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Study objective The transfusion of older packed RBCs may be harmful in critically ill patients. We seek to determine the association between packed RBC age and mortality among trauma patients requiring massive packed RBC transfusion. Methods We analyzed data from the Pragmatic, Randomized Optimal Platelet and Plasma Ratios trial. Subjects in the parent trial included critically injured adult patients admitted to 1 of 12 North American Level I trauma centers who received at least 1 unit of packed RBCs and were predicted to require massive blood transfusion. The primary exposure was volume of packed RBC units transfused during the first 24 hours of hospitalization, stratified by packed RBC age category: 0 to 7 days, 8 to 14 days, 15 to 21 days, and greater than or equal to 22 days. The primary outcome was 24-hour mortality. We evaluated the association between transfused volume of each packed RBC age category and 24-hour survival, using random-effects logistic regression, adjusting for total packed RBC volume, patient age, sex, race, mechanism of injury, Injury Severity Score, Revised Trauma Score, clinical site, and trial treatment group. Results The 678 patients included in the analysis received a total of 8,830 packed RBC units. One hundred patients (14.8%) died within the first 24 hours. On multivariable analysis, the number of packed RBCs greater than or equal to 22 days old was independently associated with increased 24-hour mortality (adjusted odds ratio [OR] 1.05 per packed RBC unit; 95% confidence interval [CI] 1.01 to 1.08): OR 0.97 for 0 to 7 days old (95% CI 0.88 to 1.08), OR 1.04 for 8 to 14 days old (95% CI 0.99 to 1.09), and OR 1.02 for 15 to 21 days old (95% CI 0.98 to 1.06). Results of sensitivity analyses were similar only among patients who received greater than or equal to 10 packed RBC units. Conclusion Increasing quantities of older packed RBCs are associated with increased likelihood of 24-hour mortality in trauma patients receiving massive packed RBC transfusion (≥10 units), but not in those who receive fewer than 10 units.

Introduction Severe trauma is accompanied by endothelial glycocalyx disruption, which drives coagulopathy, increasing transfusion requirements and death. This syndrome has been termed endotheliopathy of trauma (EOT). Some have suggested EOT results from endothelial cellular damage and apoptosis. Endothelial microvesicles (EMVs) represent cellular damage. We hypothesized that EOT is associated with endothelial damage and apoptosis resulting in an increase in circulating EMVs. Methods Prospective, observational study enrolling severely injured patients. Twelve patients with EOT, based on elevated Syndecan-1 levels, were matched with 12 patients with lower levels, based on Injury Severity Score (ISS), abbreviated injury scale profile, and age. Thrombelastography and plasma levels of biomarkers indicative of cellular damage were measured from blood samples collected on admission. EMVs were determined by flow cytometry using varied monoclonal antibodies associated with endothelial cells. Significance was set at P Results Admission physiology and ISS (29 vs. 28) were similar between groups. Patients with EOT had higher Syndecan-1, 230 (158, 293) vs. 19 (14, 25) ng/mL, epinephrine, and soluble thrombomodulin levels. Based on thrombelastography, EOT had reductions in clot initiation, amplification, propagation and strength, and a greater frequency of transfusion, 92% vs. 33%. There were no differences in EMVs irrespective of the antibody used. Plasma norepinephrine, sE-selectin, sVE-cadherin, and histone-complexed DNA fragments levels were similar. Conclusion In trauma patients presenting with EOT, endothelial cellular damage or apoptosis does not seem to occur based on the absence of an increase in EMVs and other biomarkers. Thus, this suggests endothelial glycocalyx disruption is the underlying primary cause of EOT.

Progressive hemorrhagic injury (PHI) is common in patients with severe traumatic brain injury (TBI) and is associated with worse outcomes. PHI pathophysiology remains poorly understood and difficult to predict. We performed an exploratory analysis aimed at identifying markers in need of further investigation to establish their predictive value in PHI following TBI.We performed a retrospective chart review of prospectively collected data from 424 highest-level activation trauma patients from January 2012 through December 2013. Patients with severe TBI, defined as head acute injury scale (AIS) score ≥3 and intracranial hemorrhage (ICH) on initial CT, were included. Stable hemorrhage (SH) and PHI was determined by measuring ICH expansion on repeat CT within 6 h. Of 424 patients evaluated, 72 met inclusion criteria. Twenty-five patients had repeated samples available and were dichotomized into SH (n = 6, 24%) and PHI (n = 19, 76%). Levels of plasminogen, urokinase and tissue plasminogen activators (uPA, tPA), plasminogen activator inhibitor-1, α2-antiplasmin (α2AP), and D-Dimers (DD) were measured upon admission and 2, 4, and 6 h later.Longitudinal models identified tPA and DD as positively associated and α2AP inversely associated with PHI. High DD levels are strongly associated with developing PHI over time. Using the full TBI cohort of N = 72, receiver operating curve analysis provided a cutoff of 3.04 μg/mL admission DD to distinguish SH from PHI patients.Our findings support a relationship between markers of fibrinolysis in polytrauma patients with severe TBI and PHI, warranting further investigation into the potential for novel, predictive biomarkers.

Background Reductions in platelet (PLT) count and function are associated with poor outcomes in trauma patients. We proposed to determine if patients expected to receive blood products have a decrease in PLT function higher than expected based on the reduction in PLT count, and if the reduction in function could be associated with the donor plasma/supernatant received. Methods PLT count and function were measured on admission to the emergency department and intensive care unit in severely injured patients expected to receive a transfusion. PLT function was measured by Multiplate aggregometry in response to five agonists. Function was corrected for alterations in count. In vitro studies were conducted in the blood of normal subjects to assess the effect of dilutions with AB donor plasma on PLT function. Results Forty-six patients were enrolled, with 87% requiring a transfusion. Median Injury Severity Score was 23 (13, 29) and mortality 15%. PLT count and function were decreased from emergency department to intensive care unit admission by 25% and 58%, respectively. Decreases in function persisted after adjustment for count. Patients requiring large volumes of blood products had reductions in function that were disproportionately greater. Reductions in PLT function were greatest after transfusion of PLTs. In in vitro studies with a 30% dilution by autologous plasma caused a relational reduction in function, whereas allogenic plasma resulted in greater decreases that were highly variable between donors. Conclusions Within hours of injury a decrease in both PLT count and function occurs, that is aggravated with the administration of blood products, with transfusion of PLTs showing the greatest effect. The effect on PLT function of allogenic transfused plasma appears to be highly donor related.

Rational There has been increased focus on hemostatic potential and function in the initial assessment of the patient with traumatic injuries, that not been extensively studied in patients with burns. We proposed to determine the hemostatic potential of patients with burns upon admission to the emergency department and contrasted their condition with that of healthy controls and patients with other traumatic injuries. In addition we assessed differences due to thermal versus electrical injury and evaluated the effect of burn size. Methods This is a patient based prospective observational study conducted with delayed consented. Subjects at the highest level of trauma activation upon admission to the ED had a blood sample collected for research purposes and were subsequently consented. Hemostatic potential was measured by rapid thromelastography (r-TEG®), thrombin generation by calibrated automated thrombogram (CAT) and platelet function by Multiplate® using five activators. Burn subjects were compared to subjects with other traumatic injuries and controls. Within the burn subjects additional analysis compared mechanism (thermal vs. electrical) and burn size. Values are medians (IQR). Results Two hundred and eighty two trauma patients (with burns n = 40, 14%) and 27 controls were enrolled. Upon admission, compared to controls, subjects with burns or trauma were hyper-coagulable based on r-TEG and CAT, with increased rates of clot formation and thrombin generation. There were no differences in burns compared to other traumatic injuries. The presence of hyper-coagulation did not appear to be related to the type of burn or the percentage of total body surface area involved. Employing previous defined cut points for R-TEG driven therapeutic interventions burn patients had similar rates of hyper- and hypo-coagulation noted in patients with traumatic injuries. Conclusion Upon admission patients with burns are in a hyper-coagulable state similar to that of other trauma patients. Employing demonstrated cut points of hemostatic potential in trauma patients associated with increased risk of poor outcomes demonstrated the incidence in burn patients to be similar, suggesting that these values could be used in the early assessment of the patient with burns to guide treatment interventions.

Increased thrombin generation in injured patients possibly contributes to early consumption of coagulation factors, exacerbating hemorrhage. Identifying optimal resuscitation products for restoring plasma homeostasis following injury is important for improving management of these patients.To determine the effects of crystalloid versus plasma resuscitation on thrombin generation in a rat model of trauma and hemorrhagic shock (HS).Rats were subjected to trauma and HS followed by resuscitation with Lactated Ringer's solution (LR) or fresh frozen plasma (FFP). Blood was collected at baseline, decompensation, and 3-h post-resuscitation. Thrombin generation was measured by calibrated automated thrombogram and antithrombin III (AT) by ELISA. In a prospective observational study, admission blood samples were collected on highest-level activation trauma patients and diluted with LR or FFP for thrombin generation analysis.Resuscitation with LR resulted in persistent hypercoagulability; however, FFP resuscitation reversed this hypercoagulability to baseline thrombin generation or below. Plasma AT levels decreased following HS and remained low in rats receiving LR, but were corrected in rats receiving FFP. Similarly, in trauma patient plasma LR increased thrombin generation while FFP reduced it. However, results with AT-deficient plasma dilution were similar to LR. In patients with admission hypocoagulability, FFP slightly increased thrombin generation.HS in rats is associated with increased thrombin generation and resuscitation with FFP, not LR, reverses hypercoagulability. Dilution of trauma patient plasma with LR or FFP yielded similar results; however, the modulatory effects of FFP were attenuated when AT was absent. Importantly, FFP reduced thrombin generation in hypercoagulable patient plasma, but slightly increased thrombin generation in hypocoagulable patient plasma. Thus, FFP restores hemostatic balance following trauma and HS which is, in part, by delivering AT.

Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis.

Endotheliopathy of trauma is characterized by breakdown of the endothelial glycocalyx. Elevated biomarkers of endotheliopathy, such as serum syndecan-1 (Synd-1) ≥ 40 ng/mL, have been associated with increased need for transfusions, complications, and mortality. We hypothesized that severely injured trauma patients who exhibit elevated Synd-1 levels shortly after admission have an increased likelihood of developing sepsis.We analyzed a subset of patients from the Pragmatic, Randomized Optimal Platelet and Plasma Ratios (PROPPR) trial who survived at least 72 hours after hospital admission, and we determined elevated Synd-1 levels (≥ 40 ng/mL) 4 hours after hospital arrival. Sepsis was defined a priori as meeting systemic inflammatory response criteria and having a known or suspected infection. Univariate analysis was performed to identify variables associated with elevated Synd-1 levels and sepsis. Significant variables at a value of p0.2 in the univariate analysis were chosen by purposeful selection and analyzed in a mixed effects multivariate logistic regression model to account for the 12 different study sites.We included 512 patients. Of these, 402 (79%) had elevated Synd-1 levels, and 180 (35%) developed sepsis. Median Synd-1 levels at 4 hours after admission were 70 ng/dL (interquartile range [IQR] 36 to 157 ng/dL) in patients who did not develop sepsis, and 165 ng/dL [IQR 67 to 336 ng/dL] in those who did (p0.001). Adjusting for treatment arm and site, multivariable analyses revealed that elevated Synd-1 status, Injury Severity Score (ISS), and total blood transfused were significantly associated with an increased likelihood of developing sepsis.Elevated Synd-1 levels 4 hours after admission in severely injured adult trauma patients who survived the initial 72 hours after hospital admission are associated with subsequent sepsis.

Objective The current study leveraged data from the Early Whole Blood (EWB) trial to explore the effects of modified whole blood (mWB) versus component (COMP) transfusions on coagulation parameters over time using longitudinal statistical methods. Study design and methods The EWB study was a single-center randomized controlled trial, approved by the local IRB. Adult patients at highest-level trauma activations were randomized into mWB or COMP groups. Coagulation status was evaluated (at times 0, 3, 6, 12, and 24 h postadmission) using thrombelastography, platelet aggregometry, and calibrated automated thrombograms. Longitudinal statistical analyses with generalized estimating equations (GEE) were used to evaluate the effects of group, time, transfusion types, and their respective interactions on changes in measured coagulation markers. Results A total of 59 patients were enrolled and adhered to protocol in the EWB trial, 25 in the mWB group, and 34 in the COMP group. Patients in both the mWB and COMP groups demonstrated a significant decline in their thrombelastography parameters during the first 3-6 h, specifically K-time, α-angle, maximum amplitude, G, and LY30. Patients receiving mWB exhibited improved thrombin potential than those receiving COMP. Platelet count and function declined over time in both mWB and COMP groups; however, platelet aggregation in response to ristocetin in the mWB group was significantly improved at 12 h compared with the COMP group. The longitudinal GEE model revealed significant group-time interactive effects on the changes in coagulation markers and significant effect of platelet transfusions on improvements in coagulation profile. Conclusions We observed significant interactive group-time effects, indicating that the types of transfusion as well as the time of transfusion significantly affect the patient's coagulation status. Our pilot data suggest that there is an improvement in platelet function with mWB, but further studies are needed. Regardless, platelet transfusions were associated with improvements in coagulation over time in both the groups.

Background Positive Focused Assessment with Sonography in Trauma examination and hypotension often indicate urgent surgery. An abdomen/pelvis computed tomography (apCT) may allow less invasive management but the delay may be associated with adverse outcomes. Methods Patients in the Prospective Observational Multicenter Major Trauma Transfusion study with hypotension and a positive Focused Assessment with Sonography in Trauma (HF+) examination who underwent a CT (apCT+) were compared with those who did not. Results Of the 92 HF+ identified, 32 (35%) underwent apCT during initial evaluation and apCT was associated with decreased odds of an emergency operation (odds ratio .11, 95% confidence interval .001 to .116) and increased odds of angiographic intervention (odds ratio 14.3, 95% confidence interval 1.5 to 135). There was no significant difference in 30-day mortality or need for dialysis. Conclusions An apCT in HF+ patients is associated with reduced odds of emergency surgery, but not mortality. Select HF+ patients can safely undergo apCT to obtain clinically useful information.

Protein C is an endogenous anticoagulant protein with anti-inflammatory properties. Single-nucleotide polymorphisms (SNPs) affect the levels of circulating protein C in European Americans. We performed a genome-wide association (GWA) scan of plasma protein C concentration with approximately 2.5 million SNPs in 2,701 African Americans in the Atherosclerosis Risk in Communities Study. Seventy-nine SNPs from the 20q11 and 2q14 regions reached the genome-wide significance threshold of 5 × 10(-8) . A missense variant rs867186 in the PROCR gene at 20q11 is known to affect protein C levels in individuals of European descent and showed the strongest signal (P = 9.84 × 10(-65) ) in African Americans. The minor allele of this SNP was associated with higher protein C levels (β = 0.49 μg/ml; 10% variance explained). In the 2q14 region, the top SNPs were near or within the PROC gene: rs7580658 (β = 0.15 μg/ml; 2% variance explained, P = 1.7 × 10(-12) ) and rs1799808 (β = 0.15 μg/ml; 2% variance explained, P = 2.03 × 10(-12) ). These two SNPs were in strong linkage disequilibrium (LD) with another SNP rs1158867 that resides in a biochemically functional site and in weak to strong LD with the top PROC variants previously reported in individuals of European descent. In addition, two variants outside the PROC region were significantly and independently associated with protein C levels: rs4321325 in CYP27C1 and rs13419716 in MYO7B. In summary, this first GWA study for plasma protein C levels in African Americans confirms the associations of SNPs in the PROC and PROCR regions with circulating levels of protein C across ethnic populations and identifies new candidates for protein C regulation.

Rodent models of hemorrhagic shock are paramount to our understanding of the pathophysiology of this disease, the effects on coagulation and in exploring the utility of resuscitative methods for managing patients in shock. These models usually require serial blood sampling during experimentation. The lack of standardized practices for these experimental models has resulted in technical variability, discordance in the literature, and incomparable results on blood coagulation analysis between researchers, hindering substantial progress in the field of hemorrhagic shock. The aim of this study was to define the effects of cardiac puncture versus arterial catheterization on coagulation in a rat model to provide data supporting standardization of one practice over another. Blood was collected from anesthetized rats via cardiac puncture or femoral artery catheterization and hemostatic potential analyzed by thrombelastography and calibrated automated thrombography. Our data show that blood collected via cardiac puncture demonstrated hypercoagulability as indicated by faster rates of clot formation and thrombin generation, increased overall clot strength, and a greater thrombin-generating capacity when compared with blood collected via femoral artery catheter. We conclude that blood collection methods have a profound effect on hemostatic potential, and standardization of these practices is necessary to define the effects of shock on coagulation in rodents.

Hemodynamic status and coagulation capacity affect blood loss after injury. The most advantageous fluid and blood pressure to optimize resuscitation and minimize perturbation of coagulation are unclear. We investigated interactions of isovolumic hemodilution on hemodynamics, coagulation, and blood loss after injury.Twenty-five male rats were randomized into three groups (Whole Blood Uncontrolled Blood Pressure [WBU], n = 7; Lactated Ringers Uncontrolled Blood Pressure [LRU], n = 10; Whole Blood Controlled Blood Pressure [WBC], n = 8) with isovolumic hemodilution of 50% blood volume, with and without control of pre-injury blood pressure. All rats underwent uniform grade IV liver injury 30 min after serial exchanges. Post-injury blood loss and coagulation function were measured.Dilution occurred, determined by hematocrit, with LRU having a greater reduction. Pre-injury mean arterial pressure (MAP) decreased compared with baseline (98 ± 7 mmHg) with LRU (62 ± 14 mmHg) and WBC (61 ± 10 mmHg), resulting in WBU (101 ± 13 mmHg) being significantly higher and not changed from baseline. Post-injury, MAP decreased from pre-injury, with LRU significantly lower than the other two groups. No differences were observed in prothrombin time/international normalized ratio or thromboelastography. Bleed volume was significantly different between groups: WBUWBCLRU and associated with the pre-injury MAP. Controlling baseline MAP, dilution with Lactated Ringers (LR) resulted in greater blood loss than whole blood (3.0 ± 0.4 versus 1.9 ± 0.3 mL).In this rat model of liver injury, blood loss was associated with baseline MAP and type of fluid used for dilution. Hemodilution with LR did not produce coagulopathy based on laboratory values. When controlling baseline MAP, dilution with LR increased bleeding, confirming a functional coagulopathic state.

