toll - like receptors ( tlrs ) are type i transmembrane receptors that are expressed on the cell membrane and are critical for the induction of downstream signals during innate immune responses to bacterial components .
tlr4 is expressed in many cells , including endothelial cells , keratinocytes , epithelial cells , macrophages , neutrophils , and dendritic cells [ 24 ] .
previous evidences demonstrated that platelet tlr4 expression modulates lipopolysaccharide- ( lps- ) stimulated platelet aggregation   and tumor necrosis factor - alpha ( tnf- ) production .
in addition , circulating platelet counts decrease precipitously during sepsis and the degree of thrombocytopenia associates with the serious consequence of sepsis .
furthermore , platelet counts decreased under sepsis because of well - established migration into the liver and lungs [ 79 ] .
demonstrated that platelet tlr4 is essential for platelet migration into the lungs in mice with lps - induced sepsis .
lps accelerates collagen- or thrombin - induced aggregation in platelets in in vitro study ; this acceleration is associated with expression of tlr4 .
it has been reported that the function of platelet tlr4 is to activate neutrophil extracellular traps in septic blood , and histones may promote the generation of thrombin via platelet tlr4 .
thus , scientists proposed that platelet tlr4 has important roles in platelet migration , adhesion , destruction , and attraction .
although the evidence indicates that tlr4 not only regulates platelet migration and shape change but also indirectly affects their aggregation , it remains to be explored whether tlr4 expression directly affects platelet aggregation and blood coagulation .
previous studies demonstrated that the level of tlr4 expression on cells is associated with the early outcome of patients undergoing major surgery . in 2002 , dybdahl et al . demonstrated that open heart surgery induces an inflammatory response and the release of heat - shock protein 70 via tlr4 signaling
recently , our group demonstrated that the level of tlr4 expression on monocytes is associated with early outcomes in coronary artery bypass graft ( cabg ) surgery patients .
it remains unclear whether the platelets regulate both blood coagulation and systemic inflammation in surgery and the expression of tlr4 on platelets is affected by the type of cardiac surgery , which may be associated with a patient 's early outcome in hospital .
cabg with conventional cardiopulmonary bypass ( cpb ) is widely used in cardiovascular surgery . although stopping the heart and temporarily replacing its function with the heart - lung machine has associated risks , conventional cpb permits surgeons to perform coronary revascularization on a still , bloodless heart .
however , evidence suggests that platelet count , aggregation , and shape change significantly decreased after conventional cpb .
platelet activation and platelet injury are well - recognized phenomena occurring after cardiac surgery with cpb that may contribute to pathological postoperative bleeding   and systemic oxidative stress / inflammation .
to control the systemic oxidative stress and inflammatory responses as well as decrease the occurrence of postoperative complications , cardiac surgeons have devoted their efforts to developing various surgical processes , such as the off - pump technique or beating - heart cpb technique for coronary surgery .
although the previous literatures indicated that off - pump technique has become an established and feasible procedure and offers a great benefit in cabg patients [ 12 , 1721 ] , the other studies also hold contrary opinion [ 22 , 23 ] .
regardless of the technique of coronary surgery , clarifying the actual molecular mechanisms of impact of the platelet function is the only way to promote the patients ' outcomes .
it is well known that calpain plays important roles in the physiological activation of platelets .
calpain activation regulates snares ( proteins involved in the degranulation of platelets ) and serca-2 ( an intracellular pump required for ca signaling in platelets ) .
previous evidences demonstrated that cardiac surgery induces oxidative stress , and oxidative stress may inhibit calpain activity .
based on our evidence , the calpain - myosin 9-rab7b axis is responsible for the regulation of tlr4 in activated platelets , which is involved in platelets function .
therefore , we evaluated the platelet tlr4 expression in patients who underwent cabg surgery with conventional cpb or off - pump technique and analyzed the correlation between platelet calpain activation , tlr4 expression , and drainage loss in hospital .
additionally , in order to confirm the critical role of tlr4 on platelets function , we analyzed the coagulation in c57bl/6 and c57bl/6-tlr mice .
this study was approved by the ethics committee at tri - service general hospital . written informed consent was obtained from 23 patients undergoing elective cabg surgery .
