Vall d’Heb on Clinical Resea ch E hics Commi ee as he coo dina ing cen e (PR[AG]80/2012) and by all pa icipa ing cen e s and was conduc ed in acco dance wi h he Decla a ion o Helsinki. All pa ien s o hei ela i es p o ided in o med consen . A de ailed his o y o ascula isk ac o s was ob ained o each pa ien . S oke se e i y a hospi al admission and 24 and 48 hou s a e admission was assessed using he Na ional Ins i u es o Heal h S oke Scale (NIHSS). The clinical ollow- up o each pa ien wi h s oke was pe o med un il he hi d mon h a e s oke. Func ional ou come was e alua ed acco ding o he modi ied Rankin Scale (mRS)19; pa ien s wi h an mRS sco e ≤2 we e classi ied as ha ing a good ou come, and pa ien s wi h an mRS sco e anging om 3 o 6 we e classi ied as ha ing a poo ou come. Blood Sample Collec ion and A1AT Measu emen Pe iphe al blood samples we e d awn om all pa ien s a hospi al admission (<6 hou s a e s oke onse ) be- o e any ea men was gi en. Blood was collec ed wi h e hylenediamine e aace ic acid ubes and cen i uged a 1500g o 15 minu es a 4 °C, and plasma aliquo s we e ozen a −80 °C un il bioma ke analysis. Fo he pilo s udy, ELISA ki s we e used, and he manu ac u e ’s ins uc ions we e ollowed o quan i y he ci cula ing le els o A1AT (ca alog numbe 108799; Abcam, Uni ed Kingdom). Op ical densi y was mea- su ed using a Syne gy Mx mic opla e eade (BioTek Ins umen s, Uni ed S a es). Each sample was es ed in duplica e, and he mean alue o bo h measu e- men s was used. Samples wi h a coe icien o a ia- ion be ween duplica es >20% we e excluded om he analysis. All samples we e wi hin he de ec able ange o he assay. In he eplica ion s udy, A1AT le els we e measu ed by a quan i a i e immunonephelome y- based assay (A ellica NEPH 630; Siemens Heal hca e Diagnos ics), a s anda d p ocedu e in ou ine hospi al es ing, ol- lowing he manu ac u e ’s guidelines. S a is ical Analysis R e sion 4.4.1 was used o he s a is ical analyses and g aph gene a ion. Desc ip i e ables p esen medians and in e qua ile anges o con inuous a iables and coun s and pe cen ages o ca ego ical a iables. Fo bi a ia e ables, di e ences among g oups we e as- sessed wi h he Wilcoxon- Mann- Whi ney ank sum es o con inuous a iables and he χ2 es o ca ego ical a iables. Analyses o bo h coho s we e conduc ed independen ly wi h no sha ed da a. In all analyses, we ea ed NIHSS and mRS sco es as con inuous a i- ables. Fo all es s, P alues <0.05 we e conside ed s a is ically signi ican . Due o he ea men imbalance p esen be ween coho s, analysis was ocused on he subse o un- ea ed pa ien s (pa ien s wi h ischemia no ea ed wi h ei he h ombolysis o mechanical h ombec omy). Ne e heless, sensi i i y analyses on esul s including he 2 ea men g oups we e also pe o med. To quan i y A1AT aw disc iminan capabili y, mea- su ed le els o he p o ein we e plo ed o ele an ou come g oups and compa ed wi h he Wilcoxon- Mann- Whi ney es . An es ima e o he C s a is ic (a ea unde he ecei e ope a ing cha ac e is ic cu e) and i s 95% CI we e calcula ed h ough 2000 s a i ied boo s ap esampling. Bina y logis ic eg ession models we e i ed in he pilo and eplica ion s udies independen ly o p ognosis (poo ou come: mRS >2 e sus good ou come: mRS ≤2) a hospi al discha ge and 3 mon hs a e he episode. All models included sex, age, p emo bid mRS sco e, and NIHSS sco e. The likelihood a io es was used o de e - mine A1AT’s added alue o e he