Immediate use of thawed fresh frozen plasma (FFP) when resuscitating hemorrhagic shock patients has become more common. According to the AABB (formerly known as American Association of Blood Banks), FFP is the preferred product that can be used up to 5 days after thawing. However, limited data exist on the clinical use and hemostatic profiles of Food and Drug Administration-approved liquid plasma (LQP), which can be stored at 1 °C to 6 °C for up to 26 days. We characterized changes in LQP hemostatic potential during 26 days of cold storage.Ten FFP and 10 LQP single-donor units, matched by sex and blood group, were analyzed. FFP was thawed and kept refrigerated for 5 days and LQP for 26 days. Plasma samples were evaluated at Days 0 and 5 for thawed plasma (TP) and 0, 5, 10, 20, and 26 for LQP, by thrombelastography, thrombogram, platelet counts, platelet microparticles, clotting factors, and natural coagulation inhibitors.LQP had a better capacity to form a clot and generate thrombin compared with TP. LQP's hemostatic potential, expressed as endogenous thrombin potential (total amount of thrombin [nM] formed over time [minute]), initially exceeded that of TP (1,425 vs. 1,184, p0.05) but decreased to levels similar to TP by Day 26 (1,201 vs. 1,103, p = 0.15). Significantly higher platelet microparticles were found in LQP on Day 26 compared with those in LQP on Day 0 (23.6 x 10(9)/L vs. 3 x 10(9)/L, p0.001) or those in TP on Day 5 (2.8 x 10(9)/L). By Day 26, the majority of clotting factors and inhibitors retained more than 88% of their initial activities in LQP, with the few exceptions of factors well known to be unstable.The hemostatic profiles of LQP were better and sustained five times longer than the more commonly used TP, indicating that never-frozen plasma can be considered for use in the United States in trauma patients requiring immediate plasma resuscitation.

Introduction To improve fresh frozen plasma (FFP) availability, thawed plasma is stored at 4 °C for up to 5 days and considered equivalent to freshly thawed FFP. However, we have shown that hemostatic potential of thawed plasma is highly variable between donors and significantly diminished during storage. We hypothesized that smaller volumes of plasma with higher hemostatic potential (FFP-H) would be needed to restore normal thrombelastogram (TEG) values compared to plasma with lower hemostatic potential (FFP-L). Materials and Methods A dilutional coagulopathy model was established from whole blood by diluting plasma with saline to 23%, while cellular components were kept unchanged. Saline was gradually replaced with equal volumes of FFPs with distinctive hemostatic potentials, which was evaluated by the calibrated automated thrombogram. Clot formation in the presence of tissue factor was evaluated by TEG at baseline and after addition of increasing concentrations of FFP-H and FFP-L. Results Blood dilution with saline in the presence of tissue factor resulted in abnormal TEGs that resemble a pattern observed in severely bleeding trauma patients. All FFPs produced similar improvements in TEG variables despite different hemostatic potentials. TEG changes were solely dependent on FFP volume and reached the normal reference range when plasma concentration increased to 40%. Conclusion Plasma dilution and tissue factor in whole blood results in an abnormal TEG with a hyperfibrinolytic pattern. A plasma concentration of at least 40% was necessary for TEG normalization after dilution with saline. An effect of FFPs’ hemostatic potential on clot formation could not be detected by TEG in this in vitro model.

Hyperfibrinolysis (HF) has been reported to occur in a range of 2% to 34% of trauma patients. Using rapid thromboelastography (r-TEG), we hypothesized that HF is (1) rarely present at admission on patients with severe injury and (2) associated with crystalloid hemodilution. To further strengthen this hypothesis, we created an in vitro hemodilution model to improve our mechanistic understanding of the early HF.The trauma registry was queried for patients who were our highest-level trauma activations and admitted directly from the scene (October 2009-October 2010). HF was defined as more than 7.5% amplitude reduction 30 minutes after maximal amplitude (LY30). Using r-TEG, we then created an in vitro hemodilution model (0.9% NS) with and without tissue injury (addition of tissue factor and tissue plasminogen activator) to identify crystalloid volumes and injury needed to achieve specific LY30 values.Admission r-TEG values were captured on 1996 consecutive admissions. Only 41 patients (2%) had HF at admission r-TEG. The groups were similar in demographics. Compared with patients without HF, the HF group had more prehospital crystalloid (1.5 vs. 0.5 L), higher median Injury Severity Score (25 vs. 16), greater admission base deficit (20 vs. 2), and higher mortality (76% vs. 10%); all p0.001. Controlling for Injury Severity Score and base deficit on arrival, prehospital fluid was associated with a significant increase in likelihood of HF. In fact, each additional liter of crystalloid was associated with a 15% increased odds of HF. The in vitro model found that hemodilution to 15% of baseline and tissue factor + tissue plasminogen activator was required to achieve an LY30 of 50%.Although uncommon immediately after injury, HF is associated with prehospital crystalloid administration and shock at admission and is highly lethal. Our in vitro model confirms that tissue injury and significant crystalloid hemodilution result in severe and immediate HF.

Activated partial thromboplastin time (aPTT) and prothrombin time (PT) are clinical tests commonly used to screen for coagulation-factor deficiencies. One genome-wide association study (GWAS) has been reported previously for aPTT, but no GWAS has been reported for PT. We conducted a GWAS and meta-analysis to identify genetic loci for aPTT and PT. The GWAS for aPTT was conducted in 9,240 individuals of European ancestry from the Atherosclerosis Risk in Communities (ARIC) study, and the GWAS for PT was conducted in 2,583 participants from the Genetic Study of Three Population Microisolates in South Tyrol (MICROS) and the Lothian Birth Cohorts (LBC) of 1921 and 1936. Replication was assessed in 1,041 to 3,467 individuals. For aPTT, previously reported associations with KNG1, HRG, F11, F12, and ABO were confirmed. A second independent association in ABO was identified and replicated (rs8176704, p = 4.26 × 10(-24)). Pooling the ARIC and replication data yielded two additional loci in F5 (rs6028, p = 3.22 × 10(-9)) and AGBL1 (rs2469184, p = 3.61 × 10(-8)). For PT, significant associations were identified and confirmed in F7 (rs561241, p = 3.71 × 10(-56)) and PROCR/EDEM2 (rs2295888, p = 5.25 × 10(-13)). Assessment of existing gene expression and coronary artery disease (CAD) databases identified associations of five of the GWAS loci with altered gene expression and two with CAD. In summary, eight genetic loci that account for ∼29% of the variance in aPTT and two loci that account for ∼14% of the variance in PT were detected and supported by functional data.

Injury leads to dramatic disturbances in coagulation with increased risk of bleeding followed by a hypercoagulable state. A comprehensive assessment of these coagulation abnormalities can be measured and described by thrombelastography. The purpose of this study was to identify whether admission rapid-thrombelastography (r-TEG) could identify patients at risk of developing pulmonary embolism (PE) during their hospital stay.Patients admitted between September 2009 to February 2011 who met criteria for our highest-level trauma activation and were transported directly from the scene were included in the study. PE defined as clinically suspected and computed tomography angiography confirmed PE. We evaluated r-TEG values with particular attention to the maximal amplitude (mA) parameter that is indicative of overall clot strength. Demographics, vital signs, injury severity, and r-TEG values were then evaluated. In addition to r-TEG values, gender and injury severity score (ISS) were chosen a priori for developing a multiple logistic regression model predicting development of PE.r-TEG was obtained on 2,070 consecutive trauma activations. Of these, 2.5% (53) developed PE, 97.5% (2,017) did not develop PE. Patients in the PE group were older (median age, 41 vs. 33 years, p = 0.012) and more likely to be white (69% vs. 54%, p = 0.036). None of the patients in the PE group sustained penetrating injury (0% vs. 25% in the no-PE group, p0.001). The PE group also had admission higher mA values (66 vs. 63, p = 0.050) and higher ISS (median, 31 vs. 19, p = 0.002). When controlling for gender, race, age, and ISS, elevated mA at admission was an independent predictor of PE with an odds ratio of 3.5 for mA65 and 5.8 for mA72.Admission r-TEG mA values can identify patients with an increased risk of in-hospital PE. Further studies are needed to determine whether alternative anticoagulation strategies should be used for these high-risk patients.Prognostic study, level III.