the patients were assigned to the off - pump technique and conventional cardiopulmonary bypass ( cpb ) groups .
cpb and off - pump technique was used in 17 and 6 patients , respectively .
patients would not be included if they experienced a decreased cardiac ejection fraction ( low than 50% ) , had undergone cardiac surgery , had a history of cardiogenic shock , had used extracorporeal membrane oxygenation ( ecmo ) , received an intra - aortic balloon pump ( iabp ) , or placed on a ventilator before surgery .
additionally , patients with chronic bronchitis , autoimmune diseases , cancer , asthma , or rheumatoid arthritis or who were receiving anti - inflammatory drugs administration were excluded .
the blood pressure was monitored using an arterial catheter . upon discontinuation of the cpb circuit or completion of proximal anastomoses ,
the blood samples were collected at two time points : ( 1 ) preincision and ( 2 ) at the end of the operation .
whole blood samples were collected from all patients at the indicated time points and analyzed immediately .
fluorescein isothiocyanate- ( fitc- ) conjugated mouse anti - human cd41a antibodies ( becton dickinson , san jose , ca , usa ) and phycoerythrin- ( pe- ) conjugated monoclonal mouse anti - human tlr4 antibodies ( clone : hta125 ; biolegend , san diego , ca , usa ) were added to 50  l whole blood and incubated in the dark for 20  min .
the cd41a - positive cells were separated and detected using a bd facscanto ii flow cytometer ( bd biosciences , mountain view , ca , usa ) with bd facsdiva software ( becton dickinson immunocytometry systems , san jose , ca , usa ) .
a total of 3  10 cd41a - positive cells were gated for the analysis of membrane tlr4 expression .
western blot analysis was performed using 50  g samples of protein to evaluate calpain-1 expression .
the protein was electrophoretically transferred to polyvinylidene difluoride membrane and then blocked with 5% milk in tris - buffered saline solution ( 20  mm tris - hcl ph 7.5 , 138  mm nacl , and 0.2% np-40 ) containing 0.2% tween-20 .
the membranes were probed using monoclonal rabbit anti - human calpain antibody ( clone : hpr3319 ; epitomics co. , burlingame , ca , usa ) , incubated with horseradish
peroxidase - conjugated anti - rabbit igg ( amersham , arlington heights , il , usa ) , and developed using the enzyme - linked chemiluminescence detection reagents ( millipore , bedford , mass .
-actin was used as a loading control ; the monoclonal mouse anti - human -actin antibody ( clone : actn05/c4 ) was obtained from genetex co. ( irvine , ca , usa ) .
six male c57bl / b6 mice were purchased from biolasco co. , ltd . , ( yi - lan , taiwan ) .
six male c57bl/6-tlr mice ( a tlr4-knockout mouse homozygous for the defective lps - response deletion allele tlr4 ) were purchased from the jackson laboratory ( jax , 003752 , bar harbor , me , usa ) .
this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health ( nih publication number 85 - 23 , revised 1996 ) .
the protocol was approved by the committee on the ethics of animal experiments of the taipei medical university ( permit number : lac-100 - 0056 ) .
all mice were kept in microisolator cages on a 12-h day / night cycle and fed a commercial mouse chow diet ( scientific diet services , essex , uk ) with water ad libitum .
the blood samples for biochemical measurements were collected without sedation from the mandibular artery in sodium citrate - containing tubes and separated by centrifugation .
whole blood was collected from the mice through cheek - pouch bleeds and anticoagulated with 3.8% sodium citrate .
platelet - rich plasma ( prp ) was obtained from the collected blood by centrifugation at 280  g for 8  min .
the prp was gently transferred to a fresh tube , and the centrifugation was repeated at 280  g for 4  min .
the platelets were isolated by filtering the resulting prp through a sepharose 2b column ( sigma - aldrich , st .
the washed platelets were counted using an automated blood cell counter kx-21n ( sysmex , kobe , japan ) .
platelet aggregation was performed using the turbidimetric method with a chrono - log model 560-ca dual sample lumi - ionize calcium aggregometer ( chrono - log , havertown , pa , usa ) .
approximately 500  l of a washed platelet solution containing 3  10 platelets/l in ca - free tyrode 's buffer was added to a silicon - treated glass cuvette for each experiment .
