hirudin and Hemolysis

The use of extracorporeal membrane oxygenation (ECMO) support for cardiac and respiratory failure has increased in recent years. Improvements in ECMO oxygenator and pump technologies have aided this increase in utilization. Additionally, reports of successful outcomes in supporting patients with respiratory failure during the 2009 H1N1 pandemic and reports of ECMO during cardiopulmonary resuscitation have led to increased uptake of ECMO. Patients requiring ECMO are a heterogenous group of critically ill patients with cardiac and respiratory failure. Bleeding and thrombotic complications remain a leading cause of morbidity and mortality in patients on ECMO. In this review, we describe the mechanisms and management of hemostatic, thrombotic and hemolytic complications during ECMO support.

Topics: Anticoagulants; Blood Coagulation; Blood Coagulation Tests; Cardiac Output, Low; Cardiac Tamponade; Extracorporeal Membrane Oxygenation; Hemolysis; Hemorheology; Hemorrhage; Heparin; Hirudins; Humans; Peptide Fragments; Purpura, Thrombocytopenic, Idiopathic; Recombinant Proteins; Respiratory Insufficiency; Thrombosis; von Willebrand Diseases

Hirudin (Hir), a thrombin direct inhibitor, was used to modify a polyethylene glycol diglycidyl ether (PEG-DE) crosslinked regenerated silk fibroin (SF) material to improve hemocompatibility. Hemolysis characteristics, platelet adhesion, platelet activity, and plasma recalcification time were investigated using absorption spectrometry, scanning electron microscopy, MTT analysis, and the time counting method. Hirudin could be grafted evenly to the silk fibroin, and the modified material was resistant to hemolysis at ratios of less than 0.5%. Scanning electron microscopy and MTT results showed that platelet adhesion and aggregation activity decreased after modificaton with trace amounts of hirudin, compared with PEG-DE crosslinked and ethanol-treated silk fibroin film. Plasma recalcification of PEG-DE crosslinked silk fibroin film was slower than with ethanol-treated material, and this increased slightly after hirudin modification. Furthermore, L929, HAVSMC, and HUVEC cells adhered to the modified material, grew well, and possessed high proliferation activity on SF/Hir blend films. This study suggests that hirudin could improve the anticoagulation properties of regenerated silk fibroin materials.

Topics: Biocompatible Materials; Cell Division; Cells, Cultured; Cross-Linking Reagents; DNA; Epoxy Resins; Fibroblasts; Fibroins; Hemolysis; Hirudins; Human Umbilical Vein Endothelial Cells; Humans; Materials Testing; Microscopy, Electron, Scanning; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Plasma; Platelet Adhesiveness; Polytetrafluoroethylene

Thermosensitive injectable hydrogels have been used for the delivery of pharmacological and cellular therapies in a variety of soft tissue applications. A promising class of synthetic, injectable hydrogels based upon oligo(ethylene glycol) methacrylate (OEGMA) monomers has been previously reported, but these polymers lack reactive groups for covalent attachment of therapeutic molecules. In this work, thermosensitive, amine-reactive and amine-functionalized polymers were developed by incorporation of methacrylic acid N-hydroxysuccinimide ester or 2-aminoethyl methacrylate into OEGMA-based polymers. A model therapeutic peptide, bivalirudin, was conjugated to the amine-reactive hydrogel to investigate effects on the polymer thermosensitivity and gelation properties. The ability to tune the thermosensitivity of the polymer in order to compensate for peptide hydrophilicity and maintain gelation capability below physiological temperature was demonstrated. Cell encapsulation studies using an H9 T-cell line (CD4+) were conducted to evaluate feasibility of the hydrogel as a carrier for cellular therapies. Although this class of polymers is generally considered to be non-toxic, it was found that concentrations required for gelation were incompatible with cell survival. Investigation into the cause of cytotoxicity revealed that a hydrolysis byproduct, diethylene glycol monomethyl ether, is likely a contributing factor. While modifications to structure or composition will be required to enable viable cell encapsulation, the functionalized injectable hydrogel has the potential for controlled delivery of a wide range of drugs.

