Hyperglycemi Induced Cererl Hemtom Expnsion is Medited y Plsm Kllikrein Authors: Ji Liu, Ben-Bo Go, Allen C. Clermont, Price Blir, Tmie J. Chilcote, Suknto Sinh, Roert Flumenhft, Edwrd P. Feener. Supplementry Figure 1 Supplementry Figure 1: Representtive imges of the dietic rt rin fter intrcererl injection of mixture of utologous lood nd Evns lue. () Dorsl surfce nd () coronl section of dietic rt rin 3 minutes fter intrcererl injection of 5 µl of mixture of utologous lood nd Evns lue dye into the right hemisphere nd utologous lood only into the left hemisphere. Scle r, 2mm. Nture Medicine doi:1.138/nm.2295
Supplementry Figure 2 75 kd 4 Klk1 +/+ Klk1 +/- Klk1 -/- mrna Level (u) 3 2 1 Klk1 +/+ Klk1 +/ Klk1 / Protein Level (u) 25 * 2 15 1 5 c d Klk1 +/+ Klk1 +/ Klk1 / 3 1 Body Weight (g) 2 1 e Klk1 +/+ Klk1 +/ Klk1 / Blood Glucose (mg/dl) 8 6 4 2 f Klk1 +/+ Klk1 +/ Klk1 / 15 12 PT(s) 1 5 Til Bleeding Time (s) 1 8 6 4 2 Klk1 +/+ Klk1 +/ Klk1 / Klk1 +/+ Klk1 +/ Klk1 / Supplementry Figure 2: Chrcteristics of Klk1 knockout mice. () Rel time PCR of totl Klk1 mrna isolted from 2-month-old Klk1 +/+, Klk1 +/, nd Klk1 / mice for determintion of totl Klk1 mrna levels. n = 3 mice per group. () Protein levels of prekllikrein from 2- month-old Klk1 +/+, Klk1 +/, nd Klk1 / mice. n = 3 mice per group. (c) Body weight, (d) lood glucose, (e) prothromin time (PT), nd (f) til leeding time from 2-month-old Klk1 +/+, Klk1 +/, nd Klk1 / mice. n = 5 28 mice per group. * P <.5. Error rs represent men ± s.e.m. Nture Medicine doi:1.138/nm.2295
PTT(s) Supplementry Figure 3 2 ** * 15 1 5 Til Bleeding Time (s) 5 4 3 2 1 *** *** NDM DM DM + 44 NDM DM DM + 44 Supplementry Figure 3: PTT nd til leeding time from nondietic (NDM), dietic (DM) nd dietic rts with ASP-44 tretment. () PTT from NDM (n = 7), DM (n = 13) nd DM rts with ASP-44 tretment (n = 6). () Til leeding time from NDM (n = 26), DM (n = 26), nd DM rts with ASP-44 tretment (n = 18). * P <.5; ** P <.1; *** P <.1. Error rs represent men ± s.e.m. Nture Medicine doi:1.138/nm.2295
Supplementry Figure 4 5 *** *** Blood Glucose (mg/dl) 4 3 2 1 seline pre-surgery post-surgery Insulin injection Supplementry Figure 4: Blood glucose levels fter insulin injection of rts with 4-weeks of streptozotocin-induced dietes. Blood glucose ws mesured efore (pre-surgery) nd fter (post-surgery) the injection of PK. *** P <.1; n = 13 rts. Error rs represent men ± s.e.m. Nture Medicine doi:1.138/nm.2295
Supplementry Figure 5 NDM DM Men Blood Pressure (mmhg) c 13 12 11 1 NDM DM 9 1 2 3 Time (min) Men Blood Pressure Chnge (mmhg) d 2 15 1 5 NDM DM 1 2 3 Time (min) Hert Rte Chnge (BPM) 6 4 2 e NDM DM 1 2 3 Time (min) Supplementry Figure 5: Blood pressure nd hert rte chnges from NDM or DM rts following cererl infusion of PK. Representtive imges of the lood pressure recording from () NDM nd () DM rts following cererl infusion of PK. (c) Men lood pressure, (d) men lood pressure chnges, nd (e) hert rte chnges from NDM or DM rts following cererl infusion of PK. n = 4 5 rts per group. Error rs represent men ± s.e.m. Nture Medicine doi:1.138/nm.2295
