Evaluating the Effect of Shear Stress on Graft-To Zwitterionic Polycarboxybetaine Coating Stability Using a Flow Cell

The effect of surface coatings on the performance of antifouling activity under flow can be influenced by the flow/coating interactions. This study evaluates the effect of surface coatings on antifouling activity under different flows for the analyses of coating stability. This was done by exposing DOPA-PCB-300/dopamine coated polydimethylsiloxane (PDMS) to physiological shear stresses using a recirculation system which consisted of dual chamber acrylic flow cells, tygon tubing, flow probe and meter, and perfusion pumps. The effect of shear stress induced by phosphate buffered saline flow on coating stability was characterized with differences in fibrinogen adsorption between control (coated PDMS not loaded with shear stress) and coated samples loaded with various shear stresses. Fibrinogen adsorption data showed that relative adsorption on coated PDMS that were not exposed to shear (5.73% ± 1.97%) was significantly lower than uncoated PDMS (100%, p \u3c 0.001). Furthermore, this fouling level, although lower, was not significantly different from coated PDMS membranes that were exposed to 1 dyn/cm2 (9.55% ± 0.09%, p = 0.23), 6 dyn/cm2 (15.92% ± 10.88%, p = 0.14), and 10 dyn/cm2 (21.62% ± 13.68%, p = 0.08). Our results show that DOPA-PCB-300/dopamine coatings are stable, with minimal erosion, under shear stresses tested. The techniques from this fundamental study may be used to determine the limits of stability of coatings in long-term experiments

The effects of continuous venovenous hemofiltration on coagulation activation

INTRODUCTION: The mechanism of coagulation activation during continuous venovenous hemofiltration (CVVH) has not yet been elucidated. Insight into the mechanism(s) of hemostatic activation within the extracorporeal circuit could result in a more rational approach to anticoagulation. The aim of the present study was to investigate whether CVVH using cellulose triacetate filters causes activation of the contact factor pathway or of the tissue factor pathway of coagulation. In contrast to previous studies, CVVH was performed without anticoagulation. METHODS: Ten critically ill patients were studied prior to the start of CVVH and at 5, 15 and 30 minutes and 1, 2, 3 and 6 hours thereafter, for measurement of prothrombin fragment F1+2, soluble tissue factor, activated factor VII, tissue factor pathway inhibitor, kallikrein–C1-inhibitor and activated factor XII–C1-inhibitor complexes, tissue-type plasminogen activator, plasminogen activator inhibitor type I, plasmin–antiplasmin complexes, protein C and antithrombin. RESULTS: During the study period the prothrombin fragment F1+2 levels increased significantly in four patients (defined as group A) and did not change in six patients (defined as group B). Group A also showed a rapid increase in transmembrane pressure, indicating clotting within the filter. At baseline, the activated partial thromboplastin time, the prothrombin time and the kallikrein–C1-inhibitor complex and activated factor XII–C1-inhibitor complex levels were significantly higher in group B, whereas the platelet count was significantly lower in group B. For the other studied markers the differences between group A and group B at baseline were not statistically significant. During CVVH the difference in the time course between group A and group B was not statistically significant for the markers of the tissue factor system (soluble tissue factor, activated factor VII and tissue factor pathway inhibitor), for the markers of the contact system (kallikrein–C1-inhibitor and activated factor XII–C1-inhibitor complexes) and for the markers of the fibrinolytic system (plasmin–antiplasmin complexes, tissue-type plasminogen activator and plasminogen activator inhibitor type I). CONCLUSION: Early thrombin generation was detected in a minority of intensive care patients receiving CVVH without anticoagulation. Systemic concentrations of markers of the tissue factor system and of the contact system did not change during CVVH. To elucidate the mechanism of clot formation during CVVH we suggest that future studies are needed that investigate the activation of coagulation directly at the site of the filter. Early coagulation during CVVH may be related to lower baseline levels of markers of contact activation

Anesthetic Management of Patients on ECMO

The management of a patient placed on extracorporeal membrane oxygenation (ECMO) is a team effort. The anesthesiology team plays an integral part during cannulation and oftentimes as well during decannulation. In addition, the management of a patient taken to the operating room on ECMO requires a degree of expertise. This chapter will review monitors, echocardiography, medications, fluid and blood management protocols, and ventilation strategies to help the anesthesiology team provide best care for this patient population

