ALBERT

Description: Key PointsA single exposure to protamine and heparin during CPB is highly sensitizing; 29% of patients develop Abs to PRT/H complexes by day 30 after CPB. PRT/H Abs share several features with platelet factor 4/heparin Abs, including high titer formation after CPB, heparin dependence, antigen specificity, and platelet activation.

Description: Abstract 3344 INTRO: Protamine (PRT) is routinely used to reverse heparin (H) anticoagulation during cardiopulmonary bypass (CPB). Previous studies have shown that PRT interacts with H to form ultra-large complexes that are immunogenic in mice (Chudasama 2010). Given the high levels of PRT and H used during CPB, we hypothesized that patients undergoing CPB will develop antibodies (Abs) to PRT/H complexes. To test this, we examined the serologic and clinical characteristics of 500 patients undergoing CPB from a recently completed prospective clinical trial (HIT 5801 study). METHOD: With IRB approval, we analyzed samples and clinical outcomes from patients at baseline, 3–7, and 30 days after CPB. Control subjects included healthy volunteers without diabetes, prior cardiac surgery, prior H exposure or chronic medications (n=101). PRT/H Abs were measured by enzyme linked-immunoassay (ELISA). RESULT: The mean A450nm for control subjects in the PRT/H ELISA was 0.39 ± 0.28, yielding a positive cut-off value (≥ mean A450nm +3 standard deviations (SD)) for the PRT/H ELISA of 1.2 (Fig A). Using this cut-off, PRT/H Abs were present in 2/101 (2%) of control subjects. New seroconversions were seen in 6/497 (1%) subjects at day 3–7 and 143/489 (29%) at day 30. The mean PRT/H reactivity of control subjects (mean A450nm ± SD; 0.39 ± 0.28) did not differ from that of patients at day 0 (0.28 ± 0.26) or day 3–7 (0.26 ± 0.37), but differed significantly from that at day 30 (1.06 ± 0.96, p 〈 0.0001). Serologic features of PRT/H Abs were examined on patients with high positivity in the screening ELISA (mean A450nm 〉 3.0, n = 32). These patients expressed high PRT/H Ab titers ranging from 1:1800–1:175,000 (mean 1:14,744). As seen in Fig B, high-titer PRT/H Abs bound to PRT alone and/or PRT/H complexes, but showed minimal binding to human platelet factor 4 (hPF4), hPF4/H complexes, or albumin (p 〈 0.0001 for PRT/H vs hPF4, PRT/H vs hPF4/H, and PRT/H vs albumin). Because Ab binding was significantly higher with PRT/H complexes than with PRT alone, we examined the effects of excess H (100 U/mL) on binding. All Abs with the exception of one, showed decreased binding in the presence of excess H (range: 8%-76%, mean inhibition = 45%). To determine the effects of Abs on platelet activation, a SRA was performed on patients (n=3) with high-titer Abs and control subjects. PRT/H seropositive plasma did not activate platelets in the presence of buffer or H (0.25 or 1 U/ml), but robustly activated platelets in the presence of PRT (1.25μg/mL) alone, with decreasing 14C release in the presence of PRT and increasing amounts of H (Fig C). Platelet activation in the presence of PRT was inhibited by IV.3, which blocks platelet activation via the platelet FcgRIIA. There was a high incidence of PF4/H seroconversion after CPB (362/500 or 72%). While the majority of PRT/H seropositive patients (n=154) were also positive for PF4/H Abs (120/154 or 78%), the converse was not true. Of the 362 PF4/H seropositive patients, only 120 (33%) were positive for PRT/H. A minority of subjects were seronegative for both (104/500 or 21%). Results in one assay were not predictive of the other. In analyzing long-term outcomes (〉30 days), there was a trend towards an association between PRT/H Ab positivity at baseline and event free survival (p = 0.07; hazard ratio (HR) = 1.65 (0.96–2.82); days to major adverse cardiac event). Seropositivity at other time points was not associated with decreased event free survival. There was no significant difference between platelet counts by Ab status. CONCLUSION: Our studies are the first to comprehensively characterize a new class of H-dependent Abs, PRT/H Abs, in a large cohort of patients undergoing CPB. We show that PRT is a highly sensitizing drug, associated with formation of high-titer, antigen-specific Abs to PRT/H complexes that are capable of platelet activation. We show that the majority of seroconversions occurred after discharge and in the absence of circulating antigen (at day 30) was not associated with adverse clinical events. However, several features of PRT/H Abs, including high-titer expression and PRT-dependent platelet activation, suggest that additional studies are needed to determine the clinical significance of these Abs in the context of PRT re-exposure. Disclosures: Arepally: Teva Pharmaceuticals: Research Funding.

Description: The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo.

Description: INTRODUCTION Prothrombin complex concentrate use (PCC) may be complicated by thrombosis, and increased factor II levels are recognized as a risk factor.1 When administering PCCs in cardiac surgery patients using K Centra ®, a 4-factor PCC for warfarin reversal, or Profilnine®, a 3-factor PCC for refractory hemorrhage, we administer a low-dose of 1000-2000 units and initially measured post-dose factor II activity, for quality control. These QC findings were the basis for a hematology/anesthesiology/surgery based standardized, refractory bleeding algorithm (Figure 1). However, the risk of thromboembolic events in this patient population is not well characterized. Therefore, we evaluated our patient population for perioperative thromboembolic events. METHODS A retrospective review of 85 patients (11 presented here, 85 to be presented in December) from January 2014 to April 2015 who activated the refractory bleeding algorithm were examined. Data include demographics, comorbidities, lab values, blood product usage, PCC and rFVIIa doses, chest tube output and postoperative thromboembolic complications i.e. (cerebral vascular accidents, myocardial infarction, pulmonary embolism or deep vein thrombosis.) Descriptive statistics: percentage, mean +/- standard deviation (SD) or median [interquartile range (IQR)] will be used to describe the results. RESULTS Initially, from March 2014 to October 2014, 11 complex cardiothoracic surgery patients received PCCs. Average age was 59(+/-14) years and 2/11(18%) died. Patients received 28 (+/-21) units per kilogram ideal body weight PCCs (K Centra 27 (+/-22), Profilnine 24 (+/-20)). The average, post-dose factor II levels were 78 (+/-16)%; 3/11 (27%) patients were below our normal reference range (72-132%), even after dosing, and two received a third dose. One patient died 3 days after a heart transplant and KCentra administration that was complicated by cardiogenic shock requiring biventricular mechanical support with late thrombosis, in the setting of blood stagnanattion and inability to anticoagulate due to poor BiVAD flows and continued (surgical) bleeding respectively. DISCUSSION Our initial findings from this on-going safety evaluation of our institutional adoption of a factor concentrate based, refractory bleeding algorithm represent an important therapeutic approach to major bleeding in a cardiac surgical setting where major bleeding is common. We noted a single episode of catastrophic thrombosis likely due to technical difficulties as post-dose INR was 1.6 and factor II activity was low at 60% (normal 72-132%), supportting a primary stasis/mechanical etiology. While (7/11) patients had a significant reduction in bleeding severity, 4/11 had ongoing hemorrhage requiring additional blood products (3/4) and rFVIIa (2/4). Low-dose PCCs barely normalize the FII activity levels and additional therapies were needed for 4/11 patients, presenting possible benefit from increased dose or repeated low-dose protocols. These preliminary data illustrate the need for careful evaluation of the results of algorithm implementation. Treatment failure may support the use of other procoagulant products such as FEIBA, if rFVIIa is required in addition to PCC for life threatening hemorrhage. Our presentation will include follow-up for thromboembolic events, blood product utilization, treatment failure rates and laboratory values for all 85 patients. Disclosures Off Label Use: Prothrombin complex concentrates and rFVIIa use in the perioperative/surgical and intensive care settings.. Ortel:Daiichi Sankyo: Consultancy; Instrumentation Laboratory: Consultancy; Instrumentation Laboratory: Research Funding; Eisai: Research Funding.

Description: Storage lesion–induced, red cell–derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV–induced coagulation activation, the ability of storage lesion–induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV–induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Description: Background: Three component prothrombin complex concentrates (PCCs) are currently approved to treat bleeding associated with Hemophilia B, and 4 component PCCs for warfarin reversal. They are increasingly used to treat bleeding in cardiac surgery patients. High dose PCC administration in the non-surgical setting has been associated with thrombotic complications, likely due to increased Factor II (Prothrombin) levels. We hypothesized that the use of low dose 3 factor PCCs (Profilnine, Grifols, CA) reduces bleeding following cardiopulmonary bypass without increasing the incidence of postoperative thrombotic complications. Methods: After IRB approval, a retrospective chart review of patients who underwent cardiac surgery and received Profilnine for refractory bleeding from February 2014 to February 2015. Demographic information, preoperative lab values, blood product use before and after PCC, use of rFVIIa, chest tube output and venous and arterial thromboembolic complications were collected. PCC was dosed according to an institutional perioperative bleeding algorithm (Figure 1) after approval by one of the senior authors (KG, JL or IW). Results: 114 patients received Profilnine¨ (Grifols, CA) for postoperative bleeding following cardiac surgery. The most common procedure that required PCC administration was elective aortic reconstruction (61, 57%) followed by acute aortic dissection (13, 12.1%). The mean dose of PCC was 15.8±7.1 IU. The chest tube output for the first hour after arriving in the ICU 150.8±214.0 ml and quickly decreased to 89.5±122.1 ml in the second hour. The mean total blood product transfusion was 12.4±9.9 units prior to PCC dose and 5±6.3 units post dose. The difference is represented in Figure 2 and was found to be statistically significant on a paired t-test. (p

