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Introduction The acute traumatic coagulopathy (ATC) which develops within 30 min following severe trauma with tissue damage and shock is defined by an increased prothrombin time (PT) and international normalized ratio (INR). While reduced thrombin might be expected in conjunction with elevated PT, recent clinical studies reveal paradoxically elevated thrombin generation potential in patients with ATC. We therefore hypothesized that the quantity of thrombin and the timing of thrombin-fibrinogen interactions both have an impact on clot quality; the exuberant production of thrombin found in trauma results in improper clot formation. Methods In vitro studies were conducted in human blood products and simplified synthetic plasma (consisting of purified human coagulation factors in HEPES buffered saline). Turbidimetry was used to observe fibrin crosslinking, while thromboelastography (TEG) was used to quantify clot formation parameters. Quantitation of fibrin(ogen) degradation products (FDPs) was conducted with the STA-R Evolution coagulation analyzer and by ELISA. A fluorogenic substrate was used to observe thrombin generation. Results Increasing the amount of prothrombin or thrombin (0-1400nM) in prothrombin-immunodepleted citrated plasma resulted in reduced clot times. The same dose response was examined in a buffered mixture of fibrinogen (300 mg/dl), FXIII (31.25nM), Ca2+ (2mM), and FXa (170nM-only used with prothrombin samples). However, while increasing prothrombin increased clot strength in both FII-deficient plasma and in the synthetic plasma, direct addition of thrombin decreased clot strength and by 3-fold at 1000nM versus 100nM (Figure 1; *p An examination of FDPs from plasmin-degraded fibrinogen ± thrombin ± FXIII showed that FDPs from both crosslinked and uncrosslinked fibrin had 4-fold higher amounts of fibrin monomer and D-dimer than those produced from plasmin digestion of pure fibrinogen. When FDPs from thrombin +fibrinogen ± FXIII were supplemented back into fresh fibrinogen (0-50% of the final mixture) and allowed to clot again, there was a concentration-dependent decrease in fibrin formation rate (control: 0.15 OD/min; 50% FDP: 0.07 OD/min; p Conclusions The dose responses of prothrombin versus thrombin reveal the significance of the timing of these reactions on proper clot formation. The conversion of prothrombin to thrombin mediated through FXa and FVa results in strong clots under normal circumstances. These results were reflected in both turbidimetry and TEG, indicating that fibrin crosslinking is being hindered by the presence of excess thrombin even in the presence of the antithrombin III. Additionally, FDPs from crosslinked substrates reduce new clot-making efficiency. Excess thrombin and significant increases in FDPs (which can result from fibrinolytic feedback loops) both contribute to an in vitro phenotype that may represent an underlying factor in the development of ATC. Disclosures No relevant conflicts of interest to declare.

Background: Platelets are required for effective treatment of severe hemorrhage. Standard-of-care storage is at room temperature (RT), but leads to a storage lesion characterized by loss of hemostatic function and increased risk of bacterial contamination. Refrigeration (4C) mitigates adverse effects; however, it results in pre-activation or priming, which may be suggestive of prothrombotic tendencies. We previously showed that 4C-stored platelets (4C-PLT) retain responses to physiological inhibitors comparable to those of fresh platelets (FR-PLT) in two static aggregation assays. To more closely mimic in-vivo conditions, we tested adhesive response in a microfluidic environment under physiologic high-shear flow. We hypothesized that 4C-PLT display superior adhesion compared to standard-of-care (RT-PLT) and that platelet hemostatic inhibition due to prostacyclin and nitric oxide (NO) would be similar to fresh. Methods: Apheresis platelets (AP) collected from 4 healthy donors were stored for 5 days at RT (22-24°C) or 4C (1-6°C). Additional whole blood was collected to obtain red blood cells (RBCs). Platelet samples were assayed on Day 1 (fresh) and Day 5 (RT-PLT and 4C-PLT) in the presence or absence of prostacyclin (10 nM Prostaglandin I2, PGI2) or an NO donor (50 uM S-Nitrosoglutathione, GSNO). Bioflux plates (Fluxion) were coated with 100 µg/ml type-1 collagen. Prior to perfusion, platelets were stained with calcein-AM (300x103PLT/ul) and RBCs were added to a hematocrit of 40%. Samples were perfused through the collagen-coated wells at an arterial shear rate of 720s-1, and compared to bovine serum album (BSA)-coated channels as a control to assess nonspecific binding. A fluorescence microscope acquired images every 30 sec for 6 min. Data were reported as fluorescence intensity units (FIU) and surface coverage (SC%) measured with Bioflux Montage (MetaMorph) software. Data were analyzed using two-way ANOVA