Dynamics of protein interactions with new biomimetic interfaces: toward blood-compatible biomaterials

2019 Fall.Includes bibliographical references.Nonspecific blood protein adsorption on the surfaces is the first event that occurs within seconds when a biomaterial comes into contact with blood. This phenomenon may ultimately lead to significant adverse biological responses. Therefore, preventing blood protein adsorption on biomaterial surfaces is a prerequisite towards designing blood-compatible artificial surfaces. This project aims to address this problem by engineering surfaces that mimic the inside surface of blood vessels, which is the only known material that is completely blood-compatible. The inside surface of blood vessels presents a carbohydrate-rich, gel-like, dynamic surface layer called the endothelial glycocalyx. The polysaccharides in the glycocalyx include polyanionic glycosaminoglycans (GAGs). This polysaccharide-rich surface has excellent and unique blood compatibility. We developed a technique for preparing and characterizing dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. The glycocalyx-mimetic surfaces were prepared by adsorbing heparin- or chondroitin sulfate-containing polyelectrolyte complex nanoparticles (PCNs) to chitosan-hyaluronan polyelectrolyte multilayers (PEMs). We then studied in detail the interactions of two important blood proteins (albumin and fibrinogen) with these glycocalyx mimics. Surface plasmon resonance (SPR) is a common ensemble averaging technique for detection of biomolecular interactions. SPR was used to quantify the amount of protein adsorption on these surfaces. Moreover, single-molecule microscopy along with advanced particle tracking were used to directly study the interaction of single-molecule proteins with synthetic surfaces. Finally, we developed a groundwork for a kinetic model of long-term protein adsorption on biomaterial surfaces. In the first chapter, we thoroughly summarize the important blood-material interactions that regulate blood compatibility, organize recent developments in this field from a materials perspective, and recommend areas for future research. In the second chapter, we report the preparation and characterization of dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. In the third chapter, we investigate how combining surface plasmon resonance, X-ray spectroscopy, atomic force microscopy, and single-molecule total internal reflection fluorescence microscopy provides a more complete picture of protein adsorption on ultralow fouling polyelectrolyte multilayer and polymer brush surfaces, over different regimes of protein concentration. In the fourth chapter, the interactions of two important proteins from the blood (albumin and fibrinogen) with glycocalyx-mimetic surfaces are revealed in detail using surface plasmon resonance and single-molecule microscopy. Finally, in the fifth chapter, the long-term protein interactions with different biomaterial surfaces are studied with single-molecule microscopy an

Silicon Membranes for Extracorporeal Membrane Oxygenation (ECMO)

In cases of severe lung or heart failure, extracorporeal membrane oxygenation (ECMO) is a life-saving therapy in which a patient’s blood is passed into a circuit outside of their body to provide respiratory support. The circuit’s main component is the membrane oxygenator that drives oxygen into the blood from a sweep gas source and removes excess carbon dioxide from the blood. At present, clinical use of ECMO is limited by its high risk profile, owing to two intertwined risks: thrombosis from the large circuit, and bleeding from the anticoagulation needed to prevent thrombosis. Improvements to the gas exchange efficiency and hemocompatibility of the oxygenator could enable the development of a longer-term supportive ECMO therapy, intended as a bridge-to-transplant or destination therapy for chronic lung failure. Here we describe a novel blood oxygenator concept based on parallel plate silicon membranes developed for high precision geometry, mechanical rigidity, and high efficiency membrane transport. Using these membranes, we create blood oxygenator prototypes consisting of arrays of silicon membranes, and endeavor to improve the efficiency and hemocompatibility of this concept.First, multiple types of silicon membranes were evaluated systematically for mechanical rigidity and oxygen exchange efficiency, indicators of suitability for a future oxygenator. The combination of a silicon micropore membrane (SµM) and a 5 µm-thick polydimethylsiloxane (PDMS) layer maximized both qualities, withstanding over 260 cmHg of applied pressure and producing 0.03 mL/min of O2 flux. These membranes were then assembled into prototype flow cells, and tested for in vitro and in vivo oxygenation, successfully yielding an oxygen permeability of 1.92 ± 1.04 ml O2 STP/min/m2/cmHg. From this benchmark, we then attempted to optimize the surface hemocompatibility of the Si-PDMS composite through application of multiple polyethylene glycol (PEG)-based coatings. Although successful application of PEG to the surfaces was demonstrated, none of the coatings appeared to reduce protein adhesion to the SµM -PDMS membranes. Finally, we inserted turbulence-inducing spacer meshes into the channels of the SµM-PDMS prototypes to disrupt the transport boundary layer adjacent to the membranes, with the goal of substantially improving oxygenation. Though a threefold increase in oxygen flux was observed in vitro with the spacer meshes, the disruptive turbulence resulted in thrombosis and channel occlusion within the channels despite heavy anticoagulation of the blood. In summary, the work in this dissertation demonstrates the successful construction and testing of SµM-PDMS oxygenator prototypes, laying the foundation for future work to optimize this concept and create a large-scale blood oxygenator that can expand the clinical use of this life-saving therapy