Aim Early death due to hemorrhage is a major consequence of traumatic injury. Transfusion practices differ among hospitals and it is unknown which transfusion practices improve survival. This report describes the experience of the PRospective Observational Multicenter Major Trauma Transfusion (PROMMTT) Study Data Coordination Center in designing and coordinating a study to examine transfusion practices at ten Level 1 trauma centers in the US. Methods PROMMTT was a multisite prospective observational study of severely injured transfused trauma patients. The clinical sites collected real-time information on the timing and amounts of blood product infusions as well as colloids and crystalloids, vital signs, initial diagnostic and clinical laboratory tests, life saving interventions and other clinical care data. Results Between July 2009 and October 2010, PROMMTT screened 12,561 trauma admissions and enrolled 1245 patients who received one or more blood transfusions within 6h of Emergency Department (ED) admission. A total of 297 massive transfusions were observed over the course of the study at a combined rate of 5.0 massive transfusion patients/week. Conclusion PROMMTT is the first multisite study to collect real-time prospective data on trauma patients requiring transfusion. Support from the Department of Defense and collaborative expertise from the ten participating centers helped to demonstrate the feasibility of prospective trauma transfusion studies. The observational data collected from this study will be an invaluable resource for research in trauma surgery and it will guide the design and conduct of future randomized trials.

We conducted a genome-wide association study to identify novel associations between genetic variants and circulating plasminogen activator inhibitor-1 (PAI-1) concentration, and examined functional implications of variants and genes that were discovered. A discovery meta-analysis was performed in 19 599 subjects, followed by replication analysis of genome-wide significant (P < 5 × 10−8) single nucleotide polymorphisms (SNPs) in 10 796 independent samples. We further examined associations with type 2 diabetes and coronary artery disease, assessed the functional significance of the SNPs for gene expression in human tissues, and conducted RNA-silencing experiments for one novel association. We confirmed the association of the 4G/5G proxy SNP rs2227631 in the promoter region of SERPINE1 (7q22.1) and discovered genome-wide significant associations at 3 additional loci: chromosome 7q22.1 close to SERPINE1 (rs6976053, discovery P = 3.4 × 10−10); chromosome 11p15.2 within ARNTL (rs6486122, discovery P = 3.0 × 10−8); and chromosome 3p25.2 within PPARG (rs11128603, discovery P = 2.9 × 10−8). Replication was achieved for the 7q22.1 and 11p15.2 loci. There was nominal association with type 2 diabetes and coronary artery disease at ARNTL (P < .05). Functional studies identified MUC3 as a candidate gene for the second association signal on 7q22.1. In summary, SNPs in SERPINE1 and ARNTL and an SNP associated with the expression of MUC3 were robustly associated with circulating levels of PAI-1.

INTRODUCTION Recent data have associated improved survival after hemorrhagic shock with the early use of plasma-based resuscitation. Our laboratory has shown that FFP5 has decreased hemostatic potential compared with freshly thawed plasma (FFP0). We hypothesized that FFP5 would increase bleeding and mortality compared with FFP0 in a rodent bioassay model of uncontrolled liver hemorrhage. METHODS Hemostatic potential of plasma was assessed with the Calibrated Automated Thrombogram (CAT) assay. Rats underwent isovolemic hemodilution by 15% of blood volume with the two human plasma groups (FFP0 and FFP5) and two controls (sham and lactated Ringers). A liver injury was created by excising a portion of liver resulting in uncontrolled hemorrhage. Rats that lived for 30 minutes after liver injury were resuscitated to their baseline blood pressure and followed for 6 hours. Hemostasis was assessed by thromboelastography. RESULTS Hemostatic potential of FFP5 decreased significantly in all areas measured in the CAT assay as compared with FFP0 (p < 0.01). In the FFP5 group, overall survival was 54%, compared with 100% in the FFP0 and sham group (p = 0.03). For animals that survived 30 minutes and were resuscitated, there was no difference in bleeding and/or coagulopathy between groups. Irrespective of treatment, animals that died after resuscitation demonstrated increased intraperitoneal fluid volume (14.85 mL ± 1.9 mL vs. 7.02 mL ± 0.3 mL, p < 0.001). CONCLUSION In this model of mild preinjury hemodilution with plasma, rats that received FFP5 had decreased survival after uncontrolled hemorrhage from hepatic injury. There were no differences in coagulation function or intraperitoneal fluid volume between the two plasma groups.

BACKGROUND Several recent military and civilian trauma studies demonstrate that improved outcomes are associated with early and increased use of plasma-based resuscitation strategies. However, outcomes associated with platelet transfusions are poorly characterized. We hypothesized that increased platelet:red blood cells (RBC) ratios would decrease hemorrhagic death and improve survival after massive transfusion (MT). METHODS A transfusion database of patients transported from the scene to 22 Level I Trauma Centers over 12 months in 2005 to 2006 was reviewed. MT was defined as receiving ≥ 10 RBC units within 24 hours of admission. To mitigate survival bias, 25 patients who died within 60 minutes of arrival were excluded from analysis. Six random donor platelet units were considered equal to a single apheresis platelet unit. Admission and outcome data associated with the low (>1:20), medium (1:2), and high (1:1) platelet:RBC ratios were examined. These groups were based on the median value of the tertiles for the ratio of platelets:RBC units. RESULTS Two thousand three hundred twelve patients received at least one unit of blood and 643 received an MT. Admission vital signs, INR, temperature, pH, Glasgow Coma Scale, Injury Severity Score, and age were similar between platelet ratio groups. The average admission platelet counts were lower in the patients who received the high platelet:RBC ratio versus the low ratio (192 vs. 216, p = 0.03). Patients who received MT were severely injured, with a mean (± standard deviation) Injury Severity Score of 33 ± 16 and received 22 ± 15 RBCs and 11 ± 14 platelets within 24 hours of injury. Increased platelet ratios were associated with improved survival at 24 hours and 30 days (p < 0.001 for both). Truncal hemorrhage as a cause of death was decreased (low: 67%, medium: 60%, high: 47%, p = 0.04). Multiple organ failure mortality was increased (low: 7%, medium: 16%, high: 27%, p = 0.003), but overall 30-day survival was improved (low: 52%, medium: 57%, high: 70%) in the high ratio group (medium vs. high: p = 0.008; low vs. high: p = 0.007). CONCLUSION Similar to recently published military data, transfusion of platelet:RBC ratios of 1:1 was associated with improved early and late survival, decreased hemorrhagic death and a concomitant increase in multiple organ failure-related mortality. Based on this large retrospective study, increased and early use of platelets may be justified, pending the results of prospective randomized transfusion data.

In an effort to administer life-saving transfusions quickly, some trauma centers maintain thawed plasma (TP). According to AABB, TP is approved for transfusion for up to five days when stored at 1-6° C. However, the alterations in microparticles (MP) contained in the plasma, which are an integral component of plasma's hemostatic capacity, are not well characterized. We report on MP changes in TP between its initial thaw (FFP-0) and five days (FFP-5) of storage.FFP units (n=30) were thawed at 37° C and kept refrigerated for five days. Phenotypes of residual cells, which include platelets, erythrocytes, leukocytes, monocytes, endothelial cells, and MP counterparts of each cell type, were analyzed by flow cytometry. Functional assays were used for MP procoagulant activity, plasma thrombin generation, and clotting properties (thromboelastography).In FFP-0 the majority (94%) of residual cells were platelets, along with significant levels of platelet MPs (4408 × 10(3)/L). FFP-5 showed a decline in MP count by 50% (p0.0001), and procoagulant activity by 29% (p0.0001). FFP-5 exhibited only 54% (p0.0001) of the potential for thrombin generation as FFP-0, while thromboelastography indicated a slower clotting response (p0.0001) and a longer delay in reaching maximum clot (p0.01). Removal of MP by filtration resulted in reduced thrombin generation, while the MP replacement restored it.Decline in MP with storage contributes to FFP-5's reduced ability to provide the hemostatic potential exhibited by FFP-0, suggesting the presence of platelet MPs in freshly TP may be beneficial and protective in the initial treatment of hemorrhage.

Background: Atherosclerosis is characterized by infiltration of inflammatory cells from circulating blood. Blood cell activation could play an important role in plaque formation. Methods: We analyzed the relationship between blood cellular markers and quantitative measures of carotid wall components in 1,546 participants from the ARIC (Atherosclerosis Risk in Communities) Carotid MRI Study. Carotid imaging was performed using a gadolinium contrast-enhanced MRI and cellular phenotyping by flow cytometry. Results: Monocyte Toll-like receptor (TLR)-2 is associated with larger plaques, while CD14, myeloperoxidase, and TLR-4 associate with smaller. Platelet CD40L is associated with smaller plaques and thinner caps, while P-selectin is associated with smaller core size. Conclusions: Blood cell activation is significantly associated with atherosclerotic changes of the carotid wall.