clinical model. Pilo s udy models a e i ed using a comple e case analysis pa adigm. In he eplica ion s udy, missing alues we e mul iple impu ed using p edic i e mean ma ching- based me hods (a egImpu e om he ms R package) wi h 40 i e a ions. Reg ession coe icien s we e a e aged ac oss all impu a ions, and a iance– co a iance ma ices we e co ec ed acco ding o he Rubin ule. All modeled a iables we e used o im- pu a ion. The adequacy o he impu a ion model was e alua ed by compa ing dis ibu ions o impu ed al- ues wi h comple e da a. Fo each model, we ob ained he C s a is ic (a ea unde he ecei e ope a ing cha ac e is ic cu e), R2 (Nagelke ke), B ie sco e, and calib a ion in e cep and slope. These me ics we e in e nally alida ed using boo s ap op imism es ima ion (2000 esamples). To examine al e na i e clinical ou comes, logis ic eg ession models o he NIHSS sco e a 48 hou s (p opo ional odds, o dinal) and exi us a 3 mon hs (di- cho omic) we e also i ed o eplica ion coho . The ag eemen o nephelome y measu emen s wi h ELISA alues was assessed using Bland- Al man ag eemen plo on a se o samples. RESULTS This s udy was conduc ed in 2 phases. Ini ially, a pilo s udy in ol ed 59 pa ien s wi h ischemic s oke. To al- ida e he indings o he pilo s udy, a eplica ion s udy Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 4 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come was conduc ed, inc easing he sample size o 527. The demog aphic cha ac e is ics and isk ac o p o iles o he pa ien s included in he pilo s udy and eplica ion s udy a e shown in Table1. Bo h s udies we e com- pa able in e ms o mos o hei demog aphic cha ac- e is ics. Howe e , in he pilo s udy, pa ien s exhibi ed highe NIHSS sco es a admission (pilo : 10 [6–16] e - sus eplica ion 4 [2–9]). Addi ionally, in he eplica ion s udy, he pe cen age o pa ien s wi h mRS=0 was double ha o he pilo s udy (60% e sus 29%, e- spec i ely). Samples o he pilo s udy we e analyzed by ELISA, whe eas samples o he eplica ion s udy we e analyzed by nephelome y. Measu emen com- pa ison on an independen se o samples demon- s a ed a co ela ion be ween esul s ob ained by bo h echniques (R2=0.928, =0.96, P<0.001) bu also a bias along he concen a ion ange (Figu eS1). To e alua e he ela ionship be ween A1AT le - els and he p ognosis o pa ien s wi h s oke, we i s pe o med bi a ia e analyses o he ou comes o he pa ien s bo h a discha ge (in- hospi al ou come) and a e 3 mon hs (mid e m ou come). In bo h s udies, pa- ien s wi h wo se ou comes bo h in- hospi al and mid- e m had highe baseline le els o A1AT and p esen ed highe NIHSS sco es a admission. In he pilo s udy, he numbe o pa ien s wi h diabe es who had poo ou comes was highe han hose wi h good ou comes (bo h a discha ge and mid e m). The e we e also di - e ences be ween he causes o s oke, wi h ca dioem- bolic s oke being he mos epo ed (in pe cen age) in he eplica ion s udy. Simila ly, in he eplica ion s udy, he pe cen ages o women wi h poo ou comes we e highe a discha ge and a he mid e m ollow- up com- pa ed wi h men. Mo eo e , he e also we e di e ences in p e ious mRS sco es and ou come in he eplica ion s udy, p esen ing highe p e ious mRS sco es in hose pa ien s wi h poo ou comes han hose wi h good ou comes, bo h a discha ge and mid e m (Table2). In in es iga ing he associa ion be ween A1AT le els and unc ional ou comes in he hospi al and 3 mon hs la e , as we ha e commen ed p e iously, we obse ed ha pa ien s wi h poo ou comes exhibi ed highe le els o A1AT a hospi al admission (Figu e1), as ob- se ed in bo h he pilo (Figu e1A and 1B) and ep- lica ion s udies (Figu e1C and 1D). In his sense, we also ound ha in he pilo s udy, 15% o he pa ien s wi h poo ou comes p esen ed abo e no mal le els o A1AT (>220 mg/dL) a mid e m (P=0.044) (Figu e1B and TableS1). In he sensi i i y analysis, we compa ed A1AT alues by ou come among g oups ha included ea ed pa ien s. A comp ehensi e desc ip ion o he ull coho is a ailable in TableS2. The concen a ion le els showed consis en s a is ical signi icance ac oss all subse s a bo h discha ge and 3 mon hs, excep o he g oup consis ing only o ea ed pa ien s (see TableS3 o de ailed esul s). Because we obse ed ha A1AT le els a e associ- a ed wi h poo e ou comes in 2 independen coho s o pa ien s wi h s oke (Figu e1), we sough o in es- iga e whe he A1AT le els we e associa ed wi h mo - ali y, ei he du ing hospi aliza ion o wi hin 3 mon hs (Figu e2). We saw di e ences in he pilo s udy, whe e highe le els o A1AT upon admission we e associ- a ed wi h dea h a discha ge (P=0.035) (Figu e2A) and 3 mon hs a e he e en (P=0.023) (Figu e2B). No s a is ical di e ences we e ound in he eplica ion s udy ei he a discha ge (Figu e2C) o 3 mon hs la e (Figu e2D). Table 1. Demog aphic and Clinical In o ma ion F om Pa ien s Included in Bo h he Pilo and Replica ion S udies Cha ac e is ic Pilo Replica ion N=59 N=527 A1AT (mg/dL) 124 (101–162) 156 (139–178) Age, y 76 (66–82) 76 (65–83) Sex (women) 29 (49%) 248 (47%) NIHSS sco e admission 10.0 (6.0–16.0) 4 (2–9) Smoke 6 (11%) 79 (15%) A e ial hype ension 44 (75%) 395 (75%) Diabe es 18 (31%) 146 (28%) Dyslipidemia 31 (53%) 246 (47%) A ial ib illa ion 17 (29%) 175 (33%) P e ious s oke 11 (19%) 114 (22%) P e ious mRS sco e 017 (29%) 305 (60%) 126 (45%) 55 (11%) 27 (12%) 41 (8.1%) 33 (5.2%) 71 (14%) 45 (8.6%) 33 (6.5%) 53 (0.6%) OCSP TACI 22 (37%) 100 (24%) PACI 25 (42%) 183 (43%) LACI 7 (12%) 102 (24%) POCI 5 (8.5%) 39 (9.2%) In a c TOAST A he o h ombo ic o g ea ase a he oscle osis 10 (17%) 63 (12%) Ca dioembolic 21 (36%) 196 (38%) Lacuna o small ase disease 6 (10%) 98 (19%) O he in equen cases 0 (0%) 9 (1.7%) Unde e mined 22 (37%) 155 (30%) Edema 3 (5.1%) 11 (2.1%) Da a a e exp essed as he n (%) o median (in e qua ile ange). A1AT indica es α- 1 an i ypsin; LACI, lacuna in a c ; mRS, modi ied Rankin Scale; NIHSS, Na ional Ins i u es o Heal h S oke Scale; OCSP, Ox o dshi e Communi y S oke P ojec ; PACI, pa ial an e io ci cula ion in a c ; POCI, pos e io ci cula ion in a c ; TACI, o al an e io ci cula ion in a c ; and TOAST, T ial o O g 10172 in Acu e S oke T ea men . Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 5 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come Table 2. Bi a ia e Analysis o Clinical Cha ac e is ics Associa ed Wi h In- Hospi al and Mid e m Func ional Ou comes Cha ac e is ic Pilo , discha ge Pilo , mo Replica ion, discha ge Replica ion, 3 mo Good ou come, mRS ≤2, N=28 Poo ou come, mRS >2, N=30 P alue* Good ou come, mRS ≤2, N=30 Poo ou come, mRS >2, N=27 P alue* Good ou come, mRS ≤2, N=301 Poo ou come, mRS >2, N=215 P alue† Good ou come, mRS ≤2, N=261 Poo ou come, mRS >2, N=114 P alue† A1AT (mg/dL) 114 (94–144) 146 (106–183) 0.025 116 (97–152) 148 (109–189) 0.025 153 (134–173) 162 (143–186) <0.001 151 (135–172) 167 (146–188) <0.001 Age, y 74 (64–81) 79 (66–82) 0.3 73 (64–80) 79 (67–83) 0.14 71 (60–80) 82 (74 –87) <0.001 71 (60–79) 82 (75–87) <0.001 Sex (women) 16 (57%) 13 (43%) 0.3 16 (53%) 11 (41%) 0.3 118 (39%) 125 (58%) <0.001 104 (40%) 65 (57%) 0.002 NIHSS sco e admission 7.5 (3.0–10.0) 15.0 (9.0–20.0) <0.001 8.0 (3.0–10.0) 13.5 (9.0–20.0) 0.002 2 (1–4) 11 (4–19) <0.001 2 (1–4) 11 (5 –19) <0.001 Smoke 4 (16%) 2 ( 7.1%) 0.4 3 (11%) 2 (8.0%) >0.9 58 (19%) 18 (8.4%) <0.001 47 (18%) 11 (9.6%) 0.039 A e ial hype ension 20 (71%) 23 (77%) 0.6 23 (77%) 20 (74%) 0.8 227 (75%) 163 (76%) >0.9 192 (74%) 88 (77%) 0.5 Diabe es 5 (18%) 13 (43%) 0.036 5 (17%) 13 (48%) 0.011 76 (25%) 67 (31%) 0.14 62 (24%) 33 (29%) 0.3 Dyslipidemia 14 (50%) 16 (53%) 0.8 15 (50%) 15 (56%) 0.7 148 (49%) 91 (42%) 0.12 123 (47%) 51 (45%) 0.7 A ial ib illa ion 5 (18%) 12 (40%) 0.064 4 (13%) 13 (48%) 0.004 72 (24%) 101 (47%) <0.001 67 (26%) 59 (52%) <0.001 P e ious s oke 5 (18%) 6 (20%) 0.8 4 (13%) 6 (22%) 0.5 50 (17%) 63 (29%) <0.001 45 (17%) 32 (28%) 0.017 P e ious mRS sco e 09 (32%) 8 (28%) 0.5 11 (38%) 6 (22%) 0.10 228 (78%) 73 (35%) <0.001 200 (80%) 28 (25%) <0.001 112 (43%) 13 (45%) 12 (41%) 12 (44%) 34 (12%) 19 (9.1%) 27 (11%) 16 (15%) 26 (21%) 1 (3.4%) 5 (17%) 2 (7.4%) 21 ( 7.2%) 20 (9.6%) 17 (6.8%) 12 (11%) 30 (0%) 3 (10%) 0 (0%) 3 (11%) 7 (2.4%) 62 (30%) 6 (2.4%) 30 (27%) 41 (3.6%) 4 (14%) 1 (3.4%) 4 (15%) 1 (0.3%) 31 (15%) 0 (0%) 22 (20%) 50 (0%) 3 (1.4%) 0 (0%) 2 (1.8%) OCSP TACI 4 (14%) 18 (60%) <0.001 4 (13%) 17 (63%) <0.001 8 (3.8%) 91 (46%) <0.001 9 (4.6%) 52 (51%) <0.001 PACI 16 (57%) 8 (27%) 18 (60%) 6 (22%) 112 (53%) 65 (33%) 100 (51%) 32 (31%) LACI 6 (21%) 1 (3.3%) 5 (17%) 2 (7.4%) 71 (33%) 27 (14%) 64 (33%) 12 (12%) POCI 2 (7.1%) 3 (10%) 3 (10%) 2 (7.4%) 22 (10%) 17 (8.5%) 22 (11%) 6 (5.9%) In a c TOAST A he o h ombo ic o g ea ase a he oscle osis 6 (21%) 3 (10%) 0.12 6 (20%) 4 (15%) 0.3 39 (13%) 23 (11%) <0.001 35 (14%) 10 (8.9%) <0.001 Ca dioembolic 7 (25%) 14 (47%) 7 (23%) 13 (48%) 89 (30%) 104 (49%) 80 (31%) 58 (52%) Lacuna o small ase disease 5 (18%) 1 (3.3%) 4 (13%) 2 (7.4%) 73 (24%) 21 (10.0%) 60 (23%) 11 (9.8%) O he in equen cases 0 (0%) 0 (0%) 0 (0%) 0 (0%) 4 (1.3%) 5 (2.4%) 4 (1.6%) 2 (1.8%) Unde e mined 10 (36%) 12 (40%) 13 (43%) 8 (30%) 94 (31%) 58 (27%) 79 (31%) 31 (28%) (Con inued) Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 6 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come We nex sough o de e mine he c ude disc imina- i e abili y o A1AT o p edic ing unc ional ou comes by cons uc ing ecei e ope a ing cha ac e is ic cu es. In he pilo s udy, he a ea unde he cu e o A1AT o poo ou comes (mRs >2) was 0.675 a discha ge (95% CI, 0.531–0.801) and 0.678 (95% CI, 0.531–0.806) in he mid e m. In he eplica ion s udy, he a ea unde he cu e es ima es o A1AT we e lowe in bo h ime poin s, a 0.601 (95% CI, 0.552–0.650) a discha ge, and 0.623 (95% CI, 0.561–0.680) a 3 mon hs a e discha ge. Nex , we e alua ed whe he A1AT could imp o e he p edic ion o wo se unc ional ou comes in com- bina ion wi h con en ional clinical isk ac o s. Fo his, we c ea ed a p edic i e model o bo h discha ge and mid e m s oke ou comes in he pilo s udy, including sex, age, p emo bid mRS sco e, and NIHSS sco e (Table3). Acco ding o he likelihood a io es , he ad- di ion o A1AT o he clinical model did no inc ease he i o he da a o he models in ei he coho . On al e na i e ou come mul i a iable models, no e - idence o s a is ically signi ican associa ion was ound on models wi h dea h o NIHSS sco e a e 48 hou s. Comple e es ima es a e in TableS4. We also obse ed ha A1AT le els p esen ed co - ela ion e idence wi h he NIHSS sco e a admission and age (Figu esS2 and S3). DISCUSSION P edic ing he ou come o pa ien s wi h ischemic s oke is c ucial o guiding decision- making and en- su ing he bes possible managemen o pa ien s a e he e en . Cu en ly, ou come p edic ion elies on clinical da a om pa ien s, including ac o s such as age, como bidi ies, and s oke se e i y.20 Se e al s udies ha e sugges ed ha inco po a ing blood bio- ma ke s in o hese clinical models o ou come p e- dic ion may enhance hei p edic i e accu acy.8,21,22 Obse a ional s udies claim ha ou come modi ica ion can be eached by (1) he disco e y o new candida es in ega d o omic echniques and (2) he assessmen o he candida e’s po en ial and u he analysis in la ge s udies.23 In his ega d, we assessed he possible as- socia ions o A1AT a he hype acu e phase o ischemic s oke wi h subacu e and mid e m p ognoses in a p e- limina y pilo s udy and hen in a la ge eplica ion s udy. Inc eased bloods eam le els o A1AT ha e p e- iously been associa ed wi h wo se ou comes in pa- ien s wi h o he diseases.24,25 Howe e , he exac ole o A1AT in s oke pa hophysiology emains unce ain. Au ho s such as Mah a e  al sugges ed he asculo- p o ec i e ole o A1AT.21 In hei s udies, hey showed ha a mu a ion in SERPINA1, he A1AT- encoding gene, is associa ed wi h acu e ischemic s oke p o oked by Cha ac e is ic Pilo , discha ge Pilo , mo Replica ion, discha ge Replica ion, 3 mo Good ou come, mRS ≤2, N=28 Poo ou come, mRS >2, N=30 P alue* Good ou come, mRS ≤2, N=30 Poo ou come, mRS >2, N=27 P alue* Good ou come, mRS ≤2, N=301 Poo ou come, mRS >2, N=215 P alue† Good ou come, mRS ≤2, N=261 Poo ou come, mRS >2, N=114 P alue† Edema 0 (0%) 3 (10%) 0.2 0 (0%) 3 (11%) 0.10 0 (0%) 11 (5.2%) <0.001 0 (0%) 11 (9.6%) <0.001 The da a a e exp essed as n (%) o median (in e qua ile ange). In ega d o he pa ien coun s shown in Table1, in he pilo s udy, 1 pa ien (1.69%) wi h mRS sco es was no egis e ed a discha ge, and 2 pa ien s (3.39%) we e egis e ed a 3 mo. In he eplica ion s udy, 11 (2.09%) had missing mRS sco es a discha ge, and 152 (28.84%) had missing mRS sco es a 3 mo. A1AT indica es α- 1 an i ypsin; LACI, lacuna in a c ; mRS, modi ied Rankin Scale; NIHSS, Na ional Ins i u es o Heal h S oke Scale; OCSP, Ox o dshi e Communi y S oke P ojec ; PACI, pa ial an e io ci cula ion in a c ; POCI, pos e io ci cula ion in a c ; TACI, o al an e io ci cula ion in a c ; and TOAST, T ial o O g 10172 in Acu e S oke T ea men . *Wilcoxon ank sum exac es , Wilcoxon ank sum es , Pea son χ2 es , Fishe exac es . †Wilcoxon ank sum es , Pea son χ2 es , Fishe exac es . Table 2. Con inued Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 7 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come la ge- a e y a he oscle osis.22 In his ega d, a pilo s udy measu ing A1AT le els wi hin he i s 24 hou s a e ischemic s oke in 18 pa ien s sugges ed he po- en ial p o ec i e ole o A1AT agains a he oscle osis.26 Howe e , hey also epo ed ha highe ci cula ing A1AT le els we e associa ed wi h la ge in a c olumes bu no wi h unc ional ou comes a hospi al discha ge. Simila ly, p eclinical s udies ha e highligh ed he possi- ble neu op o ec i e ole o A1AT, epo ing ha he apy wi h human