Liquid-Infused Surfaces for Anti–Thrombogenic Cardiovascular Medical Devices

Tethered-Liquid Perfluorocarbon (TLP) are a class of lubricant-infused surface coatings that, once infused with perfluorinated lubricants, show promise to reduce adverse reactions in medical devices implanted into the body such as reducing blood clot formation (thrombosis). A vapour phase silanisation reaction and the self-assembling properties of a fluorinated silane are exploited to form tethered perfluorocarbon (TP) layers containing nanostructured, bumpy aggregates. The vapour phase method compares favourably to the previously established liquid phase deposition method (LPD) to reproducibly create slippery coatings, without the need to control humidity conditions that often plague LPD methods. The TP layer retains perfluorinated lubricants when exposed to flow conditions seen in some medical devices, with a higher viscosity lubricant being more resistant to shear flow-induced depletion. TLP infused with more viscous lubricant, was equally effective in reducing adhesion of fibrin from human whole blood. Further in vitro biological assays revealed the wettability dependence of the intrinsic pathway of coagulation is applicable to TLP, based on factor XIIa activity and rate of plasma coagulation. Reduced adhesion of blood and loose packing of fibrin fibers on TLP coatings is attributed to the combined effects of low contact activation and enhanced mobility at the lubricant interface. Lubricant depletion dynamics under external flow was tested with a microfluidic device, combined with a dual-wavelength reflection interference contrast microscopy technique, enabling quantitative analysis of the lubricant on the nanoscale. The microfluidic platform also revealed reduced fibrin and platelet adhesion to TLP coatings exposed to blood flow. Optimised TLP coatings and greater understandings of anti-thrombogenic mechanisms open new avenues to assess TLP under blood flow for further translational development towards the next generation of blood-contacting medical devices

