Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis.

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.

Impact of blood products on platelet function in patients with traumatic injuries: a translational study.

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.

Genetic markers associated with plasma protein C level in African Americans: the atherosclerosis risk in communities (ARIC) study.

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 (ß = 0.49 µg/ml; 10% variance explained). In the 2q14 region, the top SNPs were near or within the PROC gene: rs7580658 (ß = 0.15 µg/ml; 2% variance explained, P = 1.7 × 10(-12) ) and rs1799808 (ß = 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.

Genetic associations for activated partial thromboplastin time and prothrombin time, their gene expression profiles, and risk of coronary artery disease.

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.

Transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma: the PROPPR randomized clinical trial.

IMPORTANCE: Severely injured patients experiencing hemorrhagic shock often require massive transfusion. Earlier transfusion with higher blood product ratios (plasma, platelets, and red blood cells), defined as damage control resuscitation, has been associated with improved outcomes; however, there have been no large multicenter clinical trials. OBJECTIVE: To determine the effectiveness and safety of transfusing patients with severe trauma and major bleeding using plasma, platelets, and red blood cells in a 1:1:1 ratio compared with a 1:1:2 ratio. DESIGN, SETTING, AND PARTICIPANTS: Pragmatic, phase 3, multisite, randomized clinical trial of 680 severely injured patients who arrived at 1 of 12 level I trauma centers in North America directly from the scene and were predicted to require massive transfusion between August 2012 and December 2013. INTERVENTIONS: Blood product ratios of 1:1:1 (338 patients) vs 1:1:2 (342 patients) during active resuscitation in addition to all local standard-of-care interventions (uncontrolled). MAIN OUTCOMES AND MEASURES: Primary outcomes were 24-hour and 30-day all-cause mortality. Prespecified ancillary outcomes included time to hemostasis, blood product volumes transfused, complications, incidence of surgical procedures, and functional status. RESULTS: No significant differences were detected in mortality at 24 hours (12.7% in 1:1:1 group vs 17.0% in 1:1:2 group; difference, -4.2% [95% CI, -9.6% to 1.1%]; P = .12) or at 30 days (22.4% vs 26.1%, respectively; difference, -3.7% [95% CI, -10.2% to 2.7%]; P = .26). Exsanguination, which was the predominant cause of death within the first 24 hours, was significantly decreased in the 1:1:1 group (9.2% vs 14.6% in 1:1:2 group; difference, -5.4% [95% CI, -10.4% to -0.5%]; P = .03). More patients in the 1:1:1 group achieved hemostasis than in the 1:1:2 group (86% vs 78%, respectively; P = .006). Despite the 1:1:1 group receiving more plasma (median of 7 U vs 5 U, P < .001) and platelets (12 U vs 6 U, P < .001) and similar amounts of red blood cells (9 U) over the first 24 hours, no differences between the 2 groups were found for the 23 prespecified complications, including acute respiratory distress syndrome, multiple organ failure, venous thromboembolism, sepsis, and transfusion-related complications. CONCLUSIONS AND RELEVANCE: Among patients with severe trauma and major bleeding, early administration of plasma, platelets, and red blood cells in a 1:1:1 ratio compared with a 1:1:2 ratio did not result in significant differences in mortality at 24 hours or at 30 days. However, more patients in the 1:1:1 group achieved hemostasis and fewer experienced death due to exsanguination by 24 hours. Even though there was an increased use of plasma and platelets transfused in the 1:1:1 group, no other safety differences were identified between the 2 groups. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01545232.

In vitro efficacy of RiaSTAP after rapid reconstitution.

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.

Association of hemodilution and blood pressure in uncontrolled bleeding.

BACKGROUND: 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. METHODS: 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. RESULTS: 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: WBU < WBC < LRU 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). CONCLUSIONS: 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.

Aged plasma transfusion increases mortality in a rat model of uncontrolled hemorrhage.

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.

Multiple levels of degradation diminish hemostatic potential of thawed plasma.

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.

Increased platelet:RBC ratios are associated with improved survival after massive transfusion.

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.

Protective effects of fresh frozen plasma on vascular endothelial permeability, coagulation, and resuscitation after hemorrhagic shock are time dependent and diminish between days 0 and 5 after thaw.