and a post hoc Tukey test for multiple comparisons. Significance was p Results: The perfusion images illustrate that 4C-PLT adhesion was similar to FR-PLT and demonstrably better than RT-PLT (Fig.1A). Measured fluorescence confirmed visual findings (Fig. 1B-E) and demonstrated that RT-PLT adhesion was considerably attenuated. Cold-stored platelet average SC% after 6 min of perfusion was similar to fresh (4C-PLT: 4.5±0.8%; FR-PLT: 4.7±0.8%; p=0.86; Figure 1F), but that of RT-PLT was significantly decreased (RT-PLT: 1.2±0.2%; p Conclusion: Although 4C-stored platelets show increased levels of activation compared to fresh, they retain comparable responses to physiological inhibitors under shear flow. Furthermore, standard-of-care RT-stored platelets are unable to adhere to collagen under flow conditions. These data suggest that 4C platelets, although primed and hemostatically more active, may respond to homeostatic signals in vivo and may not pose a risk of promoting unregulated clot formation. RT-PLT function is significantly reduced and did not recover activity in this physiologically relevant model. Testing 4C-PLT in a small animal model of trauma could further elucidate these findings. Figure 1. Platelet adhesion under high shear with and without inhibitors. (A) Micrographs of FR-PLT, 4C-PLT, and RT-PLT samples are shown for a single donor after 6 minutes of perfusion. Representative fluorescence intensity unit (FIU) traces are shown for (B) FR-PLT, (C) 4C-PLT, and (D) RT-PLT (Untreated •, PGI2 ■, and GSNO ▲). Data for (E) FIU (n=4) and (F) SC% (n=4) after 360 s are mean ± SEM (Untreated = black, PGI2= gray, and GSNO = white). The micrographs were cropped and enlarged; however image size proportionality was maintained between groups and timepoints. * p Disclosures No relevant conflicts of interest to declare.

Introduction Severe trauma with tissue damage and shock can rapidly ( aPC inactivates factor Va (FVa) as part of normal hemostasis, but our previous results indicate that aPC at trauma levels is not sufficient to prevent coagulation, particularly when platelets are present, as platelet fVa is resistant to aPC [Campbell JE, PLoS ONE 9:e99181 (2014); Camire RM, Blood 91:2818 (1998)]. Tissue Factor Pathway Inhibitor (TFPI) is an endothelial-bound protein which inhibits coagulation through effects on factor Xa and the factor VIIa-tissue factor complex. It is released in the plasma (normal level 2.5nM, pathophysiologic states including trauma up to 10nM) and is typically cleared by the liver or kidneys. TFPIα accounts for 20% of circulating TFPI and binds FV and FVa [Ndonwi M, J Thromb Haemost 10:1944 (2012)]. Recently, a truncated FV (termed FV-short) produced by an autosomal dominant mutation was found to form complexes with TFPIα, resulting in retention of TFPIα in a 10-fold increase over normal levels in affected individuals [Vincent LM, J Clin Invest 123:3777 (2013)]. FV-short has significantly reduced thrombogenic potential and, by concomitantly raising TFPIα, causes a bleeding disorder. We hypothesize that the activation of PC in acute trauma may result in the production of FV-degradation products which could stabilize TFPIα similarly to the effect of FV-short. The combination of reduced FVa and increased TFPIα may contribute to ATC. Methods Whole blood from healthy volunteers was drawn by phlebotomy into ACD-containing tubes. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were collected by centrifugation (200g for 10 min and 1000g for 15 min, respectively). Calibrated automated thrombogram (CAT) assays and thromboelastography (TEG) were conducted using PRP and PPP with exogenously delivered aPC (0, 100pM, or 1nM) and TFPI (0, 2.5nM, or 10nM). An immunodepleted FV-deficient ( Results CAT assays verified that aPC and TFPI each delay and suppress thrombin generation in PPP in a dose-dependent manner, but the combination of aPC and TFPI had a synergistic effect at the highest doses tested. The endogenous thrombin potential (ETP) was eliminated in PPP (control: 1663 nM-min; 1nM aPC + 10nM TFPI: 0 nM-min; P60 min; P60 min, P Conclusions In vitro studies suggest that PC activation is not the sole cause of ATC; the concentrations of aPC measured in trauma patient blood have little effect on the coagulation potential of plasma with or without platelets when aPC is delivered exogenously. The results here demonstrate that there is a synergistic effect between aPC and TFPI. When TFPI is added to FV deficient plasma, the thrombin generation is delayed and suppressed; however, in normal plasma digested with aPC (containing fragmented FVa), the addition of TFPI is sufficient to suppress the generation of thrombin beyond the window of measurement (>60 min). The addition of platelets to the milieu eliminated the effects of aPC, TFPI, and the combination on thrombin generation, highlighting the central role of platelets in maintaining hemostatic function. Disclosures No relevant conflicts of interest to declare.