Role of thrombin in coronary artery bypass grafting

Thrombin is a multifunctional protease, which has a central role in the development and progression of coronary atherosclerotic lesions and it is a possible mediator of myocardial ischemia-reperfusion injury. Its generation and procoagulant activity are greatly upregulated during cardiopulmonary bypass (CPB). On the other hand, activated protein C, a physiologic anticoagulant that is activated by thrombomodulin-bound thrombin, has been beneficial in various models of ischemia-reperfusion. Therefore, our aim in this study was to test whether thrombin generation or protein C activation during coronary artery bypass grafting (CABG) associate with postoperative myocardial damage or hemodynamic changes. To further investigate the regulation of thrombin during CABG, we tested whether preoperative thrombophilic factors associate with increased CPB-related generation of thrombin or its procoagulant activity. We also measured the anticoagulant effects of heparin during CPB with a novel coagulation test, prothrombinase-induced clotting time (PiCT), and compared the performance of this test with the present standard of laboratory-based anticoagulation monitoring. One hundred patients undergoing elective on-pump CABG were studied prospectively. A progressive increase in markers of thrombin generation (F1+2), fibrinolysis (D-dimer), and fibrin formation (soluble fibrin monomer complexes) was observed during CPB, which was further distinctly propagated by reperfusion after myocardial ischemia, and continued to peak after the neutralization of heparin with protamine. Thrombin generation during reperfusion after CABG associated with postoperative myocardial damage and increased pulmonary vascular resistance. Activated protein C levels increased only slightly during CPB before the release of the aortic clamp, but reperfusion and more significantly heparin neutralization caused a massive increase in activated protein C levels. Protein C activation was clearly delayed in relation to both thrombin generation and fibrin formation. Even though activated protein C associated dynamically with postoperative hemodynamic performance, it did not associate with postoperative myocardial damage. Preoperative thrombophilic variables did not associate with perioperative thrombin generation or its procoagulant activity. Therefore, our results do not favor routine thrombophilia screening before CABG. There was poor agreement between PiCT and other measurements of heparin effects in the setting of CPB. However, lower heparin levels during CPB associated with inferior thrombin control and high heparin levels during CPB associated with fewer perioperative transfusions of blood products. Overall, our results suggest that hypercoagulation after CABG, especially during reperfusion, might be clinically important.Trombiini on hyytymisjärjestelmän keskeinen entsyymi, jolla on tärkeä osuus sepelvaltimotaudin synnyssä ja etenemisessä. Lisäksi trombiini on mahdollinen välittäjäaine hapen puutteeseen ja sen jälkeiseen verenkierron palautumiseen liittyvässä sydänlihasvauriossa. Trombiinin tuotanto ja veren hyytymistä aiheuttava vaikutus lisääntyvät voimakkaasti kehon ulkoisen verenkierron aikana. Aktivoitu proteiini C puolestaan on elimistön luonnollinen hyytymistä estävä aine, eli antikoagulantti, joka aktivoituu trombiinin vaikutuksesta. Tutkimuksen tarkoituksena oli selvittää, liittyvätkö trombiinin tuotanto tai proteiini C:n aktivoituminen sepelvaltimo-ohitusleikkauksen jälkeiseen sydänlihasvaurioon tai verenkierron muutoksiin. Trombiinin säätelyn selvittämiseksi tutkimme lisäksi, lisääntyykö trombiinin tuotanto tai sen vaikutukset potilailla, joilla on veren hyytymistä lisääviä tekijöitä, sekä vertasimme uutta hyytymistutkimusta [prothrombinase-induced clotting time (PiCT)] vakiintuneisiin laboratoriomenetelmiin kliinisesti sydänleikkauksessa käytetyn antikoagulantin, hepariinin, vaikutusten mittaamisessa. Tutkimusaineistona oli sata elektiivistä potilasta, joille tehtiin sepelvaltimo-ohitusleikkaus sydämen pysäytystä ja kehon ulkoista verenkiertoa käyttäen. Trombiinin tuotannon, trombiinin hyytymistä aiheuttavan vaikutuksen ja fibrinolyysin merkkiaineiden (protombiinin fragmenttien F1+2, liukoisten fibriinimonomeerikompleksien ja D-dimeerin) pitoisuudet lisääntyivät voimakkaasti leikkauksen aikana erityisesti sydämen verenkierron palauttamisen jälkeen. Tämän ns. reperfuusiovaiheen trombiinin tuotanto liittyi leikkauksen jälkeiseen keuhkoverenkierron vastuksen kohoamiseen ja leikkauksen jälkeen todettuun sydänlihasvaurioon. Proteiini C aktivoitui viiveellä trombiinin tuotantoon nähden ja aktivoitu proteiini C liittyi leikkauksen jälkeiseen verenkierrolliseen toipumiseen eri tavoin leikkausta edeltävästi ja sen eri vaiheissa. Vaikka perinnöllinen veren hyytymisalttius lisää yleisesti veritulppariskiä, leikkausta edeltävissä tutkimuksissa todettuun hyytymisalttiuteen ei liittynyt lisääntynyttä leikkauksen aikaista hyytymisaktivaatiota. Siitä huolimatta, että erilaisten hepariinin vaikutusten mittaamisessa käytettyjen laboratoriomenetelmien tulosten vastaavuus oli huono, osoitettiin heparinisaation asteen liittyvän kääntäen verensiirtojen tarpeeseen ja hyytymisaktivaatioon. Kaikkiaan tutkimuksessa osoitettiin hyytymisaktivaatiolla olevan haitallisia vaikutuksia sydänleikkauksen yhteydessä

A Tortuosity-Driven Microfluidic Thrombosis and Hemostasis Monitoring Device Applied in a Pediatric Critical Care Unit

