ALBERT

Description: Background. It has been observed that trauma patients often display an elevated procoagulant activity. This activity has been hypothesized to be caused in part by tissue factor (TF) located on the endothelium, blood cells and microparticles. We also observed in our previous studies that trauma patients with thermal, blunt and penetrating injuries have active FIXa and FXIa in their plasma, often persistent for several days to weeks. What is not known is the effect of injury type and severity on TF release after trauma. In the current study we evaluated how the severity of an injury with or without accompanying shock affects the frequency and concentration of TF, FIXa and FXIa in plasma from trauma patients. Methods. Eighty trauma patients presenting to a major urban level 1 trauma center (47 with blunt trauma and 33 with penetrating trauma) were enrolled. 62 patients were male and 18 female. The age of patients varied between 18 and 90 years (average 42.6±19.2 years) and the Injury Severity Score (ISS) varied between 1 and 75 (average 19.3±17.2). Blood was collected immediately upon emergency department arrival prior to any resuscitation or blood product transfusion. For analysis our cohort was divided into 4 groups (20 patients/group) based on their ISS and presence of shock (base deficit BD): Group 1: Non-severe injury, no shock (ISS≤15; BD〉-6) Group 2: Non-severe injury, with shock (ISS≤15; BD≤-6) Group 3: Severe injury, no shock (ISS〉15; BD〉-6) Group 4: Severe injury, with shock (ISS〉15; BD≤-6) Citrate plasma was prepared, frozen and stored at -80°C. No additional freeze/thaw cycles were involved prior to FXIa, FIXa and TF activity assays. The assays were based on a response of thrombin generation to corresponding monoclonal inhibitory antibodies (αFXIa-2, αFIXa-91 and αTF-5, respectively; all at 0.1 mg/mL). Results. The frequency of TF in less injured patients was relatively low (table) and increased by at least 3-fold in patients with severe injury (groups 3 and 4). The concentration of TF varied in a wide range in all 4 groups, but was the lowest in group 1 patient samples and the highest in groups 3 and 4. FXIa was observed in 91% of plasma samples and was high in all 4 groups of patients. Similar to TF, the concentration of FXIa varied in a wide range in the entire study population. Unlike TF however, FXIa was present in patients with shock at a higher concentration and did not correlate with the severity of injury. Only 9 of 20 group 1 plasma samples had detectable FIXa, whereas in groups 2-4 only 1 or 2 plasma samples had no FIXa activity. The median concentration of this protein varied in a relatively narrow range between all 4 groups, although the lowest was observed in group 1. For the entire cohort, there was a good correlation between FIXa and FXIa concentrations (R2 =0.33), but no correlation between TF and FIXa or FIXa. These data suggest that FXIa in trauma patient blood is generated primarily through the contact pathway, although the input of the TF pathway to FXI activation cannot be excluded. Table 1.Patient GroupTFFXIaFIXaFrequencyConcentration (pM)FrequencyConcentration (pM)FrequencyConcentration (pM)RangeMedianRangeMedianRangeMedian14/20 (20%)0.1 - 〉6.40.2818/20 (90%)0.9 - 〉647.39/20 (45%)73 - 〉100017523/20 (15%)0.5 - 〉6.41.0019/20 (95%)3.2- 〉6423.418/20 (90%)38 - 〉1000270312/20 (60%)0.3 - 〉6.43.5017/20 (85%)0.7 - 〉6411.118/20 (90%)75 - 〉1000252414/20 (70%)0.2 - 〉6.42.4819/20 (95%)1.8 - 〉6426.519/20 (95%)26 - 〉1000205Overall33/80 (41%)0.1 - 〉6.41.0173/80 (91%)0.7 - 〉10017.064/80 (80%)26 - 〉1000250 Conclusions. 1) Frequency and concentration of TF is higher in patients with a higher trauma severity, but it is independent of shock; 2) The vast majority of plasma samples from trauma patients contain active FIXa and FXIa; 3) Concentration of FXIa is higher in patients with shock and does not appear to be affected by the trauma severity. Taken together, these data suggest separate mechanisms for contact pathway activation after injury driven by shock and TF pathway activation driven by tissue injury. Disclosures No relevant conflicts of interest to declare.

Description: Background. Histones are components of nucleosomes and can be released from activated immune cells as well as from necrotic and apoptotic cells. The release of histones is associated with an increased prothrombotic activity. It has been suggested that this activity is caused by the stimulation of platelets, interference with the protein C pathway, changes in red blood cell (RBC) function, etc. Studies published so far were done in plasma or using purified cells and proteins. We extended these studies by evaluating thrombin (FIIa) generation in fresh and "reconstituted" blood triggered with histone H4 and examined contribution of several proteins on this process. Methods. Blood from 8 healthy donors (4 male and 4 female, none of them on oral contraceptives; one male and one female donor were drawn twice) was drawn into 3.2% sodium citrate, 0.1 mg/mL corn trypsin inhibitor (CTI; prevents contact pathway activation) and 20 µg/mL H4 were added followed by recalcification. FIIa generation (TG) was monitored in a fluorogenic assay derived from the CAT method. When desired, either monoclonal inhibitory antibodies to factor (F)XIa, FIXa and tissue factor (TF) (αFXIa-2, αFIX-91 and αTF-5, respectively; all at 0.1 mg/mL) or kallikrein inhibitor aprotinin (10 µM) were added to blood. In parallel, TG in washed RBC reconstituted (50/50) either with platelet poor (PPP) or platelet rich (PRP) plasma ("reconstituted blood") was analyzed at the same conditions. FIIa generation in an autologous plasma was also analyzed. Results. A. Whole blood. In the absence of any exogenous activator, the lag phase and peak value of TG in CTI blood varied in a wide range (see the table). An addition of H4 to the blood shortened the lag phase and increased peak FIIa concentration for all donors. An addition of αFXIa-2 slightly prolonged the lag phase and slightly suppressed other parameters of FIIa generation. Aprotinin had a somewhat more pronounced effect on the lag phase but only marginally affected other parameters of FIIa generation. αFIX-91 almost completely suppressed H4-initiated FIIa generation, whereas αTF-5 had almost no effect on the process. Table 1.ConditionsLag (min)Max FIIa (nM)Max Rate (nM/min)AUC (nM*min)RangeAverage ± SD RangeAverage ± SD RangeAverage ± SD RangeAverage ± SD CTI(n=10)9 - 2313.4 ± 4.236.6 - 109.563.7 ± 25.114.5 - 92.648.9 ± 26.6309.3 - 687.8487.6 ± 130.1CTI + H4(n=10)2.6 - 7.44.7 ± 1.466.3 - 134.799.4 ± 22.436.8 - 83.357.9 ± 15.4417.5 - 865.8571.1 ± 145CTI + H4 + αFXIa-2(n=9)2.4 - 8.55.2 ± 1.956.6 - 103.983.6 ± 13.222.9 - 69. 444.9 ± 13.2409.9 - 680.3522 ± 90.4CTI + H4 + aprotinin(n=9)3.3 - 8.15.5 ± 1.662.3 - 144.295.1 ± 25.736 - 94.458.1 ± 19.4409.2 - 1056.2599.5 ± 203.6CTI + H4 + αFIXa-91(n=6)NQNQNQNQNQNQNQNQCTI + H4 + αTF-2(n=6)3.2 - 7.44.9 ± 1.765.1 - 128.490.1 ± 21.843.4 - 16973.9 ± 47.6434.1 - 867.7543.1 ± 166*NQ = Not Quantifiable (in 4 of 6 experiments no FIIa generation was observed and in 2 experiments it was negligible). B. RBC. In the absence of any exogenous activator, no FIIa generation was observed in 50 min. in RBC reconstituted with PPP. An addition of H4 led to FIIa generation after a lag phase of 17.7±4.3 min with the maximum active FIIa concentration (66.5±12.4 nM) lower than that observed in whole blood. Additions of either αFXIa-2 or aprotinin had no pronounced effect on FIIa generation, whereas no FIIa was observed in the presence of αFIX-91. When RBC were reconstituted with PRP, H4-induced FIIa generation parameters became similar to those observed in whole blood. C. Autologous PPP. No FIIa generation was observed in CTI PPP in the absence of any exogenous activator. An addition of H4 led to a delayed (25-30 min lag phase) and inefficient FIIa generation (maximum 10-15 nM). αFXIa-2 had no effect on FIIa generation, αFIX-91 completely suppressed it and aprotinin had an intermediate effect. Conclusions. 1). H4 has a pronounced effect in enhancing FIIa generation in fresh blood and washed RBC reconstituted with PPP and PRP; 2). Inhibition of contact pathway proteins has minor effect on H4-induced FIIa generation; 3). Inhibition of FIXa almost completely abolishes H4-induced FIIa generation; 4). RBC reconstituted with PPP can support H4-induced FIIa generation and platelets enhance this process; 5). H4-induced FIIa generation in PPP is negligible. Disclosures Mann: Haematologic Technologies, Inc; Baxter, Diagnostica Stago, Bayer, CSL Behring, Alnylam.: Consultancy, Equity Ownership.

Description: Background. Hemostatic tests have been utilized to clarify the blood coagulation potential of both healthy and diseased individuals. They include tests for whole blood and plasma clotting times, coagulation and fibrinolytic factors and, recently, thrombin generation (TG). TG assays provide explicit information and remain the most physiologically-relevant hemostatic tests ex vivo. We have currently been using a TG assay in whole blood, described by Ninivaggi et al. (2012), for the evaluation of several hemostatic agents and disorders of blood coagulation. Methods. Whole blood from either healthy donors or trauma patients was drawn into 3.2% sodium citrate [± 0.1 mg/mL corn trypsin inhibitor (CTI) prior to the assay; prevents contact pathway activation] or into 3.2% sodium citrate/0.1 mg/mL CTI. Selected agents were added to blood prior to recalcification and TG was monitored in a fluorogenic assay. Packed red blood cells (pRBC) from healthy donors were reconstituted with platelet-poor plasma (PPP) and treated with 0.1 mg/mL CTI. TG was monitored in the absence of any exogenous initiation, in the presence of 5 pM TF initiation and also in the presence of synthetic phospholipids. Results. Whole Blood A. Blood from 10 healthy donors was collected into both citrate (with CTI added prior to the assay) and CTI/citrate and TG was monitored ± 5 pM relipidated tissue factor (TF) initiation. In the absence of TF, blood collected into citrate led to a lag phase ~3-fold longer than that in the presence of TF and peak thrombin ~1.4-fold lower. Blood collected into CTI/citrate, in the absence of TF, led to a lag phase ~6.4-fold longer than that in the presence of TF and peak thrombin ~4.3-fold lower. B. Titrations of relipidated TF and FXIa into citrated blood containing CTI led to concentration-dependent changes in the duration of the lag phase. C. Several TG antagonists were evaluated in citrated blood containing CTI triggered with 5 pM TF. a. 0.5 U/mL unfractionated heparin prolonged the lag phase ~2-fold and suppressed the peak thrombin ~3-fold. b. Hemophilia A and B were "induced" via the addition of inhibitory antibodies to FVIII and FIX, respectively, and although the lag phase was not altered both additions led to significant TG suppression with peak thrombin dropping ~3-fold. c. Several thrombin and FXa inhibitors were evaluated at their pharmacologic concentrations. Fondaparinux slightly prolonged the lag phase, whereas dabigatran increased it by 2.6-fold. Rivaroxaban doubled the lag phase while bivalirudin was slightly less efficient. None of these inhibitors had a pronounced effect on the rate and peak value of TG. d. Activated protein C (APC) at 10 nM slightly prolonged the lag phase and suppressed the peak thrombin. D. Citrate blood from a trauma patient was tested for endogenous activity. In the presence of CTI and absence of an initiator, α-TF antibody did not alter TG, indicating the absence of functional TF. α-FXIa antibody prolonged the lag phase and suppressed TG and α-FIXa antibody completely abolished TG, indicating the presence of active FXIa and FIXa. Packed Red Blood Cells (pRBC) In the absence of an initiator, no TG was observed in pRBC/CTI reconstituted with PPP. The addition of 5 pM TF led to TG after ~8 min with a peak value slightly lower than in blood (described in A).The addition of phospholipids did not change the TG profile. Conclusions. This continuous whole blood TG assay requires 15 µL of blood for a triplicate analysis of a single condition and has the potential for the evaluation of TG in disorders relevant to blood coagulation and for the monitoring of treatments administered in response to these disorders. Disclosures Mann: Haematologic Technologies, Inc.: Employment, Equity Ownership; Baxter: Consultancy; Bayer: Consultancy; Biogen IDEC: Consultancy; CSL Behring: Consultancy; Merck: Consultancy; Pfizer: Consultancy; The Medicines Company: Consultancy; XO1: Consultancy; Vascular Solutions: Consultancy; Stago: Consultancy.

Description: Background. Cancer patients often display procoagulant activity and are at a risk for developing venous thromboembolism (VTE). Multiple mechanisms may explain this procoagulant state, such as tumor cell expression of tissue factor (TF), host tissue response to tumor formation and also contributions from comorbid factors and treatment. We previously observed elevated levels of traditional biomarkers of coagulation (such as D-dimer, CRP, etc.) in patients with advanced cancer on therapy enrolled in a randomized trial comparing rosuvastatin versus placebo. In this study, we evaluated the frequency and concentration of endogenous plasma TF, Factor (F)XIa and FIXa in the cancer patient population analyzed for traditional coagulation biomarkers. The choice of new analytes was based on previous studies in which these 3 proteins were detected and quantitated in patients with cardiovascular diseases, inflammation, and trauma - often present over the course of several days to even weeks. By our knowledge, there are no published studies of the presence of FXIa and/or FIXa in cancer patients. Methods. Thirty-seven adult cancer patients receiving systemic therapy were originally enrolled in a randomized crossover trial comparing effects of rosuvastatin versus placebo on biomarkers of VTE. Inclusion criteria included locally-advanced or metastatic cancer, estimated survival time of greater than 6 months, and anticipated duration of therapy of at least 3 months. Patients on antithrombotic or statin therapy were excluded. Blood was collected on up to 4 occasions; at the start and end of each of 2 treatment periods corresponding to 2 cycles of systemic therapy with a 3-4 week washout period. Citrate plasma was prepared, frozen and stored at -80C. The assay, performed in previously unthawed and contact-pathway inhibited plasma, is based on a response of the lag phase of thrombin generation to corresponding monoclonal inhibitory antibodies (αFXIa-2, αFIXa-91 and αTF-5; all at 0.1 mg/mL). Concentrations of TF, FXIa and FIXa were calculated from corresponding calibration curves built by titrating purified proteins into multi-donor pooled plasma from healthy individuals. Results. A total of 116 blood samples were collected (not all patients were able to complete their regime) and analyzed for endogenous levels of TF, FXIa and FIXa (see table). Overall, rosuvastatin treatment did not have any significant impact on the levels and frequency of these 3 proteins. For the samples obtained from each patient's first blood draw (n = 37) there was a weak correlation between FXIa and FIXa levels (R = 0.37, P = 0.02) and a moderate correlation between FXIa and TF levels (R = 0.57, P 〈 0.001), while no correlation between FIXa and TF was observed (R = 0.02, P = 0.91). Conclusions. 1) A portion of patients with advanced cancer had active TF in their plasma while the majority, for the first time reported, had active FXIa and FIXa; 2) No pronounced differences were observed between the frequency and concentrations of these proteins at baseline versus later time-points; 3) There was a weak correlation between FXIa and FIXa and a moderate correlation between FXIa and TF, suggesting the TF-pathway is driving the majority of FXIa generation, although contribution from the contact pathway cannot be excluded; 4) Rosuvastatin treatment did not lead to any significant changes in the levels of TF, FXIa and/or FIXa. Table Table. Disclosures No relevant conflicts of interest to declare.