Background A subset of severe trauma victims suffering from uncontrolled hemorrhage present to the emergency room with an acute coagulopathy of trauma (ACoT). This condition occurs within the first 30 min following injury, is not the result of resuscitation-associated dilutional effects, and is defined by an INR > 1.2. The mechanisms behind this coagulopathy have not been completely identified, but recent studies posit that the exuberant activation of protein C is a key contributor to ACoT. Protein C is activated through the interaction of thrombin and thrombomodulin on the endothelial surface. Activated protein C (APC) has anti-inflammatory and cytoprotective functions, in addition to regulating coagulation by inactivating factor Va (FVa). Evidence that APC is implicated in ACoT includes: 1) circulating APC levels are elevated 3-5 fold in patients with high injury severity scores (>15) and base deficit (>6); 2) high APC levels (>6 ng/ml, or 105 pM) in these patients are correlated with an increase in prothrombin time (PT) and partial thromboplastin time [Cohen et al. Ann Surg 2012; 255(2): 379-85]. FVa exists in a soluble plasma fraction and in a platelet-associated fraction (in the membrane or alpha-granules). The platelet pool of FVa is more resistant to APC degradation [Camire et al. J Biol Chem 1995; 270(35): 20794-800] and is sufficient to maintain thrombin generation even in patients with severe congenital FV-deficiencies [Duckers et al. Blood 2010; 115(4): 879-86]. We hypothesize that healthy platelets in sufficient quantity will serve as an effective promoter of hemostasis in patients with ACoT, overcoming the effects of APC. Methods and Results We studied the effects of APC on coagulation in commercially obtained FV-deficient plasma (FVdp) and platelets/plasma from healthy volunteers. Tests included prothrombin time, turbidimetric assessment of fibrin cross-linking, and thromboelastography (TEG). Prothrombin time in platelet-free plasma or FVdp subjected to exogenous APC display a dose-dependent response where clotting is only significantly delayed at APC >1 nM, much higher than the systemic concentrations found in the ACoT patients (105pM) or even in subjects treated with drotrecogin alfa, a recombinant APC product intended for the treatment of sepsis (steady-state concentration was found to be ∼790pM) [Macias et al. Clin Pharmacol Ther 2002; 72(4): 391-402]. Examining fibrin crosslinking in a static 96-well turbidimetric plate assay demonstrated that concentrations of APC of 1nM or below had no effect on the initiation time, rate, or strength of clotting in FVdp or PFP from healthy volunteers. Titration of plasma FV into FVdp illustrated that clotting time and rate (as measured by TEG) were not significantly affected at 10nM FV, less than 50% of the “normal” 23nM concentration. Clot strength was unaffected by FV depletion. To examine the contributions of platelet FVa, isolated and washed platelets were introduced in increasing amounts to FVdp and PFP; the rate of clotting and clot strength aligned between the two plasmas given equivalent numbers of platelets, emphasizing the importance of platelet FVa. At lower platelet concentrations (particularly 10,000 to 100,000 / µl), there were statistically significant differences in clotting time between FVdp and normal plasma, but these differences diminished with increasing platelet concentration. To evaluate the resistance of the platelet fraction to APC effects, 200,000 platelets / µl were suspended in FVdp and treated with a titration of APC from 1pM to 100nM. The samples were unaffected by APC up to a dose of at least 10nM as measured by TEG clotting parameters, well above the amounts found in the trauma patients or drotrecogin alfa-treated subjects. Conclusions FV-deficiency affects clotting only in cases of severe depletion (1nM). Healthy platelets in sufficient quantity are capable of rescuing FV-deficiency and are resistant to APC effects. APC is unlikely to be the main driver of ACoT. Disclosures: No relevant conflicts of interest to declare.

