M-MDSCs mediated trans-BBB drug delivery for suppression of glioblastoma recurrence post-standard treatment.

We found that immunosuppressive monocytic-myeloid-derived suppressor cells (M-MDSCs) were more likely to be recruited by glioblastoma (GBM) through adhesion molecules on GBM-associated endothelial cells upregulated post-chemoradiotherapy. These cells are continuously generated during tumor progression, entering tumors and expressing PD-L1 at a high level, allowing GBM to exhaust T cells and evade attack from the immune system, thereby facilitating GBM relapse. αLy-6C-LAMP is composed of (i) drug cores with slightly negative charges condensed by cationic protamine and plasmids encoding PD-L1 trap protein, (ii) pre-formulated cationic liposomes targeted to Ly-6C for encapsulating the drug cores, and (iii) a layer of red blood cell membrane on the surface for effectuating long-circulation. αLy-6C-LAMP persistently targets peripheral, especially splenic, M-MDSCs and delivers secretory PD-L1 trap plasmids, leveraging M-MDSCs to transport the plasmids crossing the blood-brain barrier (BBB), thus expressing PD-L1 trap protein in tumors to inhibit PD-1/PD-L1 pathway. Our proposed drug delivery strategy involving intermediaries presents an efficient cross-BBB drug delivery concept that incorporates live-cell targeting and long-circulating nanotechnology to address GBM recurrence.

Protamine Test Dose: Impact on Activated Clotting Time and Circuit Integrity.

BACKGROUND: Recurrent observation of clot in the cardiopulmonary bypass circuit after the administration of a protamine test dose (PTD) prompted concern over the effects of PTDs on patient activated clotting times (ACTs). METHODS: Data were prospectively collected on 120 patients who had cardiopulmonary bypass while undergoing a variety of cardiac surgical procedures from July to October 2018 at the Toronto General Hospital, Toronto, Canada. ACTs were documented before cardiopulmonary bypass termination, after PTDs, and after protamine full doses. Statistical analysis was completed using a paired t test. RESULTS: The average PTD was calculated to be 36 ± 21 mg or 11% ± 7% of the full protamine dose of 367 ± 153 mg. This "test" dose ranged from 1% to 67% of full dose depending on the anesthetist. Post-PTD ACTs were widely variable. On average, there was a 40% ± 25% drop from the last ACT during cardiopulmonary bypass (650 ± 155 seconds) to the ACT after PTD (376 ± 153 seconds) (P < .0001). In fact, 81% ± 5% of the patients' post-PTD ACTs were lower than the institutional ACT standard of 480 seconds for safe cardiopulmonary bypass initiation. CONCLUSIONS: Regardless of the PTD, there is no reliable way to predict how a patient's ACT will respond to a PTD. Clot formation is possible and circuit integrity is at risk when pump suction devices are continuously in use during PTD administration. Therefore, the study investigators strongly recommend that the direct recovery of mediastinal shed blood into the pump circuit be discontinued before any amount of protamine is administered to the patient.

Optimal protamine dosing after cardiopulmonary bypass: The PRODOSE adaptive randomised controlled trial.