platelet aggregation was measured for 5 minutes following the addition of 0.1  u / ml thrombin .
the percent aggregation was calculated using aggro / link software ( chrono - log , havertown , pa , usa ) .
all buffers were prewarmed before use and platelets were kept warm at 37c all along the study . in accordance with previous studies ,
two rotem analyses were performed within minutes after the sample was collected , as indicated by the manufacturer 's instructions ( pentapharm , munich , germany ) .
we performed both an extrinsically activated assay using recombinant tissue factor ( extem ) and an extrinsically activated test using recombinant tissue factor with cytochalasin d added ( fibtem ) .
the following parameters were measured using rotem : clotting time ( ct , the time from the start of the assay to clot formation with an amplitude of 2  mm ) , clot formation time ( cft , the time from the end of ct ( amplitude of 2  mm ) to a clot firmness of 20  mm ) , maximum clot firmness ( mcf , the peak strength of the clot ) , and maximum clot elasticity ( mce , which is commonly used to assess clot strength ) . the platelet component of clot strength was calculated using the following equation : mcfplatelet = mcfextem  mcffibtem , mceplatelet = mceextem  mcefibtem , and mce = ( mcf  100)/100  mcf .
statistical comparisons between groups were computed using student 's t - tests and one - way anova followed by the dunnett 's test . for all statistical evaluations , differences in data with p values of < 0.05
all analyses were performed using the spss 16 statistical package ( spss inc . , chicago , il , usa ) .
the preoperative characteristics of the two groups were similar , including in age , body weight , height , hypertension , hypercholesterolemia , and peripheral vascular disease .
the percentages of ef in the conventional cpb and off - pump technique groups were 59.7  9.2% and 63.5  8.3% , respectively . none of the patients in the off - pump group had diabetes mellitus or peripheral vascular disease .
in addition , no patient who underwent elective cabg with conventional cpb had previously experienced stroke or myocardial infarction .
for patients in the cpb group , the mean total cpb time was 119.1  12.4  min , the aortic clamping ( ischemia ) time was 65.6  5.2  min , and the minimal esophageal temperature was maintained at 26.1  0.4c .
additionally , the patients in the conventional cpb group received 30476.2  1708.0 units of heparin .
the patients who underwent the off - pump technique maintained a minimal esophageal temperature of 35.8  0.3c and were administered 28142.9  4532.6 units of heparin .
there were no significant differences in the number of grafts or amount of heparin used between the two groups . 

the percentages of circulating lymphocytes , monocytes , basophils , and neutrophils were not significantly different between the conventional cpb and off - pump technique groups before surgery .
the percentages of neutrophil numbers increased significantly in both the conventional cpb group ( from a basal level of 60.9  9.2% to 85.9  3.6% ) and the off - pump technique group ( from a basal level of 64.7  8.0% to 86.2  4.3% ) .
the number of lymphocytes decreased significantly after coronary surgery with conventional cpb ( from a basal level of 28.3  9.2% to 7.5  3.2% , resp . ) and with the off - pump technique ( from a basal level of 26.1  7.7% to 6.8  2.7% , resp . ) .
the eosinophils , basophils , and monocytes did not significantly differ between the two groups after surgery .
biochemical analyses indicated that liver function ( levels of glutamate oxaloacetate transaminase , ast , and glutamic pyruvic transaminase , alt ) , kidney function ( levels of blood urea nitrogen , bun , and creatinine ) , and inflammation ( level of c - reactive protein , crp ) indices did not deteriorate after cardiac surgery .
patients who underwent conventional cpb exhibited higher levels of troponin i after surgery , compared with the off - pump technique group .
however , the mean value of creatine kinase - mb isoform ( ck - mb ) was not significantly different between the conventional cpb and off - pump technique groups . washed platelets were isolated from all patients .
flow cytometry was performed to analyze the tlr4 expression , and western blot analysis was used to analyze the calpain activity .  
table 4 indicates that patients in the conventional cpb group possessed lower tlr4 expression ( cpb : 68.48  5.64% of preincision versus off - pump technique : 89.0  4.8% of preincision , p < 0.05 ) , less drainage loss ( cpb : 68.5  5.6% of preincision versus off - pump technique : 89.0  4.8% of preincision , p < 0.001 ) , and higher prbc transfusion volume ( cpb : 5.5  0.5 units versus off - pump technique : 1.1  1.1 units , p = 0.001 ) compared with the off - pump technique group .