The interaction of circulating monocytes and platelets may contribute to thrombosis and inflammation in heart failure. We studied platelet and monocyte activation in 15 patients with end-stage heart failure who underwent left ventricular assist device (LVAD) placement. Blood samples were collected before and at 3, 7, 14, 21, 30, 60, 90, and 180 days after LVAD implantation. We used flow cytometry to measure the expression of platelet surface glycoprotein receptors, platelet activation markers, monocyte markers, the formation of platelet complexes with monocytes (MPC), granulocytes, and lymphocytes, and platelet glycoprotein (GP) IIIa (PLA1/A2) polymorphism. The average preoperative percentage of CD62P-positive platelets was 27% +/- 17%; CD63-positive platelets, 9.7% +/- 8.1%; thrombospondin-positive platelets, 9.9% +/- 6.8%; and MPCs, 10.3% +/- 4.3%. No significant changes were noted in the percent of activated platelets with the three markers. Percentage of MPCs increased over time and peaked at day 21 (26.3% +/- 10.6%, p = 0.0028). In about 40% of patients, activation markers remained high up to 60 days after implantation. We found a significant positive correlation between MPC and CD14 (R = 0.60, p = 0.011), and a negative correlation between MPC and P-selectin glycoprotein ligand-1 (PSGL-1) (R = -0.84, p < 0.0001), and between CD14 and PSGL-1 (R = -0.46, p = 0.022) indicating monocyte activation. These results indicate increased platelet and monocyte activation and interactions in patients undergoing long-term LVAD support.

Background: P-selectin (SELP) and its ligand, P-selectin glycoprotein ligand 1 (SELPLG), play key roles in both the inflammatory response and the atherosclerotic process. Previous studies have shown genetic variation in the SELP gene [selectin P (granule membrane protein 140kDa, antigen CD62)] to be associated with plasma SELP concentrations; however, the major biological function of SELP (and SELPLG) is at the cell surface. We therefore investigated the association of SELP polymorphisms with platelet SELP measures and polymorphisms in the SELPLG gene (selectin P ligand) with lymphocyte, granulocyte, and monocyte SELPLG measures among 1870 participants in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI Study.Methods: Whole-blood flow cytometry was used to analyze leukocyte and platelet markers in the ARIC Carotid MRI Study. The allele frequencies for the SELP and SELPLG polymorphisms of whites and African Americans were markedly different; therefore, all analyses were race specific.Results: SELP T715P was significantly associated with lower values for platelet SELP measures in whites (P = 0.0001), whereas SELP N562D was significantly associated with higher values for SELP measures in African Americans (P = 0.02). SELPLG M62I was significantly associated with lower granulocyte and monocyte SELPLG measures in African Americans (P = 0.003 and P = 0.0002, respectively) and with lower lymphocyte SELPLG measures in whites (P = 0.01).Conclusions: Specific SELP and SELPLG polymorphisms were associated with cell surface measures of SELP and SELPLG in both whites and African Americans in the ARIC Carotid MRI Study. To our knowledge, this study is the first to examine the association of SELP and SELPLG genetic variation with measures of cell surface SELP and SELPLG.

Little is known about prostaglandin synthesis and function in embryonic stem cells. We postulated that mouse embryonic stem (mES) cells possess enzymes to synthesize protective prostaglandins. Compared with differentiated adult cells, mES cells were less susceptible to H2O2-induced apoptosis. However, their apoptosis was enhanced by indomethacin or SC-236, a selective inhibitor of cyclooxygenase (COX)-2. Analysis of COX pathway enzymes by Western blotting revealed expression of COX-2 and cytosolic and microsomal prostaglandin E2 (PGE2) synthases. COX-1 and prostacyclin (PGI2) synthases were undetectable. mES cells produced PGE2 but not PGI2. Importantly, PGE2 rescued mES cells from apoptosis. To elucidate the signaling mechanism by which PGE2 inhibits apoptosis, we analyzed E-type prostaglandin (EP) receptors by Western blots. All EP isoforms were detected except EP4. Butaprost, a specific EP2 agonist, rescued mES cells from apoptosis, whereas sulprostone, an EP1/EP3 agonist, had no effect, suggesting selective interaction of PGE2 with EP2. The antiapoptotic effect of PGE2 was abrogated by Ly-294002 or wortmannin but not H-89 or a specific inhibitor of protein kinase A, suggesting signaling via phosphatidylinositol-3 kinase (PI-3K). Akt was constitutively active in mES cells, which were inhibited by indomethacin and rescued by PGE2. The rescuing effect of PGE2 was abrogated by Ly-294002. These results indicate that mES cells constitutively express COX-2 and PGE synthases and produce PGE2, which confers resistance to apoptosis via EP2-mediated activation of PI-3K to the Akt pathway. Disclosure of potential conflicts of interest is found at the end of this article.

There have been major advances in our understanding of thrombosis and antithrombotic drugs. This review focuses on the molecular aspects of thrombus formation and antithrombotic therapy. Molecules involved in arterial thrombosis are derived from inflammatory cells in the atherosclerotic plaque and blood platelets. These molecules work in concert to promote plaque instability and thrombogenicity. Thrombus formation on the ruptured plaque is mediated by platelet and coagulation activation. By contrast, molecules involved in venous thrombosis are derived from the activated coagulation cascade. Platelets appear to play a secondary role. The antithrombotic drugs are classified according to their targeted constituents: antiplatelet agents and anticoagulants; the latter are further divided into non-specific anticoagulants, such as vitamin K antagonists and heparin, and direct thrombin inhibitors, including hirudin and argatroban. Currently available antiplatelet agents target glycoprotein IIbIIIa (abciximab, tirofiban, eptifibatide), cyclooxygenase-1 (aspirin) or adenosine diphosphate receptor, P2Y12 (clopidogrel).

Animal studies unequivocally support the indispensable role of prostaglandin (PG) and cyclooxygenase (COX) in ovulation and implantation. Available data also suggest that PG and COX may be important in the transport of embryos. The effects of PGE(2) and PGF(2alpha) on the contractility of human tubal muscle have been studied extensively; the expression of COX in human fallopian tubes was also reported. Despite all these, two fundamentally important questions remained to be answered: 1) which PGs are produced by human fallopian tubes; and 2) which COX isoform(s) is expressed by the fallopian tubes. We used reverse-phase HPLC to study the metabolism of [1-(14)C] arachidonic acid by the fallopian tubes. We found that 6 keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI), and PGE(2) constituted 56% +/- 10% and 35% +/- 10% (mean +/- SEM, four samples), respectively, of total eicosanoids synthesized. Western blot analysis revealed the expression of both COX isoforms. Immunohistochemistry study showed that both COX-1 and -2 were localized to nonciliated epithelia and tubal smooth muscle. In addition, COX-2 was also expressed in ciliated epithelial cells. Western blot analysis revealed the expression of PGI synthase (PGIS) and PGI receptor by fallopian tubes. Immunohistochemistry confirmed the expression of PGIS by luminal epithelia, tubal smooth muscle, vascular endothelial cells, and vascular smooth muscle cells. Iloprost, a PGI analog, inhibited the activities of circular and longitudinal muscles of the fallopian tube. Thus, the fallopian tube expresses both COX isoforms and PGIS. Furthermore, it is a source and a target of PGI. PGI and COX may be important to gamete function, embryo transport, and embryo development.

Trauma-induced coagulopathy following severe injury is associated with increased bleeding and mortality. Injury may result in alteration of cellular phenotypes and release of cell-derived microparticles (MP). Circulating MPs are procoagulant and support thrombin generation (TG) and clotting. We evaluated MP and TG phenotypes in severely injured patients at admission, in relation to coagulopathy and bleeding.As part of the Prospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study, research blood samples were obtained from 180 trauma patients requiring transfusions at 5 participating centers. Twenty five healthy controls and 40 minimally injured patients were analyzed for comparisons. Laboratory criteria for coagulopathy was activated partial thromboplastin time (APTT) ≥ 35 sec. Samples were analyzed by Calibrated Automated Thrombogram to assess TG, and by flow cytometry for MP phenotypes [platelet (PMP), erythrocyte (RMP), leukocyte (LMP), endothelial (EMP), tissue factor (TFMP), and Annexin V positive (AVMP)].21.7% of patients were coagulopathic with the median (IQR) APTT of 44 sec (37, 53), and an Injury Severity Score of 26 (17, 35). Compared to controls, patients had elevated EMP, RMP, LMP, and TFMP (all p0.001), and enhanced TG (p0.0001). However, coagulopathic PROMMTT patients had significantly lower PMP, TFMP, and TG, higher substantial bleeding, and higher mortality compared to non-coagulopathic patients (all p0.001).Cellular activation and enhanced TG are predominant after trauma and independent of injury severity. Coagulopathy was associated with lower thrombin peak and rate compared to non-coagulopathic patients, while lower levels of TF-bearing PMPs were associated with substantial bleeding.

Objective— Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some populations, elevated plasma levels of tPA have been associated with myocardial infarction and other cardiovascular diseases. We conducted a meta-analysis of genome-wide association studies to identify novel correlates of circulating levels of tPA. Approach and Results— Fourteen cohort studies with tPA measures (N=26 929) contributed to the meta-analysis. Three loci were significantly associated with circulating tPA levels ( P −8 ). The first locus is on 6q24.3, with the lead single nucleotide polymorphism (SNP; rs9399599; P =2.9×10 −14 ) within STXBP5 . The second locus is on 8p11.21. The lead SNP (rs3136739; P =1.3×10 −9 ) is intronic to POLB and PLAT . We identified a nonsynonymous SNP (rs2020921) in modest linkage disequilibrium with rs3136739 ( r 2 =0.50) within exon 5 of PLAT ( P =2.0×10 −8 ). The third locus is on 12q24.33, with the lead SNP (rs7301826; P =1.0×10 −9 ) within intron 7 of STX2 . We further found evidence for the association of lead SNPs in STXBP5 and STX2 with expression levels of the respective transcripts. In in vitro cell studies, silencing STXBP5 decreased the release of tPA from vascular endothelial cells, whereas silencing STX2 increased the tPA release. Through an in silico lookup, we found no associations of the 3 lead SNPs with coronary artery disease or stroke. Conclusions— We identified 3 loci associated with circulating tPA levels, the PLAT region, STXBP5 , and STX2. Our functional studies implicate a novel role for STXBP5 and STX2 in regulating tPA release.