Bleeding and thrombosis episodes in children are often encountered during ECMO/VAD. Pediatric patients on ECMO are known to have compromised platelet function and depleted plasma factors, making them susceptible to bleeding when anticoagulated with heparin. More concerning is the fact that hematology tests (ACT, aPTT, Anti-Xa) often report positive even when bleeding risk is present. TEG and ROTEM do provide greater information about whole blood coagulation, however a major deficiency with these tests is that they measure clotting characteristics under conditions that are not physiologically-relevant, thus limiting their ability to assess platelet function. To overcome these limitations, we developed a rapid and ultra-low volume tortuosity-activated microfluidic device for whole blood coagulation monitoring in pediatric critical care patients. The microfluidic device contains channels made of soft lithography that mimics a stenosed arteriolar blood vessel network and incorporates bio-rheological parameters known to induce clot formation such as blood vessel tortuosity and shear gradients. When the device is connected to a pressure sensor and whole blood is perfused it provides a tool to quantitatively determine clotting time. We demonstrate that the clotting times increase when clinically-relevant doses of heparin and bivalirudin is added to healthy blood samples. In addition, we validate these measurements by showing fluorescent images of that fibrin inhibition. Furthermore, we found that the clotting times were similar for devices coated with rat or human collagen, yet significantly different for horse collagen treated devices. Also, we saw that clotting time is dependent on platelet count. Lastly, we did not measure any microchannel occlusion when the device was treated with blood from pediatric ECMO patients. We then found that this lack of occlusion is due to poor platelet adhesion. We test if platelet adhesion can be restored with the addition of von Willebrand factor (VWF)

Extracorporeal membrane oxygenation in trauma patients with hypovolaemic shock

Lifesaving or Life-threatening? If the worst thing happens, and you get seriously injured in an accident, your body has a pronounced ability to immediately stop bleedings and start the process of healing. This unique capacity only works to a certain degree. Our body cannot by itself handle life-threatening bleedings from the heart, large blood vessels or severe injuries to highly vascularized organs. Transfusion of blood products, acute surgery and intensive care are necessary to support the body. In devastating injuries even this may not be enough and new ways of treating massive bleeding needs to be explored. Sometimes our body’s rescue system gets overloaded and starts to counteract the intended positive reaction. Excessive coagulation with a resulting occlusion of a blood vessel is one example. In that case anticoagulation is needed. Another example is if inflammation, the process necessary to start healing, derails. This may cause an overwhelming inflammation in the lungs that abolish their ability of saturating our blood. If the lungs take time off, a heart lung machine can buy time for the healing and save the life. A heart lung machine (ECMO) needs anticoagulation since the plastic tubings can cause clotting. Anticoagulation is associated with the risk of dangerous bleeding. If the system clots, and the machine shut down, a life saving procedure can suddenly change to a life-threatening situation. This Thesis highlights both sides of the hemostatic and anticoagulative coin. We have attacked the mentioned challenges from four different angles. In Article I venoarterial ECMO’s effect on central blood pressure was investigated. The reason for this is that we had found an unexpected effect of ECMO, during trauma resuscitation. A young girl’s severe liver bleeding suddenly could be controlled when ECMO was initiated because of lung failure. Swine were used in the study and we found that VA ECMO reduced the central venous pressure while mean arterial pressure was improved. In Article II a novel way of anticoagulation or “thromboprotection” in an ECMO system was evaluated. 3F7, an antibody that inhibits the activated form of coagulation factor XII was studied on rabbits connected to ECMO. 3F7 could prevent clotting in the ECMO system as effective as heparin but did not impair the hemostatic capacity and did not increase wound bleedings. In Article III venoarterial ECMO’s effect on rabbits in lethal traumatic bleeding shock was evaluated. Focus was on how the ECMO treatment affects central circulation, temperature, acid-base balance and the coagulation ability. ECMO efficiently increased the temperature, stabilized the circulation, improved the pH and ameliorated the hemostatic capacity. In Article IV Polyphosphate (PolyP), a substance that is released from activated platelets and that induces coagulation by activating FXII was investigated. Liver injuries in swine were treated with PolyP, Kaolin and a non-active substance. PolyP efficiently initiated thrombin formation and terminated bleeding as efficiently as Kaolin but with less inflammation