the platelet transfusion volume , duration of icu stay , and duration of fever were not significantly different between the conventional cpb and off - pump technique groups after cabg surgery . according to our recent results , tlr4 expression on platelets is mediated by the activity of calpain .  
figure 1 shows the calpain activity in all patients before and after the cabg surgery was performed
. in general , inactivated calpain appears as a 80  kda of single band ; in contrast , calpain has a higher mobility 78  kda band resulted from autolysis under activated situation , which is believed to be the more active form of calpain . before cabg surgery , all patients in both the off - pump technique and conventional cpb groups expressed active calpain . while undergoing cabg surgery , all patients in the off - pump technique group continued to express active calpain .
in contrast , 76.47% of patients ( patient numbers 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 10 , 11 , 13 , 15 , and 17 ) in the conventional cpb group expressed inactive calpain after cabg surgery .
additionally , the ratio of platelets tlr4 expression after surgery was demonstrated in figure 1 .
combined with the data of tr4 expression and calpain expression ( figure 1 ) , the information shown in table 5 indicates that patients in the conventional cpb group with active calpain possessed higher levels of tlr4 on platelets , less drainage loss , lower platelet transfusion volumes , shorter icu stay durations , and shorter fever duration after cabg surgery ( g1 versus g2 , p < 0.05 were considered statistically significant ) .
although the calpain remained in an active state , the amounts of platelet transfusion were reduced in the off - pump technique group compared with the conventional cpb group ( g1 versus g3 , p < 0.05 , considered statistically significant ) . interestingly , compared with patients who underwent the off - pump technique , the patients possessed a lower level of tlr4 , higher volumes of drainage loss , and prbc / platelet transfusion as well as longer icu stay duration and shorter fever duration in the conventional cpb group with inactive calpain ( g2 versus g3 , p < 0.05 , considered statistically significant ) .
these results indicate that in cabg surgery with either off - pump technique or conventional cpb , patients with higher level of tlr4 may experience better early outcomes ( duration of icu stay and fever , volume of drainage loss , and prbc / platelet transfusion ) in hospital than the patients with lower level of tlr4 , which may be mediated by activation of calpain on platelets .
a previous study demonstrated that platelet tlr4 modulates coagulation by recognizing extracellular histones that promote thrombin generation .
additionally , tables 15 indicate that calpain and tlr4 expression in platelets are associated with patients ' drainage loss in cardiac surgery .
therefore , we further explored whether the tlr4 on platelets is associated with coagulation in mice .
the wild - type c57bl/6 , c57bl/6-tlr , and c57bl/6-tlr mice were used in this study . the endogenous level of tlr4 expression in the mouse platelets
was first measured using western blot analysis and flow cytometry ( figure 2(a ) ) .
incubating wild - type c57bl/6 mouse platelets with 0.1  u / ml thrombin resulted in approximately 47.4  5.4% aggregation within 10 minutes , whereas treating platelets with pbs did not induce aggregation ( figure 2(b ) ) .
interestingly , platelets from c57bl/6-tlr or c57bl/6-tlr mice had reduced aggregation abilities after thrombin stimulation compared with those from wild - type mice ( 28.1  3.7% for c57bl/6-tlr and 18.5  2.5% for c57bl/6-tlr ) . in recent years , rotem has been used to provide a comprehensive overview of the entire clotting process by measuring the coagulation function of platelets .
an extrinsically activated assay using recombinant tissue factor ( extem ) indicated no significant differences in the coagulation time or clot formation time between c57bl/6 , c57bl/6-tlr , and c57bl/6-tlr mice .
the amplitude at 5 to 30 minutes and mcf were significantly lower in c57bl/6-tlr compared with c57bl/6 ( table 6 ) .
an extrinsically activated test using recombinant tissue factor plus cytochalasin d ( fibtem ) demonstrated that c57bl/6-tlr mice have a lower coagulation time , lower amplitude at 15 to 30 minutes , and lower mcf than c57bl/6 mice ( table 7 ) . according to a previous report , mcf and mce are commonly used to assess the platelet component of clot strength .  