Clot lysis values (LY30) determined by rapid thrombelastography (rTEG) predict postinjury transfusion needs and mortality risk. However, the first derivative velocity curve values generated by rTEG measuring lysis—maximum rate of lysis (MRL) and total lysis (TL)—have not been evaluated. Although recent data support use of antifibrinolytics in trauma, the population that would benefit remains poorly defined. The purpose of this study was to determine if velocity curves more accurately predict large volume transfusions and early mortality than conventional rTEG values.Conventional and velocity curve admission rTEG values of adult trauma patients were retrospectively evaluated for their ability to predict early transfusion of RBC and plasma, substantial bleeding, massive transfusion, and mortality. Patient outcomes were compared according to hyperfibrinolysis diagnosed by velocity curve values and the conventional LY30 cutoff.There were 1,625 patients included. Clot lysis values predicted early transfusion of RBC (p = 0.003), but not plasma (p = 0.298), within 3 hours of arrival. With respect to velocity curves, MRL and TL predicted both early RBC and plasma transfusion (p0.05). All 3 parameters predicted massive transfusion, but only MRL and TL predicted substantial bleeding (odds ratio [OR] 3.1 and 2.9, respectively). In addition, MRL was a stronger predictor of 24-hour and 30-day mortality (p0.001) and was also available earlier after arrival than LY30 (p0.001).Velocity curve measures of fibrinolysis are stronger predictors of early transfusion of blood components, bleeding, and mortality after trauma compared with conventional rTEG values. In addition, the MRL is more rapidly available after arrival, which may facilitate earlier diagnosis and treatment of clinically significant hyperfibrinolysis.

Severe hyperfibrinolysis after trauma is a poorly understood phenomenon associated with profound shock, serious anatomic injuries, increased transfusions, and high mortality rates. Molecular mechanisms driving hyperfibrinolysis in trauma have not been completely delineated. The authors aimed to determine the relationship between severe hyperfibrinolysis and outcomes in trauma patients and characterize the role of the plasminogen activator (PA) system in this condition. A prospective observational study was performed in 163 adult level I trauma patients admitted between April and August 2012. Blood was collected on admission, and fibrinolysis was determined by plasmin-α2 antiplasmin (PAP) levels. Tissue-derived and urokinase PA (tPA and uPA, respectively), PA inhibitor (PAI-1), fibrinogen, and antithrombin levels were also measured. Patient demographics, vital signs, laboratory values, mechanisms and severity of injuries, transfusions, and outcomes were collected at admission or from patient records. Moderate fibrinolysis was defined as PAP level 1,500 to 20,000 μg/L and severe hyperfibrinolysis as PAP level more than 20,000 μg/L. Severe hyperfibrinolysis was observed in 10% of patients and associated with increased injury severity, greater transfusions, fewer ventilator and hospital-free days, and higher mortality. Plasmin-α2 antiplasmin level was directly correlated with tPA level and inversely correlated with PAI-1 level. Patients with both elevated tPA and reduced PAI-1 were more severely injured, received more transfusions, and experienced fewer ventilator and hospital-free days. In conclusion, Severe hyperfibrinolysis is observed in a small percentage of trauma patients and is associated with severe injuries, greater transfusions, and worse outcomes. This condition is mediated, in part, by excessive upregulation of profibrinolytic tPA in the absence of concomitant increases in antifibrinolytic PAI-1.

Background Fibrinogen is the first coagulation factor to reach critical levels during hemorrhage. Consequently, reestablishing normal fibrinogen levels is necessary to achieve adequate hemostasis. Fibrinogen is supplemented through administration of fresh frozen plasma, cryoprecipitate, or human fibrinogen concentrate, RiaSTAP. RiaSTAP is potentially the most advantageous fibrinogen replacement product because it offers the highest fibrinogen concentration, lowest volume, and most consistent dose. Unfortunately, RiaSTAP is limited by a protocol reconstitution time of 15 min. Conversely, physicians in emergency settings frequently resort to a forceful and rapid reconstitution, which causes foaming and possible protein loss and/or damage. This study aims to address the in vitro effectiveness of protocol-reconstituted RiaSTAP versus rapidly reconstituted RiaSTAP versus cryoprecipitate. Methods Three fibrinogen treatments were prepared: protocol-reconstituted RiaSTAP, rapidly reconstituted RiaSTAP, and thawed cryoprecipitate. Each treatment was added in theoretical doses of 0–600 mg/dL to fibrinogen-depleted plasma (normal fibrinogen level is 150–450 mg/dL). Samples were generated in triplicate, and each sample was subjected to rapid thrombelastography and Clauss assays. The rapid thrombelastography assay measures the hemostatic potential of a blood and/or plasma sample. The maximum amplitude (MA) parameter indicates overall clot strength and is a reflection of fibrinogen efficacy. The Clauss assay measures the time to clot formation in response to a known concentration of thrombin, and the amount of functional fibrinogen is then determined from a standard curve. Results For all fibrinogen treatments, increasing fibrinogen dose resulted in an increase in MA. There was no significant difference in MA between both RiaSTAP reconstitutions (slope of RiaSTAP [protocol], 10.85 mm/[100 mg/dL] and slope of RiaSTAP [rapid], 10.54 mm/[100 mg/dL]). However, both protocol-reconstituted RiaSTAP and rapidly reconstituted RiaSTAP have higher MA values than cryoprecipitate in doses of ≥100 mg/dL. Moreover, each replicate of cryoprecipitate showed a higher variance in fibrinogen efficacy (coefficient of variance [CV] = 44.7%) at a fibrinogen dose of 300 mg/dL. RiaSTAP, however, showed a lower variance in fibrinogen efficacy for both reconstitutions (RiaSTAP [protocol], CV = 3.3% and RiaSTAP [rapid], CV = 2.7%), indicating a consistent fibrinogen dose. Conclusions RiaSTAP (either reconstitution method) has greater hemostatic potential and less variability in fibrinogen concentration compared with cryoprecipitate. Rapidly reconstituted RiaSTAP does not compromise hemostatic potential and can be used to potentially facilitate hemostasis in rapidly bleeding patients.

Background In the PRospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study, waiver of consent was used because previous literature reported low response rates and subsequent bias. The goal of this article was to examine the rationale and tradeoffs of using waiver of consent in PROMMTT. Methods PROMMTT enrolled trauma patients receiving at least 1 U of red blood cells within 6 hours after admission at 10 US Level 1 trauma centers. Local institutional review boards (IRBs) from all sites approved the study. Site 8 was required by their IRB to attempt consent but was allowed to retain data on patients unable to be consented. Results Of 121 subjects enrolled at Site 8, 55 consents were obtained (46%), and no patient or legally authorized representative refused to give consent. Of the patients, 36 (30%) died, and 6 (5%) were discharged before consent could be attempted. Consent was attempted but not possible among 24 patients (20%). Of the 10 clinical sites, 6 of the local IRBs approved collection of residual blood samples, 1 had previous approval to collect timed blood samples under a separate protocol, and 3 reported that their local IRBs would not approve collection of residual blood under a waiver of consent. Conclusion Waiver of consent was used in PROMMTT because of the potential adverse impact of consent refusals; however, there were no refusals. If the IRB for Site 8 had required withdrawal of patients unable to consent and destruction of their data, a serious bias would likely have been introduced. Other tradeoffs included a reduction in sites participating in residual blood collection and a smaller than expected amount of residual blood collected among sites operating under a waiver of consent. Noninterventional emergency research studies should consider these potential tradeoffs carefully before deciding whether waiver of consent would best achieve the goals of a study.