BACKGROUND: The dose of protamine required following cardiopulmonary bypass (CPB) is often determined by the dose of heparin required pre-CPB, expressed as a fixed ratio. Dosing based on mathematical models of heparin clearance is postulated to improve protamine dosing precision and coagulation. We hypothesised that protamine dosing based on a 2-compartment model would improve thromboelastography (TEG) parameters and reduce the dose of protamine administered, relative to a fixed ratio. METHODS AND FINDINGS: We undertook a 2-stage, adaptive randomised controlled trial, allocating 228 participants to receive protamine dosed according to a mathematical model of heparin clearance or a fixed ratio of 1 mg of protamine for every 100 IU of heparin required to establish anticoagulation pre-CPB. A planned, blinded interim analysis was undertaken after the recruitment of 50% of the study cohort. Following this, the randomisation ratio was adapted from 1:1 to 1:1.33 to increase recruitment to the superior arm while maintaining study power. At the conclusion of trial recruitment, we had randomised 121 patients to the intervention arm and 107 patients to the control arm. The primary endpoint was kaolin TEG r-time measured 3 minutes after protamine administration at the end of CPB. Secondary endpoints included ratio of kaolin TEG r-time pre-CPB to the same metric following protamine administration, requirement for allogeneic red cell transfusion, intercostal catheter drainage at 4 hours postoperatively, and the requirement for reoperation due to bleeding. The trial was listed on a clinical trial registry (ClinicalTrials.gov Identifier: NCT03532594). Participants were recruited between April 2018 and August 2019. Those in the intervention/model group had a shorter mean kaolin r-time (6.58 [SD 2.50] vs. 8.08 [SD 3.98] minutes; p = 0.0016) post-CPB. The post-protamine thromboelastogram of the model group was closer to pre-CPB parameters (median pre-CPB to post-protamine kaolin r-time ratio 0.96 [IQR 0.78-1.14] vs. 0.75 [IQR 0.57-0.99]; p < 0.001). We found no evidence of a difference in median mediastinal/pleural drainage at 4 hours postoperatively (140 [IQR 75-245] vs. 135 [IQR 94-222] mL; p = 0.85) or requirement (as a binary outcome) for packed red blood cell transfusion at 24 hours postoperatively (19 [15.8%] vs. 14 [13.1%] p = 0.69). Those in the model group had a lower median protamine dose (180 [IQR 160-210] vs. 280 [IQR 250-300] mg; p < 0.001). Important limitations of this study include an unblinded design and lack of generalisability to certain populations deliberately excluded from the study (specifically children, patients with a total body weight >120 kg, and patients requiring therapeutic hypothermia to <28°C). CONCLUSIONS: Using a mathematical model to guide protamine dosing in patients following CPB improved TEG r-time and reduced the dose administered relative to a fixed ratio. No differences were detected in postoperative mediastinal/pleural drainage or red blood cell transfusion requirement in our cohort of low-risk patients. TRIAL REGISTRATION: ClinicalTrials.gov Unique identifier NCT03532594.

Large cardiac wall hematoma with rapid growth: a case report of a potentially catastrophic complication during cardiac surgery.

BACKGROUND: Rapid growth of cardiac wall hematoma is a rare but potentially fatal complication of cardiac surgery. However, its pathophysiology and optimal management remain undefined. CASE PRESENTATION: Here we present a rare case of a large cardiac wall hematoma in the right ventricle during a thoracic aortic and valvular surgery. The hematoma expanded rapidly with epicardial rupture during cardiopulmonary bypass. We could establish non-surgical hemostasis and prevent further expansion of hematoma by early weaning of the cardiopulmonary bypass, followed by the administration of protamine and manual compression by hemostatic agent application. His postoperative recovery was uneventful and upon computed tomography analysis, the hematoma was observed to have absorbed completely at 1 week postoperatively. The patient is doing well 1 year after the surgery without evidence of recurrent cardiac wall hematoma on follow-up computed tomography. CONCLUSIONS: Cardiovascular surgeons should bear in mind this potentially catastrophic complication during cardiac surgery. Because of the vulnerability of the cardiac wall at the area of the hematoma, we believe that a hemostatic approach without sutures may be effective for this lethal complication.

Andexanet Alfa, the Possible Alternative to Protamine for Reversal of Unfractionated Heparin.

The recent shortage of protamine prompted an investigation of alternatives for reversal of unfractionated heparin. Heparin is an anticoagulant utilized in the hospital setting. Available options for anticoagulation include direct oral anticoagulants, vitamin K antagonists, thrombin inhibitors, low-molecular-weight heparins, and heparin. Protamine is the approved reversal agent for heparin with few alternatives under investigation. Although andexanet was designed as an antidote for apixaban and rivaroxaban, in vitro studies show that in a dose-dependent technique, andexanet had near full reversal of heparin, reversed anti-factor Xa activity, and neutralized anticoagulant effects of activated partial thromboplastin time and thrombin time induced by heparin.

Protamine and BSA-dextran complex emulsion improves oral bioavailability and anti-tumor efficacy of paclitaxel.