Description: Abstract 1159 Background. Heparin induced thrombocytopenia (HIT) is an immune disorder where platelets are activated by antibodies to a complex of platelet factor 4 antigen and heparin (PF4/H), leading to thrombocytopenia (HIT) and, potentially, thrombosis (HITT). Documentation of anti-PF4/H antibodies in addition to the appropriate clinical findings is essential for making a diagnosis of HIT. In the post-cardiac bypass surgery setting, however, the frequency of elevated anti-PF4/H antibodies is high, whereas the frequency of clinical HIT or HITT is relatively uncommon. Several studies have shown that the presence of anti-PF4/H antibodies may be associated with an increased frequency of adverse outcomes, even in the absence of clinical HIT. The primary objective of this study was to determine the relationship between a positive PF4/H antibody in the postoperative setting with adverse thromboembolic events occurring up to 3 months after cardiac surgery. Methods. Patients undergoing cardiac surgery who were not going to be treated with chronic anticoagulation postoperatively were eligible for this multi-center prospective cohort study. Data were collected daily during hospitalization, and then at 30 and 90 days after surgery using a structured interview format with a standardized questionnaire that included all thrombotic as well as hemorrhagic events, platelet counts, and utilization of antithrombotics in the postoperative setting. The primary outcome variable was a composite endpoint comprising arterial and venous thrombotic events and other miscellaneous events compatible with HIT, as well as death attributable to an event compatible with HIT. Citrated plasma was collected at baseline, pre-discharge (∼4–5 days after surgery), and the 30 day follow-up visit, processed, and stored at −80°C for testing. Laboratory analyses included an anti-PF4/H antibody ELISA (GTI, Waukesha, WI) on all samples, a high-heparin confirmatory test on samples with an OD reading 〉0.40, and a serotonin release assay (SRA) on all postoperative samples with an OD reading 〉0.40. A sample size of 800 patients was estimated in order to detect a 3% difference in thromboembolic events assuming a 2 to 10-fold increase risk attributable to seropositivity. Chi-squared testing was used to test the relationship between the primary outcome and postoperative anti-PF4/H levels. Results. Informed consent was obtained from 1030 eligible patients between August 2006 and May 2009, and laboratory and follow-up data were analyzable for 1016 patients. Thirty-day antibody data were available for 888 patients, and fully complete laboratory and 90-day follow-up data were available for 815 patients. The average age was 62 ± 12 years, and 73% of participants were male. A total of 769 patients underwent coronary artery bypass grafting and 237 underwent valve repair or replacement. During the entire study period, there were 17 (1.7%) deaths, 46 thromboembolic events in 44 patients (4.3%), and 25 hemorrhagic events in 24 patients (2.4%). Using an OD cutoff of 0.40 for the ELISA, 339 patients (33.4%) were positive for anti-PF4/H antibodies at the time of discharge, and 630 patients (62%) were positive by day 30. There was no correlation between seropositivity for anti-PF4/H antibodies at the day of discharge or at day 30 and the primary outcome (p=0.47 and 0.73, respectively). Incorporating the high-heparin confirmatory step did not improve the relationship between positive antibody results and the primary outcome. Using a higher cut-off value for the anti-PF4/H antibody ELISA of 1.0 decreased the number of patients with positive results (96 patients at the time of discharge [9.4%] and 221 patients at the 30-day follow-up visit [21.8%]), but this did not improve the relationship between antibody positivity at the day of discharge or day 30 and the primary clinical endpoint, since most patients with the primary endpoint had an ELISA OD below 1.0 (75th percentile of 0.90; 90th percentile of 1.22). Similarly, using the SRA did not identify a relationship between assay results and outcome. Conclusions. The presence of anti-PF4/H antibodies in the postoperative setting following cardiac bypass surgery is not associated with an increased risk for thromboembolic complications. Positive anti-PF4/H results in this clinical setting should be interpreted with caution and only in the context of clinical suspicion for HIT. Disclosures: Ortel: Instrumentation Laboratory: Consultancy; Eisai: Research Funding; GSK: Research Funding. Welsby:CSL Behring: Speaker; CSL Behring: Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Principal Investigator. Heit:Daiichi Sankyo: Honoraria; Ortho-McNeil Janssen: Honoraria; Covidien: Honoraria.

Description: Abstract 1435 Platelet Factor 4 (PF4)/heparin (H) multimolecular complexes initiate an immune response that can ultimately lead to complications of Heparin-Induced Thrombocytopenia (HIT), a life-threatening prothrombotic disorder. We have previously shown that PF4:H multimolecular complexes assemble through non-specific electrostatic interactions and that other unrelated positively-charged proteins such as protamine (PRT) and lysozyme (Lys) exhibit similar biophysical interactions with heparin (ASH 2009; abstract # 1316). In these earlier studies, we showed that PRT/H and Lys/H, like PF4/H, show heparin-dependent binding over a range of heparin concentrations and that formation of multimolecular complexes occurs at distinct stoichiometric ratios (PRT/H at 3:1 and Lys/H at 5:1 molar ratios). We now extend these observations in vivo to show relevance to human disease. Using a murine immunization model, we show that mice injected with PRT/H and Lys/H multimolecular complexes, but not PRT alone, Lys alone or buffer, develop antigen-specific immune responses. In additional studies, we show that the immune response to PRT/H or Lys/H shares important biologic similarities with the humoral response to murine (m) PF4/H multimolecular complexes. Specifically, we demonstrate that antibody formation to PRT/H and Lys/H is heparin-dependent (occurs optimally at certain stoichiometric ratios) dose-dependent (requires threshold amounts of multimolecular complexes) and shows serologic transience. To demonstrate the clinical relevance of our findings, we examined patients undergoing cardiopulmonary bypass (CPB) for development of PRT/H antibodies. For these studies, we assayed the plasma from healthy subjects (n=45) and patients undergoing CPB (n=15) at three time points {baseline (BL), 5 days (5D) and 30 days (30D) after CPB} for the presence of PRT/H antibodies. As shown Figure 1A, plasma from normal subjects and patients undergoing CPB patients at BL and D5 displayed minimal reactivity in the PRT/H ELISA. However, by 30D, we observed that 4/15 patients (27%) developed significantly elevated levels of antibodies to PRT/H as compared to normals, or their respective samples obtained at baseline or 5D after surgery. Seropositive patients (filled symbols, n=4) as compared to seronegative patients (open symbols, n=3) recognized PRT/H and to some extent, PRT alone, but did not cross-react with other antigens including PRT/H, BSA, Lys, Lys/H or human PF4/H, Figure 1B; p

Description: Introduction: Early studies identified a prothrombotic state associated with novel coronavirus disease 2019 (COVID-19) as well as a survival benefit observed with heparin use in severely ill COVID-19 patients. There is a need to clarify the incidence of thromboembolic events (TEs), as well as major hemorrhage in COVID-19 patients in the context of an escalated-dose thromboprophylaxis strategy. Methods: We conducted a single center, retrospective study of 192 consecutive patients with COVID-19 admitted to the hospital between March 26th and May 8th 2020. Our study aimed to investigate the rates of thromboembolic events (TEs), hemorrhage and mortality of in the context of an escalated-dose thromboprophylaxis strategy implemented early in our experience with hospitalized patients with COVID-19. Results: The incidence of radiographically-confirmed venous thromboembolism (VTE) was 7.3% (n=14), and the rate of combined TEs was 12% (n=23). The rate of major hemorrhage was 6.3% (n=12), including one fatal CNS bleed. The overall mortality rate was 27.6% (n=53). Conclusion: The rate of VTE and overall TE was much lower than was reported in early studies, and the majority of VTEs occurred in ambulatory patients. Our data suggest that an escalated-dose thromboprophylaxis strategy may help reduce the incidence of inpatient VTEs, and that ambulatory COVID-19 patients may benefit from primary thromboprophylaxis. However, the risk of bleeding was not negligible, and must therefore be assessed on an individual and continual basis when using a more aggressive thromboprophylaxis strategy. Disclosures No relevant conflicts of interest to declare.