Acute traumatic coagulopathy (ATC) occurs after severe injury and shock and is associated with increased bleeding, morbidity, and mortality. The effects of ATC and hemostatic resuscitation on outcome are not well-explored. The PRospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study provided a unique opportunity to characterize coagulation and the effects of resuscitation on ATC after severe trauma.Blood samples were collected upon arrival on a subset of PROMMTT patients. Plasma clotting factor levels were prospectively assayed for coagulation factors. These data were analyzed with comprehensive PROMMTT clinical data.There were 1,198 patients with laboratory results, of whom 41.6% were coagulopathic. Using international normalized ratio of 1.3 or greater, 41.6% of patients (448) were coagulopathic, while 20.5% (214) were coagulopathic using partial thromboplastin time of 35 or greater. Coagulopathy was primarily associated with a combination of an Injury Severity Score (ISS) of greater than 15 and a base deficit (BD) of less than -6 (p0.05). Regression modeling for international normalized ratio-based coagulopathy shows that prehospital crystalloid (odds ratio [OR], 1.05), ISS (OR, 1.03), Glasgow Coma Scale (GCS) score (OR, 0.93), heart rate (OR, 1.08), systolic blood pressure (OR, 0.96), BD (OR, 0.92), and temperature (OR, 0.84) were significant predictors of coagulopathy (all p0.03). A subset of 165 patients had blood samples collected and coagulation factor analysis performed. Elevated ISS and BD were associated with elevation of aPC and depletion of factors (all p0.05). Reductions in factors I, II, V, VIII and an increase in aPC drive ATC (all p0.04). Similar results were found for partial thromboplastin time-defined coagulopathy.ATC is associated with the depletion of factors I, II, V, VII, VIII, IX, and X and is driven by the activation of the protein C system. These data provide additional mechanistic understanding of the drivers of coagulation abnormalities after injury. Further understanding of the drivers of ATC and the effects of resuscitation can guide factor-guided resuscitation and correction of coagulopathy after injury.

Hyperfibrinolysis has been identified as a mechanism of trauma coagulopathy associated with poor outcome. The aim of the study was to create a trauma coagulopathy model (TCM) with a hyperfibrinolysis thrombelastography (TEG) pattern similar to injured patients and test the effects of different resuscitation fluids and antifibrinolytics on fibrinolysis. TCM was established from whole blood by either 15% dilution with isotonic saline, lactated Ringer's, Plasma-Lyte, 5% albumin, Voluven, Hextend, 6% dextran in isotonic saline or 30% dilution with lactated Ringer's plus Voluven and supplementation with tissue factor and tissue plasminogen activator (tPA). These combinations resulted in a TCM that could then be 'treated' with tranexamic acid (TXA) or 6-aminocaproic acid (ACA). Clot formation was evaluated by TEG. Whole-blood dilution by 15% with crystalloids and albumin in the presence of tissue factor plus tPA resulted in an abnormal TEG pattern and increased fibrinolysis, as did dilution with synthetic colloids. TXA 1 μg/ml or ACA 10 μg/ml were sufficient to suppress fibrinolysis when TCM was diluted 15% with lactated Ringer's, but 3 μg/ml of TXA or 30 μg/ml of ACA were needed for fibrinolysis inhibition induced by simultaneous euvolemic dilution with lactated Ringer's plus Voluven by 30%. A total of 15% dilution of whole blood in the presence of tissue factor plus tPA results in a hyperfibrinolysis TEG pattern similar to that observed in severely injured patients. Synthetic colloids worsen TEG variables with a further increase of fibrinolysis. Low concentrations of TXA or ACA reversed hyperfibrinolysis, but the efficient concentrations were dependent on the degree of fibrinolysis and whole-blood dilution.

To relate in-hospital mortality to early transfusion of plasma and/or platelets and to time-varying plasma:red blood cell (RBC) and platelet:RBC ratios.Prospective cohort study documenting the timing of transfusions during active resuscitation and patient outcomes. Data were analyzed using time-dependent proportional hazards models.Ten US level I trauma centers.Adult trauma patients surviving for 30 minutes after admission who received a transfusion of at least 1 unit of RBCs within 6 hours of admission (n = 1245, the original study group) and at least 3 total units (of RBCs, plasma, or platelets) within 24 hours (n = 905, the analysis group).In-hospital mortality.Plasma:RBC and platelet:RBC ratios were not constant during the first 24 hours (P.001 for both). In a multivariable time-dependent Cox model, increased ratios of plasma:RBCs (adjusted hazard ratio = 0.31; 95% CI, 0.16-0.58) and platelets:RBCs (adjusted hazard ratio = 0.55; 95% CI, 0.31-0.98) were independently associated with decreased 6-hour mortality, when hemorrhagic death predominated. In the first 6 hours, patients with ratios less than 1:2 were 3 to 4 times more likely to die than patients with ratios of 1:1 or higher. After 24 hours, plasma and platelet ratios were unassociated with mortality, when competing risks from nonhemorrhagic causes prevailed.Higher plasma and platelet ratios early in resuscitation were associated with decreased mortality in patients who received transfusions of at least 3 units of blood products during the first 24 hours after admission. Among survivors at 24 hours, the subsequent risk of death by day 30 was not associated with plasma or platelet ratios.

Introduction: The development of acute coagulopathy of trauma (ACoT) is associated with a significant increase in mortality. However, the contributory mechanisms behind ACoT have yet to be clearly defined. The purpose of this study was to evaluate the influence of multiple variables, including base deficit and injury severity, on development of ACoT within a subset of critically ill trauma patients. Methods: A retrospective review of all trauma laparotomies between 01/2004-12/2009 was performed. ACoT (+) was defined as an arrival INR ≥1.5, ACoT (-) defined as INR

Background: Severe bleeding after injury requires transfusion of blood products, including fresh frozen plasma (FFP). Many centers are keeping thawed plasma (TP) ready for massively transfused patients. According to the American Association of Blood Banks Standards, TP is approved for transfusion up to 5 days after thawing, when stored at 1°C to 6°C. However, there are no clinical data analyzing the effects of the approved 5-day storage on plasma. We hypothesize that the hemostatic potential (HP) of freshly thawed (FFP-0) was superior to plasma stored for 5 days (FFP-5). Methods: FFP from 30 single donors were thawed at 37°C and kept at 1°C to 6°C for 5 days. HP was evaluated at day 0 and 5 by measuring kinetics of thrombin generation (TG), kinetics of clot formation by thromboelastography, clotting factors and inhibitors, and cell-derived microparticles (MPs) by flow cytometry. Results: When comparing FFP-5 to FFP-0, FFP-5 exhibited only 40% of the potential of FFP-0 for TG (6.2 nM/min vs. 14.3 nM/min, p < 0.0001), a slower clotting response via thromboelastography (reaction time: 4.3 minutes vs. 3.2 minutes, p < 0.0001) and a longer delay in reaching maximum thrombus generation (5.7 minutes vs. 4.6 minutes, p < 0.01). Diminished HP was accompanied by a significant decline in multiple coagulation proteins, including FV, VII, VIII, von Willebrand factor, and free Protein S, by up to 30%, and a decrease of 50% in MP counts. Conclusion: The HP and clot forming ability of TP significantly declined with storage. Hence, freshly TP may have a greater ability to restore hemostasis and correct coagulopathy compared with FFP-5. The clinical consequences for transfused patients deserve further exploration.

Markers of monocyte activation play a critical role in atherosclerosis, but little is known about the genetic influences on cellular levels. Therefore, we investigated the influence of genetic variants in monocyte differentiation antigen (CD14), toll-like receptor-4 (TLR4), toll-like receptor-2 (TLR2), and myeloperoxidase (MPO) on monocyte surface receptor levels. The study sample consisted of 1,817 members of a biracial cohort of adults from the Atherosclerosis Risk in Communities Carotid MRI Study. Monocyte receptors were measured using flow cytometry on fasting whole blood samples. TLR2 rs1816702 genotype was significantly associated with CD14+/TLR2+ percent of positive cells (%) and median fluorescence intensity (MFI) in whites but not in blacks (p < 0.001). Specifically, the presence of the minor T-allele was associated with increased receptor levels. In blacks, TLR4 rs5030719 was significantly associated with CD14+/TLR4+ monocytes (MFI) with mean ± SE intensities of 16.7 ± 0.05 and 16.0 ± 0.14 for GG and GT/TT genotypes, respectively (p < 0.001). Variants in TLR2 and TLR4 were associated with monocyte receptor levels of TLR2 and TLR4, respectively, in a biracial cohort of adults. To our knowledge, this is the first study to look at associations between variants in the toll-like receptor family and toll-like receptor levels on monocytes.

We evaluated the association of blood monocyte and platelet activation markers with the risk of peripheral artery disease (PAD) in a multicenter study of atherosclerosis among African American and Caucasian patients. Flow cytometric analysis of blood cells was performed in 1791 participants (209 cases with PAD and 1582 noncases) from the cross-sectional Atherosclerosis Risk in Communities Carotid Magnetic Resonance Imaging ([MRI] ARIC Carotid MRI) Study to assess platelet glycoproteins IIb and IIIa, P-selectin, CD40 ligand, platelet—leukocyte aggregates, monocyte lipopolysaccharide receptor, toll-like receptors (TLRs) 2 and 4, P-selectin glycoprotein ligand 1, cyclooxygenase 2, and myeloperoxidase. Multivariate regression analyses evaluated the association of cellular markers with the risk of PAD. After adjusting for age, race, and gender, platelet CD40L, and monocyte myeloperoxidase (mMPO) levels were significantly lower (P < .001), and monocyte TLR-4 levels were higher (P = .03) in patients with PAD. With additional adjustments for conventional risk factors, mMPO remained inversely and independently associated with the risk of PAD (odds ratio [OR]: 0.35, P = .01).

After major traumatic injury and hemorrhage, patients often require multiple transfusions of blood products, including fresh frozen plasma (FFP). Traditional use of FFP in trauma and hemorrhagic shock (HS) has been to provide a source of clotting factors that commonly become depleted with hemorrhage, massive transfusion (MT), and fluid resuscitation. Recent clinical studies have shown that resuscitation with FFP is associated with improved outcome after severe HS.1–4 Data emerging from casualty care in the war in Iraq revealed that there is an inverse correlation between the ratio of units of plasma to units of red blood cells (RBCs) transfused and mortality.1 Civilian studies have shown mixed results but predominantly support the use of FFP in massively transfused patients.5–12 Concerns over the use of FFP involve its rare association with transfusion-related acute lung injury and, the even more uncommon, transmission of viral and bacterial infections. Although some trauma centers have advocated the sparing use of FFP to prevent side effects, such as transfusion-related acute lung injury,13 recent data on FFP and its use in MT patients have initiated patterns of change in blood product administration in many trauma centers around the country. Unfortunately, little is known about the mechanism of action of FFP, and it is widely believed that most of FFP's clinical benefit in HS treatment is due to its hemostatic properties.14 According to American Association of Blood Banks guidelines, thawed FFP is stored between 1°C and 6°C and can be used within a 24-hour period. If not used within the 24 hours, the FFP is relabeled and designated as thawed plasma (TP). TP is available for clinical use if stored between 1°C and 6°C for up to 5 days.15–17 Storage of FFP for 5 days has obvious advantages from a standpoint of reducing waste and allowing for rapid administration when needed in an emergency situation. As with other blood components, the question of storage stability has been addressed for FFP. Most studies have focused on whether postthaw storage of FFP maintains adequate coagulation factor levels, specifically factors V and VIII. Both factors V and VIII have been shown to decrease after 5 days of storage, but remain within acceptable levels for hemostasis.18–22 Unfortunately, there are no studies conclusively documenting what constitutes adequate or acceptable levels of these labile factors in patients with HS. Furthermore, no studies are available to explain the changes that occur in the more than 1,000 proteins found in a unit of plasma.23 Importantly, there are no published studies correlating the age of plasma and clinical outcomes. Plasma proteins comprise a highly heterogeneous class of biological macromolecules. Many are unstable when not in their native environments, which can vary considerably among organ compartments and extracellular fluids. If certain buffer conditions are not maintained, extracted proteins may not function properly or remain soluble. Although optimal conditions for storage are distinctive to each protein, it is well known that proteins generally lose structural integrity and activity as a result of storage at 4°C because of proteolysis, aggregation, and suboptimal buffer conditions.24 Consequently, to prevent rapid degradation of proteins, preserve function, and prevent variable results, standard techniques in the basic science laboratories mandates –80°C storage of proteins used in experiments. Studies on the effects of storage on other blood components have revealed that a myriad of biochemical, metabolic, and molecular alterations can occur over time. The “storage lesion” for RBCs has been defined as a spectrum of changes that can potentially affect the quality of the product for clinical use.1,25,26 Routine storage of blood has been shown to prime the neutrophil NADPH oxidase, suggesting a proinflammatory change in aged RBCs.27 Recent work by other groups shows that storage of platelets increases growth factors including soluble CD40 ligand, transforming growth factor-β, vascular endothelial growth factor-A, platelet derived growth factor, and bfibroblast growth factor. All these proteins have potential to destabilize the vasculature and adversely affect clinical outcome.27,28 Furthermore, plasma from stored platelets and RBCs has been shown to activate human pulmonary endothelial cells (PECs).29 In a multivariate analysis, Silliman and coworkers demonstrate that the age of transfused blood was an independent predictor of multiple organ failure after injury.30–32 As mentioned previously, it is universally recognized that most proteins lack stability at 4°C. Proteins need to be stored appropriately to inhibit degradation and preserve consistent function, which would hypothetically vary depending on the age of the blood product used. Taken together, these findings suggest that the age of the stored blood products (RBCs, plasma, and platelets) has the potential to alter the consistency of the clinical outcome, an undesirable effect in critically injured patients. Although resuscitation with FFP has been associated with improved outcome after HS,33 the mechanism of action of FFP is wholly unknown. Traditionally, clinicians have been taught to use plasma to replace consumed, depleted, or diluted coagulation proteins and to reverse an assumed or documented coagulopathy. However, we question whether the effects of FFP are solely related to replacing coagulation proteins because clinical observations suggest that there are other effects. With the increasing use of plasma and decreasing use of crystalloid, most clinicians have observed a decrease in the edema that was common in resuscitated trauma patients. Preclinical data in animals suggest that plasma is less inflammatory than artificial colloid, albumin, or lactated ringer's (LR) solution when transfused into swine and rats undergoing HS.34,35 We think that it is simplistic to assume that the beneficial effects of plasma are solely related to replenishment of coagulation factors. Thus, we hypothesized that the beneficial effects of FFP are due, in part, to its ability to globally promote systemic vascular stability, as defined by decreased endothelial permeability and improved hemodynamic stabilization after HS. Our studies are based around the central hypothesis that FFP has the capacity to “normalize” injured endothelium by inhibiting and repairing the damage from a number of detrimental processes induced by HS. These processes include increased and prolonged vascular permeability, endothelial basement membrane breakdown, exposure of subendothelium, nonspecific initiation of coagulation, endothelial contraction and death, interstitial edema, leukocyte infiltration, inflammation, and tissue hypoxia.36 Normalization of these damaging effects should result in restoration of the normal structure and function of the vessel, improved vascular endothelial function, decreased permeability, targeted hemostasis, and overall better clinical outcome. Integrated into our central hypothesis is our secondary hypothesis that the beneficial effects of FFP on the vascular endothelium, hemostasis, and resuscitation will diminish with increased storage time from day 0 through day 5 at 4°C. In this article, we attempt to begin to address these questions by studying the effects of FFP both in vitro and in vivo in a rat model of HS and resuscitation.

Specific foods and overall dietary patterns are associated with soluble biomarkers of systemic inflammation and endothelial activation. However, no large epidemiological studies have evaluated relationships between such dietary factors and cell-specific markers of activation and inflammation as measured by flow cytometry.Cell aggregates and multiple platelet and leukocyte markers were quantified by flow cytometry in fresh whole blood from 1101 white adults participating in the Carotid Artery MRI Study, a subset of the larger Atherosclerosis Risk in Communities (ARIC) Study. Two dietary patterns ("Healthy" and "Western") were empirically derived via principal components analysis using data collected by food frequency questionnaire. Cross-sectional associations between dietary patterns and flow cytometry-measured biomarkers were evaluated, adjusting for demographics and lifestyle factors, including medications use.After multivariable adjustment, monocyte lipopolysaccharide receptor (CD14), monocyte toll-like receptor-2, and platelet glycoprotein IIb (CD41) showed inverse associations with the Healthy dietary pattern (p=0.01, 0.04, and 0.01, respectively). In contrast, the Western dietary pattern was positively associated with CD41 and platelet-granulocyte aggregates (p=0.01 and 0.04, respectively). Independent of other dietary factors, alcohol consumption was inversely associated with levels of pan-leukocyte marker (CD45), P-selectin (CD62P) on PLA1 and on PLA2 platelets, and platelet-monocyte, platelet-granulocyte, and platelet-lymphocyte aggregates.Dietary patterns and alcohol intake were each cross-sectionally associated with select markers of cellular activation and inflammation measured by flow cytometry. These data are consistent with the hypothesis that holistic measures of dietary intake are associated with inflammation.

Mechanisms by which hemorrhagic shock leads to hemodynamic disturbances are not fully understood. Nuclear factor kB (NF-kB) is a nuclear transcription factor that regulates expression of genes crit...

To understand the role of prostacyclin (PGI2) in protecting endothelial cells from apoptosis, we evaluated the effects of carbaprostacyclin (cPGI2) on H2O2-induced human umbilical vein endothelial cell (HUVEC) apoptosis. cPGI2 suppressed H2O2-induced annexin V-positive cells in a concentration- and time-dependent manner. Pre-treatment of HUVEC with 50 µM cPGI2 for 4 h produced the maximal anti-apoptotic effect. Authentic PGI2 generated by adenoviral transfer of PGI2 synthetic genes exerted a similar protective effect. cPGI2 inhibited Smac/DIABLO release from mitochondria, caspase 3 activation, focal adhesion protein degradation, and cell detachment. cPGI2 selectively protected X-linked inhibitor of apoptosis protein (X-linked IAP, XIAP) from H2O2-induced ubiquitination, and preserved XIAP protein levels. PD-98059 but not H-89 abrogated the protective action of cPGI2. cPGI2 increased ERK phosphorylation which was blocked by PD-98059. HUVEC stably transfected with dominant negative Ras abrogated XIAP preservation by cPGI2 while constitutive active Ras increased ERK phosphorylation and protected XIAP from degradation. Our results demonstrate for the first time that PGI2 inhibits XIAP ubiquitination and degradation via the Ras/MEK-1/ERK signaling pathway. Preservation of XIAP proteins represents a key mechanism by which PGI2 protects endothelial cells from oxidant-induced apoptosis. J. Cell. Physiol. 212:840–848, 2007. © 2007 Wiley-Liss, Inc.