Despite a large body of literature regarding Acute Coagulopathy of Trauma and Massive Transfusion, there is little consensus on the appropriate diagnostic approach to establish the cause of associated uncontrolled bleeding. Thromboelastography (TEG) is widely used to characterize trauma-associated bleeding. However, the use of whole blood in this assay may obscure important changes in blood component hemostatic function. Blood varies in hematocrit from a typical level of 40% in large vessels to 10% in capillaries. Patients with persistent bleeding may present with prolonged prothrombin time (PT)/activated partial thromboplastin time (aPTT) measured in plasma, but unremarkable TEG tracings measured in whole blood drawn from a large vein. As one of the only ubiquitously accepted causes of a bleeding coagulopathy associated with trauma, acidemia was modeled in TEG to investigate 1) the differential coagulopathy detection obtained by separation of blood components before TEG testing, 2) the range of acidemia effects at various levels of the vascular tree through variable hematocrit testing, and 3) effects of replacing RBCs with plasma versus normal saline. Male donor blood (n=5) was drawn into citrate and the contact pathway was immediately further inhibited with corn trypsin inhibitor. TEG cups were preloaded with appropriate volumes of [morpholino]ethanesulfonic acid buffered saline (MBS) for pH 7.4, pH 7.1, pH 6.8 and were recalcified (15mM). R-time parameters were not significantly different when whole blood (WB) or red blood cells combined with platelet poor plasma (RBC/PPP) were activated to clot with tissue factor (1:5000 dilution induced a normal 4 minute clot time) in the simulated acidemia, whereas platelet poor plasma (PPP) showed a significant delay at pH 7.1 (p Hematocrit levels were chosen to model typical changes from large to small vessels, in the presence of 200,000 platelets/microliter, where the volume of RBCs was replaced with plasma. Results indicate that zero hematocrit conditions result in a significant R-time delay (pH 7.4 vs pH 7.1; p=0.0306, pH 7.1 vs pH 6.8; p Subsequent biochemical evaluation of whole blood (n=5) activated to clot in conditions of acidemia (pH 7.4, pH 7.1, pH 6.8) showed no significant delay or reduced peak of thrombin generation, no significant difference in factor V activation or fibrinopeptide A cleavage. Data indicate that 1) separation of blood components allows a more sensitive parsing mechanism for TEG evaluation of coagulation in acidemia, 2) simulated capillary blood containing low hematocrit demonstrates the anti-coagulant effects of acidemia obscured in whole blood from large vessels with normal hematocrit, 3) resuscitation with normal saline may exacerbate microvascular bleeding in the acidemic patient, and 4) RBCs prevent delays in clot initiation and loss of platelet force generation in the presence of acidemia. This work provides a mechanism to explain the observation that increased hematocrit reduces microvascular bleeding. Disclosures: No relevant conflicts of interest to declare.

Abstract 3438 Massive transfusion is defined as infusion of 10 units of red blood cells in 24 hours during treatment of uncontrolled bleeding. Mortality rates are 28.3% higher for patients that undergo massive transfusion. Administered blood products are collected from donors using citrate inhibition for anti-coagulation. Citrate chelates calcium, thus preventing its interactions with clotting enzymes and eliminating coagulation activity. Severe trauma patients suffer compromised liver function slowing citrate clearance and further reducing calcium availability. Under this scenario, calcium is empirically replaced due to concerns regarding hemostasis, however magnesium, which is also chelated by citrate, is not empirically replaced. Our goal was to describe both platelet aggregation and coagulation under conditions of increasing magnesium concentration following citrate chelation to ascertain the true spectrum of magnesium influence upon hemostasis. To analyze platelet activation/aggregation, de-identified whole blood (WB) was anti-coagulated with citrate (10.9mM) and corn trypsin inhibitor (CTI) (0.1mg/mL), or citrate and 50μL pPACK (final). Platelet-rich plasma (PRP) was prepared through centrifugation at 200g for 10 minutes. Subsequent spin at 2000g for 10 minutes yielded platelet-poor plasma (PPP). Remaining packed red blood cells were combined with PRP (platelets calcein-tagged to 10μM final) to simulate 40% hematocrit. Shear rate of 920/sec was induced for arterial flow conditions for 10 minutes at 37°C through a microchannel (350μm W × 70μm H × 5000μm L) pre-coated with a solution of 100μg/mL type-I collagen blocked with 0.5% BSA/1X PBS. Fluorescent signal intensity of calcein-tagged platelets was quantified through sequential image capture every 30 seconds using Fluxion BioFlux 1000. For coagulation analyses, citrate/CTI inhibited WB, PRP, and PPP was analyzed via thromboelastograph (TEG) following large volume pre-mixing with tissue factor (1:5000). TEG cups were preloaded with varying concentrations of calcium chloride (0M, 5.5mM, 10.9mM, 15mM), or calcium chloride (15mM) with magnesium sulfate (0M, 3mM, 3.5mM, 4.5mM) before blood or component addition. TEG data were collected for 90 minutes. Statistical analyses were performed with GraphPad Prism 5 software. Comparisons were drawn using linear analysis and area under the curve. Data indicate that re-attainment of physiological magnesium concentration in citrated blood: (1) facilitates platelet activation/aggregation, and (2) augments coagulation parameters through accelerated clot time and increased clot strength. Rate of platelet aggregation was significantly increased at final magnesium concentrations of 1.5mM (p Disclosures: No relevant conflicts of interest to declare.