A1AT in animal models amelio a es ischemic damage and imp o es animal ou comes a e ischemic s oke.27 Ne e heless, inc eased le els o A1AT ha e been ound in he ischemic co e o he b ain compa ed wi h hose in he heal hy con ala e al a ea28 and a e associa ed wi h a g ea e isk o de eloping s oke in child en.16 In ega d o neu ological ou comes, ano he s udy epo ed ha ci cula ing le els o A1AT measu ed wi hin he i s 24 hou s a e a he o h ombo ic ischemic s oke we e g ea e in pa ien s wi h poo neu ological ou comes 1 mon h a e he e en .29 In ou pilo s udy, we ound ha pa ien s wi h highe le els o A1AT had wo se ou comes (mRs >2) a dis- cha ge and 3 mon hs a e he e en . Subsequen ly, upon analyzing A1AT le els in a la ge eplica ion co- ho , we con i med hese esul s. Simila ly, a p e ious s udy epo ed ha ci cula ing le els o A1AT measu ed wi hin he i s 24 hou s a e a he o h ombo ic isch- emic s oke we e g ea e in pa ien s wi h poo neu- ological ou comes 1 mon h a e he e en .29 Taken oge he , hese indings would indica e ha he se um A1AT concen a ion measu ed ea ly a e s oke could in o m clinicians abou pa ien ou comes. We also ound ha pa ien s who died in he hospi- al o be ween discha ge and 3 mon hs a e he e en had highe le els o A1AT in he acu e phase (<6 hou s om symp om onse ). Ou esul s a e suppo ed by wha was p e iously desc ibed, which is ha high le - els o A1AT a e ela ed o a majo isk o mo ali y.30 Ne e heless, hese esul s a e no eplica ed in ou la ge coho , which is why we canno con i m whe he Figu e 1. A1AT le els a admission and hei associa ion wi h unc ional ou come. Associa ions be ween A1AT le els a admission and in- hospi al (P<0.001) and mid e m (P<0.001) unc ional ou comes in he pilo s udy (A and B) and in he eplica ion s udy (C and D). Shaded ibbon ep esen s he no mal ange o A1AT in he heal h popula ion, which anges om 80 o 220 mg/dL. Box plo s ep esen he median and IQR. A1AT indica es α- 1 an i ypsin; and mRS, modi ied Rankin Scale. Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 8 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come high se um le els o A1AT a e ela ed o u u e mo bid- i y o mo ali y. Bo h s udies p esen ed he e ha e shown ha pa ien s who p esen wi h wo se ou comes ha e in- c eased baseline A1AT plasma le els. Howe e , he pa hophysiological easons o his ela ionship a e complex and challenging o explain. To da e, limi ed li e a u e indica es ha A1AT could ac by modula ing he ac i i y o immune cells. A1AT educes he elease o p oin lamma o y cy okines and inc eases he se- c e ion o an i- in lamma o y ac o s a e oxygen and glucose dep i a ion, hence p o ec ing neu ons om nec o ic and apop o ic cell dea h.31 These esul s in- dica e ha high le els o A1AT can also p o ec glial cells om oxygen and glucose dep i a ion- induced cell dea h, ein o cing i s impo an ole in neu op o- ec ion. O he p eclinical s udies ha e shown ha he adminis a ion o A1AT a e ce eb al ischemia exe s neu op o ec i e e ec s by educing in a c size27 and modula ing he ac i i y o immune cells.32 Simila ly, A1AT has been shown o exe a p o ec i e e ec agains ascula inju y in o he in lamma o y diseases by inhibi ing Tumo Nec osis Fac o (TNF)- α- induced apop osis.32,33 Based on p e ious li e a u e and ou indings, we hypo hesize ha ele a ed A1AT le els de ec ed in pa- ien s wi h s oke wi h poo ou comes may ep esen a p o ec i e mechanism aimed a balancing he ha m ul p ocesses ini ia ed by ce eb al ischemia. Ne e heless, we canno ule ou ha his up egula ion in A1AT migh con ibu e o he expansion o he ischemic a ea, he eby exace ba ing poo s oke ou comes. Fu he esea ch is necessa y o elucida e he unc ion o A1AT in s oke pa hophysiology. Al hough he ela ionship be ween inc eased le els o A1AT, age, and neu ological sco e a e no explici ly discussed in he cu en published li e a u e, ou e- sul s sugges co ela ion. In ega d o p edic i e models o poo ou comes a hospi al discha ge and mid e m a e s oke, A1AT did no imp o e he p edic ion cu en ly done only wi h clinical in o ma ion. We ound nei he a p ognos ic Figu e 2. A1AT le els a admission and hei associa ion wi h mo ali y. Associa ions be ween A1AT le els a admission and in- hospi al and mid e m mo ali y in he pilo s udy (A and B) and in he eplica ion s udy (C and D). Shaded ibbon ep esen s he no mal ange o A1AT in he heal h popula ion, which anges om 80 o 220 mg/dL. Box plo s ep esen he median and in e qua ile ange. A1AT indica es α- 1 an i ypsin. Downloaded om h p://ahajou nals.o g by on June 2, 2025 J Am Hea Assoc. 2025;14:e036727. DOI: 10.1161/JAHA.124.036727 9 Ga cía- Rod íguez e al A1AT in he Con ex o S oke Ou come alue o A1AT in p edic i e models o NIHSS sco e a 48 hou s a e s oke no exi us. Fo ha , based on ou esul s, we canno claim ha A1AT complemen s clini- cal c i e ia o diagnosing ou comes ollowing ischemic s oke. The main s eng hs o he p esen s udy a e he la ge sample size and analysis o 2 dis inc coho s o pa ien s wi h s oke, enhancing he obus ness and gene aliz- abili y o he indings. Howe e , some limi a ions need o be no ed. Fi s , we analyzed he whole coho wi h- ou conside ing di e ences in s oke cause. As men- ioned abo e, a he o h ombo ic s okes ha e been he mos s udied ype o s oke in he A1AT esea ch ield. The e o e, ou esul s should be alida ed in an inde- penden , s a i ied analysis be o e solid conclusions can be d awn. Second, we analyzed plasma samples om di e en coho s using di e en echniques. Be o e doing s a is ical analysis, we e alua ed he co ela ion be ween bo h echniques. The esul s demons a ed a s ong co ela ion be ween ELISA and nephelome y. Howe e , o minimize po en ial echnical bias, he use o he same echnique would be ad ised. In his sense, nephelome y appea s o be he mos used me hod o quan i ying p o ein le els in hospi al acili ies.34 In b ie , we aimed o es ablish A1AT as an objec i e and eliable bioma ke o p edic ing pa ien ou comes a e a s oke e en . Al hough ou indings do no allow us o con i m A1AT as an independen and in o ma- i e bioma ke o s oke p ognosis, ou da a show ha A1AT has a ole in s oke pa hophysiology and subse- quen ly in pa ien ou comes. This insigh highligh s he need o u he esea ch in o A1AT in he con ex o s oke p ognosis and sugges s i could be explo ed as a po en ial he apeu ic a ge . ARTICLE INFORMATION Recei ed Augus 19, 2024; accep ed Decembe 19, 2024. A ilia ions Neu o ascula Resea ch Labo a o y, Vall d’Heb on Ins i u e o Resea ch (VHIR), Uni e si a Au ònoma de Ba celona, Ba celona, Spain (P.G., M.L., D.R.G., A.P., L.R., J.F., A.B., J.M.); Ce eb o ascula Resea ch Labo a o y, Ins i u o de In es igaciones Biomédicas de Ba celona (IIBB), Consejo Supe io de In es igaciones Cien í icas (CSIC), Ba celona, Spain (A.S.); S oke Uni , Hospi al Uni e si a i Ge mans T ias i Pujol, Ge mans T ias i Pujol