table 8 demonstrates that both c57bl/6-tlr and c57bl/6-tlr mice have significantly lower mcfs ( 48.80  3.27  mm and 48.50  3.00  mm , resp . ) and mces ( 190.89  20.79  mm and 218.37  19.54  mm , resp . ) than c57bl/6 mice ( mcf : 58.80  4.67  mm ; mce : 290.85  10.99  mm ) .
these results suggest that platelet tlr4 is associated with aggregation and clot strength in c57bl/6 mice .
tlr4 plays important role in platelet migration , adhesion , destruction , and attraction . in 2005 , andonegui et al .
first determined that platelets express tlr4 on their surfaces and that this phenomenon is associated with the occurrence of thrombocytopenia , indicating that the tlr4-mediated signaling pathway is involved in the cellular function of platelets .
additionally , platelets may respond to endotoxin and ensnare bacteria through tlr4 to activate neutrophil extracellular traps [ 11 , 29 ] , promote migration into the lung or liver [ 8 , 10 ] , and decrease the production of rantes , angiogenin , and pdgf - ab from platelets in severe septic blood .
although previous investigators demonstrated that platelets regulate inflammation via tlr4 surface expression , few studies investigated the relationship between platelet tlr4 and coagulation .
explored adp - induced heat shock protein ( hsp ) 27 secretion and phosphorylation from granules in platelets , which mediates platelet aggregation . moreover
interestingly , lps did not increase human platelet aggregation in vitro ; nevertheless , lps accelerates the collagen / thrombin - stimulated aggregation of platelets .
this mechanism was mediated by tlr4 expression on platelets , and the effect of lps on platelets was indirect .
in addition to tlr4 , platelets also express downstream components of the tlr4-related signaling pathway complex , including tlr4/md2 and myeloid differentiation primary response gene ( myd)88 . through the production of interleukin- ( il- ) 6 , prostaglandin e2 , and tumor necrosis factor ( tnf)- ,
lps induces platelet aggregation mediated by tlr4/md2/myd88 complex formation , mitogen - activated protein kinases ( mapks ) , nuclear factor - kappa b ( nf-b ) activation , and cgmp production [ 5 , 7 , 34 ] .
although many reports speculated that tlr4 affects platelet aggregation and regulates platelet function , we are the first group to indicate that the calpain - myosin 9-rab7b axis is liable to the regulation of tlr4-containing -granule trafficking in thrombin - induced platelets .
furthermore , we demonstrated the differences of platelet function in c57bl/6 , c57bl/6-tlr , and c57bl/6-tlr mice in this text , indicating the critical roles of tlr4 in platelets .
we speculate that the probable mechanisms of the impact of tlr4 on clot strength in animal experiments may be mediated by hsp27 or others , and we are currently investigating the cellular and molecular mechanisms involved in this phenomena . in clinic ,
patients who underwent cabg surgery with inactive calpain and lower platelet tlr4 expression experienced worse early outcomes in hospital than those with active calpain and higher platelet tlr4 expression .
these results indicate that the levels of tlr4 and calpain play pivotal roles in platelets function .
oxidative stress - mediated cytokines production was considered to be the main factor causing inflammation after cardiac surgery .
the expression of many cytokines , including tnf- , il-6 , and monocyte chemotactic protein- ( mcp- ) 1 , increased during and after cpb . except for cytokine production , stimulations such as aortic - clamping ischemia , ischemia - reperfusion injury , blood cell attachment to the foreign surfaces of the oxygenator
/ cpb tube , and artificial surgical trauma may play important roles in postoperative inflammation ; therefore , off - pump bypass graft operation significantly reduces oxidative stress and inflammation .
although clinicians still have differences of opinion on the advantages / disadvantages of the off - pump technique and conventional cpb for patients who undergo cardiac surgery [ 22 , 23 , 36 , 37 ] , at least , the patients in both the conventional cpb and off - pump groups had good early outcomes ( little drainage loss , short icu stay , and fever durations ) in hospital as long as they expressed the active form of calpain after cabg surgery rather than the inactive form of calpain .
this indeed indicates that maintaining active calpain is an important factor in patient recovery after cardiac surgery .