Supplementry Figure 6 Hemtom re (%) 3 2 1 Hemtom re (%) 1 Shm BK 8 P=.8 P=.7 6 4 2 Blood Blood + Hoe + Des-Hoe Sline Glucose Supplementry Figure 6: Effects of BK ntgonists or BK on hemtom expnsion. () The surfce hemtom re s percentge of hemisphere re of rins 2 h fter intrcererl co-injections of utologous lood mixed with 2 µm [des-arg 1 ]-Hoe14 (Des- Hoe) nd Hoe-14 (Hoe) into right hemisphere or lood only into left hemisphere of DM rts. n = 7 rts. () The surfce hemtom re s percentge of hemisphere re of rins.5 h fter intrcererl injection of 5 µm BK or PBS (shm) from sline or glucose intrperitonelly injected rts. n = 7 1 rts per group. Error rs represent men ± s.e.m. Nture Medicine doi:1.138/nm.2295
Supplementry Figure 7 Plsminogen + - + + + + + + + Plsmin - + - - - - - - - tpa (nm) - - 5 - - - - - - PK (nm) - - - 16 32 64 16 32 64 Glucose (mm) - - - - - - 25 25 25 Protein level (fold) 2.5 2. 1.5 1..5 Control 25 mm Glucose OD 34 c.6.4.2 Thromin Thromin + tpa Thromin + PK OD 34. PK (nm) d.6.4.2 16 32 64 Thromin Thromin + tpa Thromin + PK Hemtom re (%). 5 1 15 Time (min) 2 15 1 5 e * NDM DM. 3 6 9 12 15 18 21 24 Time (min) PK Plsmin tpa Supplementry Figure 7: Effect of PK-induced plsmin ctivtion on clot formtion, clot lysis, nd hemtom expnsion. () Representtive western lot nd () densitometric quntittion of the ctivtion of plsminogen y PK (16 64 nm) in the sence or presence of 25 mm glucose compred with the ctivtion y 5 nm tpa. (c) Effects of 5 nm tpa or 32 nm PK on throminmedited clot formtion in the presence of 25 mm glucose. Dt represent the men of triplicte wells from 3 independent experiments. (d) Effects of tpa or PK on clot lysis in the presence of 25 mm glucose. Dt represent the men of triplicte wells from 3 independent experiments. (e) The surfce hemtom re s percentge of hemisphere re of rins from NDM nd DM rts 3 min fter intrcererl injection of PK, plsmin (3 µg), or tpa (1 µg). n = 6 8 rts per group. * P <.5. Error rs represent men ± s.e.m. Nture Medicine doi:1.138/nm.2295
Supplementry Figure 8 PK Activity (% of Control) Mximum ggregtion (%) PK Activity (% of Control) 12 1 8 6 4 2 c 1 e 8 6 4 2 12 1 8 6 4 2 PK De-PK Prekllikrein ns ns *** ** + PK + PK NDM DM 1 5 1 5 1 ASP-44 ( M) PK Activity (% of Control) Mximum ggregtion (%) 12 1 8 6 4 2 6 4 2 d 12.5 25 5 Glucose (mm) 1 *** ns ns 8 ns Collgen + PK + PK + PK + PK ASP-44 ( M) 1 2 5 Supplementry Figure 8: Hydrolytic ctivity of PK nd effect of ASP-44 on PK-induced inhiition of collgen-stimulted pltelet ggregtion. () Hydrolytic ctivity of PK, dectivted PK (De-PK), nd plsm prekllikrein ginst the fluorogenic sustrte H-D-Vl-Leu-Arg-AFC. () Effect of glucose on PK ctivity. (c) Effect of PK (16 nm) on collgen-stimulted ggregtion of pltelets from NDM nd DM rts. (d) Effect of ASP-44 on PKinduced inhiition of collgen-stimulted pltelet ggregtion. (e) Effect of ASP-44 on PK ctivity. n = 3 independent experiments. ** P <.1; *** P <.1. Error rs represent men ± s.e.m. Nture Medicine doi:1.138/nm.2295
SUPPLEMENTARY METHODS Western lotting. We mesured the protein concentrtion of the rin lyste using the Brdford protein ssy kit (Bio-Rd). 5 2 µg protein from ech smple ws seprted y 4 2% SDS-PAGE nd immunolotted using primry ntiodies ginst cronic nhydrse-i (Acm, A6619), plsminogen (Acm, A6189) nd plsm kllikrein 1B (Acm, A16). Results were visulized y enhnced chemiluminescence (Cell Signling) nd quntified using ImgeQunt 5. (Moleculr Dynmics). Genertion of Klk1 / mice, genotyping nd expression nlysis. The Klk1 / mice were provided y Texs Institute for Genomic Medicine. In rief, Klk1 / mice (129/SvEv x C57BL/6 ckground) mde y deletion of the first non-coding nd the first coding exons vi homologous recomintion. The deleted genomic sequence is s follows: ATGCCAGAAACCCAGTGTAAACACTGGAGCCAAGCAAAGACCGCCCTCGGTGCCATATTC AGAGGGCTTGAAGACCATCTTCATGTGAAGACTCCCTCTCCTCCAGAACCACAACGTGAC CATCCTTCCAGGTAGCTGCTTTCTACCGGTCTTGTTATTTCCTGTGTCTTGGGTTTTTTT TTTTCAATATAACTATTTCTGCATGAACAAAAGCTCACTGGTATAATGCACTAATTTCCT GAGTTTTTAGAAAATCTACAAGGAGTTGTTTTTCTTTCTATACAAATATATTAATGTAAA ATATTTTAAAACCAAGAATAAGTTTTTGATTCTTTTCAAAGATGTCCTTTCTGTGAAGGT TGTGGGTGATATTATTGTCCTTATTCATAATAATTTTGCATTAATCTGGAAATTTAATAA GTGTTTTTAATTATTTGTATTAACTCTTTACAAAATTATTAGAAATAAATCTTCAAAATT AACAATAAATACACTAATACACACTGATAGTAATCTAGAGGTCTCAATTTGGATCTTGGG AAGCATGATAAATATTTTAATTTTCTATATACAAAATATCCCACAGCCAAATCTTCCTTC TCCTGGTGATTATGTGCTGTGATTTGCAACTTAGACATTTATCAAAAAGGTGAAGTCTAC ATGAAGTTAAATTGTCTATTAAATACATGGTAAACATGATCTCAACCTAGTAGTTATATG TATATTTTTTTCTTTCAAAGGATGATTTTATTCAACCGAGTGGGTTATTTTGTTTCCTTG TTTGCTACCGTCTCCTGTGGTAAGTATTAGTTTAAGGAGTTCAAATTAATAGTGTGTGAG AGAGGAATGGTCTT Trgeting strtegy is s follows: Nture Medicine doi:1.138/nm.2295
Klk1Trgeting Strtegy ATG TGA HindIII ApI HindIII ATG ApI 1 k 1 2 5 externl proe 3 externl proe 3 f r X 1 2 pkos-59 (Trgeting Vector) G GT LcZ/C Neo cssette ApI HindIII Southern Strtegies Proe 5 externl 3 externl Enzyme ApI HindIII Wildtype 4. k 8.5 k Trgeted 9.1 k 11.2 k PCR Strtegies Strtegy Wildtype Trgeted Primers f+r 3+GT Wildtype 245 ps ----------- Trgeted -------- 391 ps We used genomic DNA from til smples for PCR genotyping under the following conditions: denturtion t 94 C for 15 seconds, nneling t 65 C for 3 seconds, nd extension t 72 C for 4 seconds, 3 cycles. We used the following two primers for genotyping of wild type-specific product: 5 CTTCCAGGTAGCTGCTTTCTACC 3 (f, forwrd) nd 5 TCACCCACAACCTTCACAGAAAGG 3 (r, reverse) nd for muttionspecific product: 5 CGCTGCTTAGGATTGGTAGGAG 3 (3, forwrd) nd 5 GCTAGACTAGTCTAGCTAGAGCGG 3 (GT, reverse). Nture Medicine doi:1.138/nm.2295
The Klk1 +/ mice were ckcrossed to wild type C57BL/6 ckground mice for t lest three genertions. The homozygous knockout (Klk1 / ) mice, heterozygous littermte controls (Klk1 +/ ), nd wild type littermte controls were derived y mting heterozygous Klk1 +/ mice (ckcross # 3). Systemic hemostsis mesurement. For mesurement of PTT nd PT, we collected lood (.5 ml from mice, 1 ml from rts) vi the hert puncture from ech niml nd trnsferred the lood into tues contining sodium citrte nticogulnt. We nlyzed freshly prepred plsm smples for PTT nd PT using PTT nd PT regent, nd Cscde M-4 Mnul Cogultion Anlyzers (Helen Lortories) ccording to the mnufcturer's instructions. We mesured til leeding time y cutting the extremity of the til (3 mm from the tip) with surgicl lde nd immeditely inserting the til into the pre-wrmed tue of sline (37 C). The time to rrest of leeding ws noted. Hemogloin nlysis. For the rt hemtom volume ssy, we homogenized identicl rin slices in 5 mm coronl section of the hemisphere encompssing the injection site from ech hemisphere in uffer consisting of: 15 mm NCl, 1 mm Tris-HCl (ph 7.4), 1% glycerol, 1% Triton X-1 nd protese inhiitors. We centrifuged the homogente t 1, rpm for 15 min t 4 C. To determine hemogloin level, we mixed 1 µl of protein lyste with 6 µl of 1% SDS (2.8 mm finl concentrtion) in 96-well plte nd gently shken. We determined the reltive mount of hemogloin spectrophotometriclly t 54 nm. The totl hemispheric hemogloin content in mice ws determined with QuntiChrom Hemogloin Assy Kit (BioAssy Systems). Nture Medicine doi:1.138/nm.2295
Arteril pressure mesurement. We nesthetized the rts with pentoritl (5 mg/kg, ip), nd implnted polyethylene ctheter into the right femorl rtery. We recorded the rteril lood pressure with trnsducer connected to PowerL system (ADInstruments Pty). Rts were sujected to intrcererl injection of 15 µg PK fter 2 min of seline lood pressure recording. Blood pressure ws recorded for dditionl 3 min. We clculted men rteril lood pressure nd hert rte (ets per minute) using PowerL Chrt softwre (ADInstruments Pty). PK ctivity ssy. We used fluorogenic PK sustrte (H-D-Vl-Leu-Arg-AFC, Cliochem) to quntify PK enzymtic ctivity. The rection kinetics were performed in 1 µl HEPES (1 mm) uffer contining 137 mm NCl, 4 mm KCl, 11 mm D-glucose,.5 mg/ml BSA, 5 µm zinc sulfte, nd.4 mm PK sustrte. We monitored PK ctivity y fluorescence (excittion 42 nm/emission 528 nm) t 37 o C on Synergy HT Multi-Detection microplte reder (BioTek Instruments). Dectivtion of PK ws induced y incution of PK in 2 mm 4-(2-Aminoethyl) enzenesulfonyl fluoride hydrochloride (AEBSF, ph 7) for 1 hr t 25 C. We removed AEBSF with 3 wshes of 4 mm sodium cette-hcl/.15 M NCl (ph 5.3) through 1 kd cut-off filter (Millipore). PK ctivity ws not detected in the residue nd the finl filtrte did not inhiit PK ctivity. Plsminogen ctivtion. We incuted 2.2 µm humn glu-type plsminogen (Sigm) with 5 nm tpa (Cliochem) or 16 64 nm PK t 37 C in Tris uffer (5 mm Tris, 1 mm CCl 2 nd.1% Tween-2, ph 7.5). Plsminogen/plsmin ws detected y Nture Medicine doi:1.138/nm.2295
Western lotting using n ntiody ginst humn plsminogen. We used reduced smples of humn glu-type plsminogen nd humn plsmin s controls 1. Continuous ssys of clot formtion nd lysis. 5 µl norml humn pooled plsm nticogulted with citrte-phosphte-dextrose (Innovtive Reserch) ws comined with n equl volume of tris uffered sline contining 2 mm CCl 2. We dded glucose to ring the finl exogenous glucose concentrtion to 5 mm or 25 mm. We dded tpa (5nM) or PK (4 32 nm) to certin smples ccordingly. We initited the clotting y dding 25 µl of thromin to give 5 nm finl concentrtion. The turidity ssocited with the clot formtion nd dissolution ws monitored t 34 nm every 6 sec t 25 C using SpectrMx Spectrophotometric plte reder (Moleculr Devices). Nture Medicine doi:1.138/nm.2295