Food protein and polysaccharide complex emulsions are safe carriers of hydrophobic drugs and nutrients. To improve oral bioavailability and therapeutic/healthy efficacy of hydrophobic drugs and nutrients, herein, protamine (PRO), a cationic cell-penetrating peptide, was introduced into protein and polysaccharide complex emulsion. The electrostatic complex of PRO and BSA-dextran conjugate (BD) produced by Maillard reaction was used as emulsifier to produce oil-in-water emulsion (@BD/PRO). The BSA molecules were crosslinked at the oil-water interface by a heat treatment and the PRO chains were simultaneously anchored in the interface. BD emulsion (@BD) without PRO was produced for comparation. Paclitaxel (PTX), a hydrophobic antineoplastic drug, was encapsulated in the emulsions with 99% loading efficiency and 6.4% loading capacity. The emulsions had long-term stability. The bioavailability and H22 tumor inhibition efficacy of PTX@BD/PRO were 40% and 70% higher than those of PTX@BD, respectively, after oral administration in the mice. More importantly, orally administrated PTX@BD/PRO had the same anti-tumor efficacy as intravenously injected commercial PTX injection. No abnormality was observed in the main organs of the mice after consecutive oral administration of PTX@BD/PRO. This study indicates that @BD/PRO is an excellent carrier of hydrophobic drugs/nutrients and is suitable for long-term oral administration.

Safety and Efficacy of Protamine Administration for Prevention of Bleeding Complications in Patients Undergoing TAVR.

OBJECTIVES: The aim of this study was to evaluate whether protamine administration for heparin reversal after transcatheter aortic valve replacement (TAVR) reduces bleeding complications and affects patient outcomes. BACKGROUND: Occurrence of major bleeding complications in patients undergoing TAVR is associated with increased morbidity and mortality. METHODS: This study included 873 patients undergoing TAVR, of whom 677 received protamine for heparin reversal. Standard access management included the use of pre-closure devices, manual compression, and percutaneous transluminal angioplasty or implantation of a covered stent graft, if necessary. The study complied with Good Clinical Practice guidelines and was approved by the local ethics committee. Written informed consent was obtained from all patients. RESULTS: The primary endpoint, a composite of 30-day all-cause mortality and life-threatening and major bleeding, occurred less frequently in the protamine administration group (3.2%) compared with the control group (8.7%) (p = 0.003). This was driven mainly by lower rates of life-threatening and major bleeding in the protamine group (0.1% vs. 2.6% [p < 0.001] and 1.0% vs. 4.1% [p = 0.008], respectively). Furthermore, protamine administration resulted in a significantly shorter hospital stay (11.1 ± 5.8 days vs. 12.7 ± 7.8 days; p = 0.05). In the overall cohort, stroke was observed in 1.9% and myocardial infarction in 0.2% of patients, with no significant difference between the groups (p > 0.05). Multivariate analysis revealed that only protamine administration (odds ratio: 0.24; 95% confidence interval: 0.10 to 0.58; p = 0.001) and acute kidney injury (odds ratio: 5.82; 95% confidence interval: 2.02 to 16.77; p = 0.001) were independently associated with the primary endpoint. CONCLUSIONS: Protamine administration resulted in significantly lower rates of life-threatening and major bleeding complications compared with patients without heparin reversal. Occurrence of stroke and myocardial infarction was not increased by protamine administration.

Verification of a thrombus induction method at the target point inside the blood pump using a fibrinogen coating for a thrombus detection study.

Although the magnetically levitated centrifugal blood pump (mag-lev pump) is considered superior to other pumps in antithrombogenicity, thrombotic complications are still reported. Research into thrombus detection inside a mag-lev pump is very important for solving this problem. Our research group has already proposed a method to detect a thrombus inside a mag-lev pump in real time without an additional sensor, which is named the impeller vibration method. To efficiently advance our research with reproducibility, a preconditioning method to induce thrombus inside the pump was thought to be necessary. Therefore, this study aimed to develop a preconditioning method that induces thrombus formation. To verify this method, in vitro experiments for thrombus detection were performed. A mag-lev pump developed at Tokyo Institute of Technology was used. A fibrinogen solution was coated on the inner surfaces of the bottom housing to induce thrombus formation at the target point inside the pump. The thrombus is detected by utilizing the phenomenon that the phase difference between the impeller displacement and input current to the magnetic bearing increases when a thrombus is formed inside a pump. Five hundred mL of porcine blood anticoagulated with heparin sodium was circulated in the mock circuit, and protamine sulfate was administered. Flow rate (1 L/min), impeller vibrational frequency (70 Hz), and vibrational amplitude (30 µm) were set to constant. The experiment was terminated when the phase difference increased by over 2° from the minimum value. The experiments were performed in fibrinogen-coated (group F, n = 5) and non-coated pumps (group N, n = 5). In group F, thrombus formation was observed at the fibrinogen-coated point of the housing. In contrast, a relatively small thrombus was observed in varying locations such as the housing or the impeller in group N. Thrombus formation time (the time from when the phase difference takes the minimum value to when the experiment is terminated) was different between the two groups. The mean time was significantly shorter in group F (44 ± 29 minutes) than in group N (143 ± 38 minutes; p = 0.0019). Therefore, a preconditioning method that induced thrombus formation at the target point inside a blood pump was successfully developed.