in contrast , the patients with active calpain in the conventional cpb group still required larger blood transfusion volumes than the patients in the off - pump technique group , which may result from the cessation of pumping blood cells . recently
, we also undertook to distinguish the influences of foreign surface contamination by the cpb tube and oxygenator , surgical incisive trauma , and cardiac ischemia by comparing cytokine production and early outcome during conventional cpb technique and the off - pump technique in patients .
however , cpb - associated immune suppression results from the changes in tlr4 capacity on monocytes and neutrophils .
it is still remained to be elucidated that the causes result in tlr4 downregulation in platelets . during the process of cardiac surgery with cpb , mechanical force ( pressure from rolling pump )
therefore , the transfusion of prbc and fresh platelet is essential for the patients who are undergoing cpb . according to the results in table 5 , patients with cpb really receipted more transfusion of prbc and platelet comparing to patients in off - pump technique group .
interestingly , patients with activated calpain in cpb group had similar level of early outcomes and platelet tlr4 expression to the patients in off - pump technique group .
in contrast , even though the patients with inactivated calpain in cpb group receipted more platelet and prbc transfusion , they also had worse early outcomes comparing to patients with activated calpain .
therefore , we can not exclude the importance and effects of blood transfusion for patients with cardiac surgery in this study , and we still have the opinion that the calpain activation and tlr4 expression in platelets are associated with early outcomes .
hyperglycemia and diabetes mellitus really induce oxidative stress which result in complex complications in many sickle situations .
the major finding of the previous study is that diabetes mellitus is associated with the activation of calpain .
however , the opposite evidence demonstrated that cardiac surgery induces oxidative stress , and oxidative stress may inhibit calpain activity
. in our results , mellitus patients ( patient numbers 3 , 5 , and 10 ) also have inactive form of calpain after cpb .
although diabetes mellitus may be associated with patient 's postsurgical outcomes , we also speculate that diabetes mellitus may not be a factor in regulation of platelets calpain activity in cpb .
calpain is responsible for platelet shape change , clotting , and aggregation , which are mediated by the activation of cytoskeleton proteins .
previous report demonstrated calpain with the important roles in regulating cytoskeletal signaling in vwf - activated platelets .
calpain also regulates the activation of the extracellular fibrinogen - binding function of iib3 and the platelet aggregation resulting in cleavage of integrin 3 .
platelet - derived growth factor may suppress oxidative stress induced calpain activation in neurons . presently , the causes of inactive calpain in the majority of patients ( 82.35% ) treated with conventional cpb are unknown ; it is also unclear why 17.64% of the patients undergo calpain activation .
there are many proteases with critical roles in the stability and assembly of cytoskeletal signaling complexes that may result in the phosphorylation of proteins , association of the receptor with the cytoskeleton , and a consequent increase in the calpain activity .
whether the pressure force of pumping , aortic - clamping ischemia , ischemia - reperfusion injury , or attachment of the foreign cpb circuit during conventional cpb disrupts these normal conditions in platelets remains to be elucidated . through further studies , we expect that patients ' early outcomes may be improved and promoted by adjusting the activation of calpain in platelets after cardiac surgery with conventional cpb .
our results highlight the important roles of tlr4 in blood coagulation and platelet function in clinic and in c57bl/6 mouse . of clinical relevance
, we also explored novel roles for calpain and tlr4 in platelets , which are associated with patients ' early outcomes in cardiac surgery .
these results also provide a basis for further controlling calpain activation and tlr4 expression as a therapeutic strategy to avoid coagulopathic and thrombocytic disorders after cardiac surgery .
 human platelets express toll - like receptors ( tlr ) 4 . however , the mechanism by which tlr4 directly affects platelet aggregation and blood coagulation remains to be explored . 
 therefore , in this study , we evaluated the platelet tlr4 expression in patients who underwent cabg surgery ; we explored the correlation between platelet tlr4 expression and the early outcomes in hospital of patients . 
 additionally , c57bl/6 and c57bl/6-tlrlps/ mice were used to explore the roles of platelet tlr4 in coagulation by platelet aggregometry and rotation thromboelastometry . in conclusion , 
 our results highlight the important roles of tlr4 in blood coagulation and platelet function . of clinical relevance 
 , we also explored novel roles for platelet tlr4 that are associated with early outcomes in cardiac surgery . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion