we induced diabetes in sprague - dawley rats by intraperitoneal injection of 55 mg / kg of stz in 10 mm citrate buffer ( ph 4.5 ) after overnight fasting , or in mice by intraperitoneal injection of 100 mg / kg of stz in citrate buffer for 2 days .
we confirmed diabetes by measuring blood glucose ( > 250 mg / dl ) 24 h after stz injection .
blood glucose at the time of surgery was 442  12 and 109  3 mg / dl for dm and ndm rats , respectively .
initial body weight of rats was 250 g and average body weight after 4 weeks was 403  10 g for ndm rats and 294  5 g for dm rats , respectively .
we performed all experiments in accordance with the guidelines of the national institutes of health guide for the care and use of laboratory animals and with approval from the animal care and use committee of the joslin diabetes center .
we anaesthetized the rats with pentobarbital ( 50 mg / kg , ip ) , surgically exposed the surface of the skull and slowly injected 50 l pbs , 50 l autologous whole blood ( from tail vein ) , 15 g purified human pk ( enzyme research laboratories ) , 30 g deactivated pk or 5 m bradykinin ( calbiochem ) in 50 l pbs into the brain at 1 mm anterior , 4 mm lateral and 6 mm in depth to the bregma using a 30 gauge needle . for mice , we slowly injected 10 l saline or autologous whole blood into the brain at 1 mm anterior , 2 mm lateral and 3.5 mm in depth to the bregma using a 30 gauge needle .
we removed the infusion needle 10 min after injection and sealed the burr hole with bone wax .
( 1 g / kg / hr ) , [ des - arg]-hoe140 ( 1 g / kg / hr ) ( sigma ) , 1-benzyl-1h - pyrazole-4-carboxylic acid 4-carbamimidoyl - benzylamide ( asp-440 ) ( 14 g / kg / hr ) or its vehicle ( 10 % polyethylene glycol in pbs ) by subcutaneous osmotic pump ( alzet ) into rats 1 day prior to injection . for the mixture of hoe 140 and [ des - arg]-hoe140 injection ,
for pk - specifc antibody injection , antibody to pk ( abcam , final concentration : 0.06 mg / ml ) or mouse igg ( final concentration : 0.06 mg / ml ) was mixed with 50 l blood prior to injection . for convulxin injection , 35 ng convulxin ( alexis biochemicals ) was mixed with 15 g pk in 50 l pbs prior to injection .
for gpvi - specific antibody injection , we injected 10 g jaq1 antibody ( amfret analytics ) or rat igg in 30 pbs into mice brains . for mannitol infusion experiment
, we injected a bolus of 15 ml / kg of 20% mannitol intravenously ( calculated plasma osmolality : 320 mosm / kg ) to the rats followed by cerebral injection of autologous blood or pk .
we continuously infused the rats intravenously with 20% mannitol at a rate of 15 ml / kg / h for 2 h to maintain constant osmolality .
we examined cerebral hematoma area or volume at 30 min , 2 h , or 48 h after injection .
we perfused the animals with saline through the left ventricle , harvested the brains , and determined hematoma areas with quantity one software ( biorad ) .
we collected blood from the vena cava of the rats and anti - coagulated with acid - citrate - dextrose buffer ( 9:1 , v / v ) .
platelet - rich plasma ( prp ) was obtained by centrifugation of the blood at 250 g for 15 min .
we then centrifuged the prp at 500 g for 10 min to obtain platelet pellets , and washed the pallets 3 times with a modified tyrode s buffer without ca ( 134 mm nacl , 3 mm kcl , 0.3 mm nah2po4 , 2 mm mgcl2 , 5 mm hepes , 5 mm glucose , 12 mm nahco3 , 1 mm egta , and 3.5 mg / ml bsa ) .
we resuspended the final pellets in tyrode s buffer with 1 mm cacl2 but no egta and adjusted to a final platelet concentration at 2x10 cells / ml . we monitored platelet aggregation using a chrono - log 680 aggregation system ( chrono - log ) with stirring at 37c .
we activated platelets with equine type i collagen ( 2 g / ml , chrono - log ) , thrombin ( 0.25 u / ml , sigma ) , or adp ( 20 m , sigma ) , and recorded the change of transmission .
briefly , the aggregation process was carried out in 100 l volume in a 96 well flat - bottom microplate .
platelets were activated with type i collagen ( 2 g ) , collagen related peptide ( crp , 10 ng , university of cambridge , uk ) , or convulxin ( 0.25 nm ) , and the readings were taken every 30 seconds over a 20-minute period at 405 nm wavelength .
we measured the interaction between pk and collagen with a biacore 3000 system ( biacore ab ) using he s buffer ( 10 mm hepes , 150 mm nacl , 2 mm edta , ph 7.4 ) at 25c .
rat tail type i collagen in 10 mm sodium acetate buffer ( ph 5.0 ) was covalently coupled to a cm5 chip using an amine coupling kit ( biacore ab ) according to the manufacturer 's instructions .
a control surface underwent the same activation and deactivation procedures in the absence of collagen .
regeneration of the collagen surface was achieved by running 10 l of 10 mm glycine ( ph 2.0 ) through the flow cell at 10 l / min .
pk solutions at several concentrations were perfused over the immobilized collagen at a flow rate of 10 l / min for 3 min , and the resonance changes were recorded .
the sensorgram of the immobilized - collagen surface was subtracted from that of the control surface , and the data thus obtained were analyzed by biaevaluation software ( biacore ab ) .
we rinsed the aorta free of blood , trimmed surrounding connective tissue and the intercostal vessels , opened longitudinally , and gently scraped the inner surface with a sterile razor blade to remove the endothelium .
we washed the vessel 3 times with ice cold pbs and divided it into approximately 4 mm pieces .
care was given to assure that the exposed aorta was covered by pbs during this procedure .
we incubated aorta pieces with buffer containing 300 nm pk and glucose ( 050 mm ) for 30 min at 25c .
we then washed the segments , and measured the activity of surface - bound pk .
statistical analysis was performed by one - way analysis of variance ( anova ) followed by bonferroni s multiple comparisons test or students t - test as appropriate .
statistically significant differences between groups were defined as p < 0.05 and are indicated in the legends of the figures .
the methods for generation and characterization of klkb1 mice , systemic hemostasis measurement , hemoglobin analysis , western blotting , cardiac output measurement , plasminogen activation , continuous assays of clot formation and lysis , and pk activity assay are provided in the supplementary methods online .
we induced diabetes in sprague - dawley rats by intraperitoneal injection of 55 mg / kg of stz in 10 mm citrate buffer ( ph 4.5 ) after overnight fasting , or in mice by intraperitoneal injection of 100 mg / kg of stz in citrate buffer for 2 days .
we confirmed diabetes by measuring blood glucose ( > 250 mg / dl ) 24 h after stz injection .
blood glucose at the time of surgery was 442  12 and 109  3 mg / dl for dm and ndm rats , respectively .
initial body weight of rats was 250 g and average body weight after 4 weeks was 403  10 g for ndm rats and 294  5 g for dm rats , respectively .
we performed all experiments in accordance with the guidelines of the national institutes of health guide for the care and use of laboratory animals and with approval from the animal care and use committee of the joslin diabetes center .
we anaesthetized the rats with pentobarbital ( 50 mg / kg , ip ) , surgically exposed the surface of the skull and slowly injected 50 l pbs , 50 l autologous whole blood ( from tail vein ) , 15 g purified human pk ( enzyme research laboratories ) , 30 g deactivated pk or 5 m bradykinin ( calbiochem ) in 50 l pbs into the brain at 1 mm anterior , 4 mm lateral and 6 mm in depth to the bregma using a 30 gauge needle . for mice , we slowly injected 10 l saline or autologous whole blood into the brain at 1 mm anterior , 2 mm lateral and 3.5 mm in depth to the bregma using a 30 gauge needle .
we removed the infusion needle 10 min after injection and sealed the burr hole with bone wax .
( 1 g / kg / hr ) , [ des - arg]-hoe140 ( 1 g / kg / hr ) ( sigma ) , 1-benzyl-1h - pyrazole-4-carboxylic acid 4-carbamimidoyl - benzylamide ( asp-440 ) ( 14 g / kg / hr ) or its vehicle ( 10 % polyethylene glycol in pbs ) by subcutaneous osmotic pump ( alzet ) into rats 1 day prior to injection . for the mixture of hoe 140 and [ des - arg]-hoe140 injection ,
for pk - specifc antibody injection , antibody to pk ( abcam , final concentration : 0.06 mg / ml ) or mouse igg ( final concentration : 0.06 mg / ml ) was mixed with 50 l blood prior to injection . for convulxin injection , 35 ng convulxin ( alexis biochemicals ) was mixed with 15 g pk in 50 l pbs prior to injection .
for gpvi - specific antibody injection , we injected 10 g jaq1 antibody ( amfret analytics ) or rat igg in 30 pbs into mice brains . for mannitol infusion experiment
, we injected a bolus of 15 ml / kg of 20% mannitol intravenously ( calculated plasma osmolality : 320 mosm / kg ) to the rats followed by cerebral injection of autologous blood or pk .
we continuously infused the rats intravenously with 20% mannitol at a rate of 15 ml / kg / h for 2 h to maintain constant osmolality .
we examined cerebral hematoma area or volume at 30 min , 2 h , or 48 h after injection .
we perfused the animals with saline through the left ventricle , harvested the brains , and determined hematoma areas with quantity one software ( biorad ) .
we collected blood from the vena cava of the rats and anti - coagulated with acid - citrate - dextrose buffer ( 9:1 , v / v ) .
platelet - rich plasma ( prp ) was obtained by centrifugation of the blood at 250 g for 15 min .
we then centrifuged the prp at 500 g for 10 min to obtain platelet pellets , and washed the pallets 3 times with a modified tyrode s buffer without ca ( 134 mm nacl , 3 mm kcl , 0.3 mm nah2po4 , 2 mm mgcl2 , 5 mm hepes , 5 mm glucose , 12 mm nahco3 , 1 mm egta , and 3.5 mg / ml bsa ) .
we resuspended the final pellets in tyrode s buffer with 1 mm cacl2 but no egta and adjusted to a final platelet concentration at 2x10 cells / ml .
we monitored platelet aggregation using a chrono - log 680 aggregation system ( chrono - log ) with stirring at 37c .
we activated platelets with equine type i collagen ( 2 g / ml , chrono - log ) , thrombin ( 0.25 u / ml , sigma ) , or adp ( 20 m , sigma ) , and recorded the change of transmission .
briefly , the aggregation process was carried out in 100 l volume in a 96 well flat - bottom microplate .
platelets were activated with type i collagen ( 2 g ) , collagen related peptide ( crp , 10 ng , university of cambridge , uk ) , or convulxin ( 0.25 nm ) , and the readings were taken every 30 seconds over a 20-minute period at 405 nm wavelength .
we measured the interaction between pk and collagen with a biacore 3000 system ( biacore ab ) using he s buffer ( 10 mm hepes , 150 mm nacl , 2 mm edta , ph 7.4 ) at 25c .
rat tail type i collagen in 10 mm sodium acetate buffer ( ph 5.0 ) was covalently coupled to a cm5 chip using an amine coupling kit ( biacore ab ) according to the manufacturer 's instructions .
a control surface underwent the same activation and deactivation procedures in the absence of collagen .
regeneration of the collagen surface was achieved by running 10 l of 10 mm glycine ( ph 2.0 ) through the flow cell at 10 l / min .
pk solutions at several concentrations were perfused over the immobilized collagen at a flow rate of 10 l / min for 3 min , and the resonance changes were recorded .
the sensorgram of the immobilized - collagen surface was subtracted from that of the control surface , and the data thus obtained were analyzed by biaevaluation software ( biacore ab ) .
we rinsed the aorta free of blood , trimmed surrounding connective tissue and the intercostal vessels , opened longitudinally , and gently scraped the inner surface with a sterile razor blade to remove the endothelium .
we washed the vessel 3 times with ice cold pbs and divided it into approximately 4 mm pieces .
care was given to assure that the exposed aorta was covered by pbs during this procedure .
we incubated aorta pieces with buffer containing 300 nm pk and glucose ( 050 mm ) for 30 min at 25c .
we then washed the segments , and measured the activity of surface - bound pk .
statistical analysis was performed by one - way analysis of variance ( anova ) followed by bonferroni s multiple comparisons test or students t - test as appropriate .
statistically significant differences between groups were defined as p < 0.05 and are indicated in the legends of the figures .
the methods for generation and characterization of klkb1 mice , systemic hemostasis measurement , hemoglobin analysis , western blotting , cardiac output measurement , plasminogen activation , continuous assays of clot formation and lysis , and pk activity assay are provided in the supplementary methods online .
 hyperglycemia is associated with increased hematoma expansion and worse clinical outcomes following intracerebral hemorrhage . 
 we demonstrate that cerebral hematoma expansion triggered by intracerebral infusion of autologous blood is increased in diabetic rats and mice , and this response is ameliorated by plasma kallikrein ( pk ) inhibition and deficiency , respectively . 
 both diabetes and hyperglycemia induced in nondiabetic rats increase hematoma expansion following intracerebral injection of purified pk , a response not observed with bradykinin , plasmin , or tissue plasminogen activator . 
 this response is rapid , prevented by co - injection of the glycoprotein vi ( gpvi ) agonist convulxin , and mimicked by gpvi inhibition or deficiency . 
 we show that pk binding to collagen and pk - mediated inhibition of collagen - induced platelet aggregation is enhanced by hyperglycemia . 
 hyperosmotic mannitol also increases hematoma expansion induced by blood and pk , and increases pk - mediated inhibition of platelet aggregation . 
 these findings suggest that hyperglycemia increases cerebral hematoma expansion by pk - mediated osmotic - sensitive inhibition of hemostasis . 