BACKGROUND: Clinical studies have shown that resuscitation with fresh frozen plasma (FFP) is associated with improved outcome after severe hemorrhagic shock (HS). We hypothesized that in addition to its effects on hemostasis, FFP has protective and stabilizing effects on the endothelium that translate into diminished endothelial cell (EC) permeability and improved resuscitation in vivo after HS. We further hypothesized that the beneficial effects of FFP would diminish over 5 days of routine storage at 4 degrees C. METHODS: EC permeability was induced by hypoxia and assessed by the passage of 70-kDa Dextran between monolayers. Thrombin generation time and coagulation factor levels or activity were assessed in FFP. An in vivo rat model of HS and resuscitation was used to determine the effects of FFP on hemodynamic stability. RESULTS: Thawed FFP inhibits EC permeability in vitro by 10.2-fold. Protective effects diminish (to 2.5-fold) by day 5. Thrombin generation time is increased in plasma that has been stored between days 0 and 5. In vivo data show that day 0 FFP is superior to day 5 FFP in maintaining mean arterial pressure in rats undergoing HS with resuscitation. CONCLUSION: Both in vitro and in vivo studies show that FFP has beneficial effects on endothelial permeability, vascular stability, and resuscitation in rats after HS. The benefits are independent of hemostasis and diminish between days 0 and 5 of storage.

SELP and SELPLG genetic variation is associated with cell surface measures of SELP and SELPLG: the Atherosclerosis Risk in Communities Carotid MRI Study.

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 140 kDa, 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.

Absences of Endothelial Microvesicle Changes in the Presence of the Endotheliopathy of Trauma.

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 < 0.05. 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.

Early Fibrinolysis Associated with Hemorrhagic Progression Following Traumatic Brain Injury.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

PLASMA RESUSCITATION PROMOTES COAGULATION HOMEOSTASIS FOLLOWING SHOCK-INDUCED HYPERCOAGULABILITY.

BACKGROUND: 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. OBJECTIVES: To determine the effects of crystalloid versus plasma resuscitation on thrombin generation in a rat model of trauma and hemorrhagic shock (HS). PATIENTS/METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Upon admission coagulation and platelet function in patients with thermal and electrical injuries.

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.

Microvesicle phenotypes are associated with transfusion requirements and mortality in subjects with severe injuries.

BACKGROUND: Severe injury often results in substantial bleeding and mortality. Injury provokes cellular activation and release of extracellular vesicles. Circulating microvesicles (MVs) are predominantly platelet-derived and highly procoagulant. They support hemostasis and vascular function. The roles of MVs in survival after severe injury are largely unknown. We hypothesized that altered MV phenotypes would be associated with transfusion requirements and poor outcomes. METHODS: This single-centre study was approved by the Institutional Review Board. The study cohort consisted of patients with major trauma requiring blood product transfusion and 26 healthy controls. Plasma samples for MVs were collected upon admission to the emergency department (n=169) and post-resuscitation (n=42), and analysed by flow cytometry for MV counts and cellular origin: platelet (PMV), erythrocyte (RMV), leukocyte (LMV), endothelial (EMV), tissue factor (TFMV), and annexin V (AVMV). Twenty-four hour mortality is the outcome measurement used to classify survivors versus non-survivors. Data were compared over time and analysed with demographic and clinical data. RESULTS: The median age was 34 (IQR 23, 51), 72% were male, Injury Severity Score was 29 (IQR 19, 36), and 24 h mortality was 13%. MV levels and phenotypes differed between patients and controls. Elevated admission EMVs were found both in survivors (409/µL) and non-survivors (393/µL) compared to controls (23/µL, p<0.001) and persisted over time. Admission levels of PMV, AVMV, RMV, and TFMV were significantly lower in patients who died compared to survivors, but were not independently associated with the 24 h mortality rate. Patients with low MV levels at admission received the most blood products within the first 24 h. AVMV and PMV levels either increased over time or stabilized in survivors but decreased in non-survivors, resulting in significantly lower levels at intensive care unit admission in non-survivors (1,048 vs. 1,880 AVMV/µL, p<0.00004 and 1,245 PMP/µL vs. 1,866 PMP/µL, p=0.003). CONCLUSION: Severe injury results in endothelial activation and altered MV phenotypes. Significant differences in specific MV phenotypes or changes over time were associated with blood product requirements and the 24 h mortality rate.

Trauma, Time, and Transfusions: A Longitudinal Analysis of Coagulation Markers in Severely Injured Trauma Patients Receiving Modified Whole Blood or Component Blood Products.

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.

Elevated tissue plasminogen activator and reduced plasminogen activator inhibitor promote hyperfibrinolysis in trauma patients.

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.