Protamine sulfate use during tibial bypass does not appear to increase thrombotic events or affect short-term graft patency.

OBJECTIVES: While the use of protamine sulfate as a heparin reversal agent has been extensively reviewed in patients undergoing carotid endarterectomy and coronary artery bypass grafting, there is a lack of literature on protamine's effects on lower extremity bypasses. The purpose of this study was to determine the risk of protamine sulfate dosing after tibial bypass on thrombotic or bleeding events, including early bypass failure. METHODS: We performed a retrospective review of our institutional database for patients undergoing primary distal peripheral bypass from January 2009 through December 2015 (contralateral bypass was considered to be a new primary bypass). Primary endpoints include composite thrombotic events (myocardial infarction, stroke, amputation at 30 days and patency less than 30 days) and composite bleeding events (bleeding or transfusion). RESULTS: A total of 152 tibial or peroneal bypasses in 136 patients with critical limb ischemia were identified. Of these, 78 (57.4%) patients received protamine sulfate intraoperatively and 58 (42.6%) did not. There were no differences in composite thrombotic or hemorrhagic outcomes. Protamine use had no effect on the rates of perioperative MI (9.0% versus 3.5%, p = 0.20), stroke (1.3% versus 1.7%, p = 0.83), or perioperative mortality (5.1% versus 3.5%, p = 0.64). There was no significant difference in composite post-operative bleeding events (20.7% versus 14.1%, p = 0.31) or composite thrombotic events (17.2% versus 18.0%, p = 0.91). Patients who received protamine undergoing bypass with non-autogenous conduit had significantly higher-recorded median operative blood loss (250 mL versus 150 mL, p = 0.0097) and median procedure lengths (265 min versus 201 min, p = 0.0229). No difference in 30-day amputation-free survival was noted (91.0% versus 91.4%, p = 0.94). Follow-up Kaplan-Meier estimation did not demonstrate a difference in 30-day patency (91.7% versus 88.5%, p = 0.52). CONCLUSIONS: Heparin reversal with protamine sulfate after tibial or peroneal bypass grafting is not associated with higher cardiovascular morbidity, bypass thrombosis, amputation, or mortality. Additionally, there was no statistically significant difference in post-operative bleeding or thrombosis complications for patients who did not receive protamine, although the findings are suggestive of a potential difference in a more adequately powered study. Our results suggest that protamine sulfate is safe for intraoperative use without increased risk of thrombotic complications or early tibial bypass graft failure.

Developing spray-freeze-dried particles containing a hyaluronic acid-coated liposome-protamine-DNA complex for pulmonary inhalation.

The liposome-protamine-DNA complex (LPD) is an effective cationic carrier of various nucleic acid constructs such as plasmid DNA and small interfering RNA (siRNA). Hyaluronic acid coated on LPD (LPDH) reduces cytotoxicity and maintains the silencing effect of LPD-encapsulated siRNA. Herein, we aim to develop LPD- or LPDH-containing spray-freeze-dried particles (SFDPs) for therapeutic delivery of siRNA to the lungs. LPD- or LPDH-containing SFDPs (LPD- or LPDH-SFDPs) were synthesized and their structure and function as gene carriers were evaluated using physical and biological methods. The particle size of LPDH, but not of LPD, was constant after re-dispersal from the SFDPs and the amount of siRNA encapsulated in LPDH was larger than that in LPD after re-dispersal from the SFDPs. The in vitro pulmonary inhalation properties of LPDH-SFDPs and LPD-SFDPs were almost the same. The cytotoxicity of LPDH-SFDPs in human umbilical vein endothelial cells (HUVEC) was greatly decreased compared with that of LPD-SFDPs. In addition, Bcl-2 siRNA in LPDH-SFDPs had a significant gene silencing effect in human lung cancer cells (A549), whereas Bcl-2 siRNA in LPD-SFDPs had little effect. These results indicate that compared with LPD, LPDH is more useful for developing SFDPs for siRNA pulmonary inhalation.