Resea ch Ins i u e (IGTP), Uni e si a Au ònoma de Ba celona, Ba celona, Spain (A.B.); Depa men o Biochemis y, Vall d’Heb on Uni e si y Hospi al, Ba celona, Spain (N.D.); and S oke Resea ch P og am, Ins i u e o Biomedicine o Se ille, IBiS/Hospi al Uni e si a io Vi gen del Rocío/CSIC/ Uni e si y o Se ille & Depa men o Neu ology, Hospi al Uni e si a io Vi gen Maca ena, Se ille, Spain (J.M.). Acknowledgmen s The au ho s acknowledge all o he pa ien s, pa ien s’ amilies, and p o es- sionals om he ec ui ing cen e s o hei kind suppo in he s udy. Sou ces o Funding The Neu o ascula Resea ch Labo a o y akes pa in he Redes de In es igación Coope a i a O ien adas a Resul ados en Salud- ICTUS En e medades Vascula es Ce eb ales Ne wo k, Ins i u o de Salud Ca los III), Spain (RD21/0006/0007), and acknowledges unding o his p ojec by a PI21/01158 g an om Fondos Semilla D Miguel Blanco o he Ins i u o de Salud Ca los III. A.B. ecei ed unding om Ins i u o de Salud Ca los III (INT22/00068), co inanced by he Fondo Eu opeo de Desa ollo Regional. Table 3. P edic i e Models o Poo Ou comes a Hospi al Discha ge and a Mid e m A e S oke In- hospi al poo unc ional ou come Mid e m poo unc ional ou come Pilo Replica ion Pilo Replica ion OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) A1AT (mg/dL) (135.5:176) 1.536 (0.861–2.737) 1.055 (0.804–1.384) 1.403 (0.806–2.441) 1.115 (0.835 –1.489) Age, y (65:83) 1.632 (0.483–5.513) 1.401 (0.950–2.067) 2.846 (0.707–11.458) 2.077 (1.332–3.239) NIHSS admission (3:14) 10.093 (2.403–42.385) 16.614 (8.710–31.688) 6.444 (1.707–24.332) 3.747 (2.185 – 6.426) P e ious mRS (0:1) 1.235 (0.681–2.240) 1.890 (1.504–2.375) 1.605 (0.881–2.924) 1.588 (1.249–2.020) Sex (women) 0.572 (0.151–2.171) 1.390 (0.837–2.311) 0.417 (0.109–1.601) 0.901 (0.506–1.604) Likelihood a io es A1AT 𝜒2 1 =2.44 𝜒2 1 =0.15 𝜒2 1 =1.56 𝜒2 1 =0.51 P alue P=0.118 P=0.702 P=0.212 P= 0.474 C s a is ic (AUC) 0.849 (0.802) 0.890 (0.884) 0.842 (0.785) 0.846 (0.838) Nagelke ke R20.458 (0.337) 0.582 (0.568) 0.439 (0.286) 0.443 (0.421) In e cep 0.000 (0.010) 0.000 (−0.019) 0.000 (0.015) 0.000 (−0.030) Slope 1.000 (0.764) 1.000 (0.964) 1.000 (0.705) 1.000 (0.953) B ie Sco e 0.159 (0.197) 0.125 (0.129) 0.163 (0.208) 0.142 (0.148) OR es ima es o con inuous a iables o in e qua ile ange shi (be ween pa en heses, i s column). G ea e ORs ep esen g ea e associa ions wi h poo e ou comes, and ORs <1 ep esen s onge associa ions wi h a good p ognosis. χ2 ep esen s he alue o he likelihood a io es s a is ic. The C s a is ic (AUC) shows he p obabili y o conco dance o he isk sco e p edic ed by he model wi h he ou come, wi h 1 indica ing comple e conco dance and 0.5 indica ing o al unce ain y. Nagelke ke R2 shows model imp o emen om a null model, and a alue o 1 indica es a pe ec i o he da a. In e cep and slope a e calib a ion measu es desc ibing he ela ion o model- p edic ed isks wi h hose obse ed; a slope o 1 and an in e cep o 0 indica e a pe ec isk desc ip ion, and he alues unde hese igu es e eal he o e es ima ion o isk g ow h o a e age isk, espec i ely. The B ie sco e is he a e age quad a ic e o sco e o he p edic ed isk. Values in pa en heses a e op imism co ec ed boo s ap es ima es, B=2000. A1AT indica es α- 1 an i ypsin; AUC, a ea unde he cu e; mRS, modi ied Rankin Scale; NIHSS, Na ional Ins i u es o Heal h S oke Scale; and OR, odds a io. Downloaded om h p://ahajou nals.o g by on June 2, 2025