The Inhibitory Effect of Protamine on Platelets is Attenuated by Heparin without Inducing Thrombocytopenia in Rodents.

Protamine sulfate (PS) is a polycationic protein drug obtained from the sperm of fish, and is used to reverse the anticoagulant effect of unfractionated heparin (UFH). However, the interactions between PS, UFH, and platelets are still not clear. We measured the platelet numbers and collagen-induced aggregation, P-selectin, platelet factor 4, ß-thromboglobulin, prostacyclin metabolite, D-dimers, activated partial thromboplastin time, prothrombin time, anti-factor Xa, fibrinogen, thrombus weight and megakaryocytopoiesis in blood collected from mice and rats in different time points.. All of the groups were treated intravenously with vehicle, UFH, PS, or UFH with PS. We found a short-term antiplatelet activity of PS in mice and rats, and long-term platelet-independent antithrombotic activity in rats with electrically-induced thrombosis. The antiplatelet and antithrombotic potential of PS may contribute to bleeding risk in PS-overdosed patients. The inhibitory effect of PS on the platelets was attenuated by UFH without inducing thrombocytopenia. Treatment with UFH and PS did not affect the formation, number, or activation of platelets, or the thrombosis development in rodents.

Activated platelet aggregation is transiently impaired also by a reduced dose of protamine.

Objectives: Protamine reduces platelet aggregation after cardiopulmonary bypass (CPB). We studied the inhibitory effect of a reduced protamine dose, the duration of impaired platelet function and the possible correlation to postoperative bleeding. Design: Platelet function was assessed by impedance aggregometry in 30 patients undergoing cardiac surgery with CPB at baseline, before protamine administration, after 70% and 100% of the calculated protamine dose, after 20 minutes and at arrival to the intensive care unit. Adenosine diphosphate (ADP), thrombin receptor activating peptide-6 (TRAP), arachidonic acid (AA) and collagen (COL) were used as activators. Blood loss was measured during operation and three hours after surgery. Results are presented as median (25th-75th percentile). Results: Platelet aggregation decreased markedly after the initial dose of protamine (70%) with all activators; ADP 89 (71-110) to 54 (35-78), TRAP 143 (116-167) to 109 (77-136), both p < .01; AA 25 (16-49) to 17 (12-24) and COL 92 (47-103) to 60 (38-81) U, both p < .05. No further decrease was seen after 100% protamine. The effect was transient and after twenty minutes platelet aggregation had started to recover; ADP 76 (54-106), TRAP 138 (95-158), AA 20 (10-35), COL 70 (51-93) U. Blood loss during operation correlated to aggregometry measured at baseline and after protaminization. Conclusions: Protamine after CPB induces a marked decrease in platelet aggregation already at a protamine-heparin ratio of 0.7:1. The impairment seems to be transient and recovery had started after 20 minutes.

Protamine may have anti-atherogenic potential by inhibiting the binding of oxidized-low density lipoprotein to LOX-1.

Oxidized low-density lipoprotein (ox-LDL) leads to atherosclerosis via lectin-like oxidized lipoprotein receptor-1 (LOX-1), one of the major receptor for ox-LDL. Inhibition of the binding of ox-LDL to LOX-1 decreases the proinflammatory and atherosclerotic events. The aim of the present study was to investigate whether protamine, a polybasic nuclear protein, interferes the binding of ox-LDL to LOX-1. Using sandwich ELISA with newly generated antibody, we measured the blocking effect of protamine on the binding of ox-LDL to LOX-1. Protamine dose-dependently inhibited the binding of ox-LDL to LOX-1. DiI-labeled ox-LDL uptake assay in two types of cultured human endothelial cells was performed with fluorescence microplate reader. Activation of extracellular-signal-regulated kinase (ERK)1/2 by ox-LDL was analyzed by immunoblotting. We found that protamine suppressed uptake of ox-LDL in endothelial cells and inhibited ERK1/2 activation by ox-LDL. These results suggest that protamine may possess anti-atherogenic potential by inhibiting ox-LDL binding to LOX-1 through electrostatic interactions.

Clinical Impact of Protamine Titration-Based Heparin Neutralization in Patients Undergoing Coronary Bypass Grafting Surgery.

OBJECTIVES: A hemostasis management system (HMS) is a point-of-care method for heparin and protamine titration. The authors hypothesized that protamine dosing over the HMS estimate would be associated with elevated activated clotting time (ACT), increased bleeding, and transfusion owing to protamine's anticoagulant activity. DESIGN: A retrospective cohort study. SETTING: Single-center university hospital. PARTICIPANTS: One hundred eighty-nine patients undergoing elective coronary artery bypass grafting surgery. INTERVENTIONS: Patients were stratified into 3 groups per ratio of actual total administered protamine versus the HMS-derived protamine estimate: (1) low-ratio (≤66% of HMS estimate), (2) moderate-ratio (66%-100% of HMS estimate), and (3) high-ratio (>100% of HMS estimate). MEASUREMENTS AND MAIN RESULTS: The primary endpoints were post-protamine ACT, and residual heparin levels on HMS among the 3 groups in addition to bleeding and transfusion. There were 54 (28.6%) patients in the low, 95 (50.3%) in the moderate, and 40 (21.2%) in the high-ratio group. The high-ratio patients who were overdosed with protamine relative to the HMS estimate had elevated ACT, international normalized ratio, and activated partial thromboplastin time values, and subsequently received more red blood cell (RBC) and non-RBC transfusions compared to lower-ratio groups. Higher actual/HMS protamine ratios were associated independently with post-protamine ACT elevations after adjustment for sex, body mass index (BMI), and cardiopulmonary bypass (CPB) time. CONCLUSION: Most patients received the protamine dose sufficiently close to the HMS estimate, but protamine dosing above the HMS estimate occurred in both obese and nonobese patients, which was associated independently with prolonged ACT after adjusting for sex, BMI, and CPB time.

Is 300 Seconds ACT Safe and Efficient during MiECC Procedures?

INTRODUCTION: The recommended minimum activated clotting time (ACT) level for cardiopulmonary bypass (CPB) of 480 seconds originated from investigations with bubble oxygenators and uncoated extracorporeal circulation (ECC) systems. Modern minimal invasive ECC (MiECC) systems are completely closed circuits containing a membrane oxygenator and a tip-to-tip surface coating. We hypothesized that surface coating and the "closed-loop" design allow the MiECC to safely run with lower ACT levels and that an ACT level of 300 seconds can be safely applied without thromboembolic complications. The aim of this study was to investigate the potential risks during application of reduced heparin levels in patients undergoing coronary surgery. METHODS: In this study, 68 patients undergoing coronary artery bypass grafting with MiECC were randomized to either the study group with an ACT target of 300 seconds or the control group with an ACT of 450 seconds. All other factors of MiECC remained unchanged. RESULTS: The study group received significantly less heparin and protamine (heparin [international units] median [min-max], Red_AC: 32,800 [23,000-51,500] vs. Full_AC: 50,000 [35,000-65,000] p < 0.001; protamine [international units], Red_AC: 18,000 [10,000-35,000] vs. Full_AC: 30,000 [20,000-45,000] p < 0.001). The ACT in the study group was significantly lower at the start of MiECC (mean ± standard deviation: study group 400 ± 112 vs. control group 633 ± 177; p < 0.0001). Before termination of CPB the ACT levels were: study group 344 ± 60 versus control group 506 ± 80. In both groups, the values of the endogenous thrombin potential (ETP) decreased simultaneously. None of the study participants experienced thromboembolic complications. CONCLUSION: Since no evidence of increased thrombin formation (ETP) was found from a laboratory standpoint, we concluded that the use of MiECC with a reduced anticoagulation strategy seems possible. This alternative anticoagulation strategy leads to significant reduction in dosages of both heparin and protamine. We can confidently move forward with investigating this anticoagulation concept. However, to establish clinical safety of ACT below 300 seconds, we need larger clinical studies.