Topics: Amines; Animals; Anticoagulants; Cell Line; Cell Survival; Cell- and Tissue-Based Therapy; Copper; Drug Carriers; Drug Compounding; Drug Delivery Systems; Hemolysis; Hirudins; Hot Temperature; Humans; Hydrogels; In Vitro Techniques; Methacrylates; Mice; NIH 3T3 Cells; Peptide Fragments; Polyethylene Glycols; Polymethacrylic Acids; Recombinant Proteins

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this study was therefore to assess the effect of bioactive coatings on biocompatibility of Nitinol coils designed for the closure of intra-atrial communications. Nitinol coils (n = 10, each) and flat Nitinol bands (n = 3, each) were treated by basic coating with poly(amino-p-xylylene-co-p-xylylene) and then coated with either heparin, r-hirudin or fibronectin. Anti-thrombogenicity was studied in vitro in a dynamic model with whole blood by partial thromboplastin time (PTT), platelet binding and thrombin generation, respectively, and cytotoxicity by hemolysis. Endothelialisation was studied on Nitinol bands with human umbilical venous endothelial cells (HUVEC) by 3-(4,5-dimethylthiazole-2yl)-2,5-triphenyl tetrazolium (MTT) assay and immnuofluorescence analysis of Ki67, vinculin, fibronectin and von Willebrand Factor. Uncoated or coated devices did not influence hemolysis and PTT. r-Hirudin (but not heparin) and fibronectin coating showed lower platelet binding than uncoated Nitinol (p < 0.005, respectively). Heparin and r-hirudin coating reduced thrombin formation (p < 0.05 versus Nitinol, respectively). HUVEC adhesion, proliferation, and matrix formation decreased in the order: fibronectin coating > uncoated Nitinol > r-hirudin coating > heparin coating > basic coating. MTT assay corroborated these findings. In conclusion, r-hirudin and fibronectin coating, by causing no acute cytotoxicity, decreasing thrombogenicity and increasing endothelialisation improve in vitro biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.

Topics: Alloys; Animals; Biocompatible Materials; Blood Platelets; Cells, Cultured; Endothelium, Vascular; Fibronectins; Hemolysis; Heparin; Hirudins; Humans; Immunohistochemistry; Microscopy, Confocal; Microscopy, Electron, Scanning; Partial Thromboplastin Time; Sheep; Tetrazolium Salts; Thiazoles; Umbilical Veins

The effect of hirudin, a known inhibitor of thrombin, was evaluated for whole blood samples in terms of platelet deposition/adhesion to a non-biological test surface (tetrafluoroethylene-propylene copolymer), adenosine diphosphate (ADP) release and reduction in platelet count during laminar shear flow for a shear rate to 5680 s-1 (corresponding to a shear stress of about 150 dynes/cm2). Experiments were done in a cone-and-plate viscometer for samples of whole blood with and without the addition of hirudin. Whole blood samples containing hirudin showed about a 50% reduction in platelet surface coverage compared with blood samples not containing hirudin. Results on low-stress, shear-induced release of ADP showed that for shear rates of 2860 s-1 and above there was an increase in ADP release for the blood samples not containing hirudin compared with the hirudin-treated samples. However, no differences in haemoglobin leakage from red blood cells as well as residual platelet count following shear were observed between both types of blood samples.

Topics: Adenosine Diphosphate; Biocompatible Materials; Blood Flow Velocity; Hemolysis; Hirudins; Humans; Platelet Adhesiveness; Platelet Aggregation; Platelet Count; Polymers; Polytetrafluoroethylene; Viscosity

Dilute whole blood clots were prepared by addition of thrombin to blood diluted 1:10 in phosphate buffer. The pH of this buffer was 7.4 and the ionic strength was 0.084. Though the ionic strength was low, there was no hemolysis of red corpuscles due to the contribution to the osmotic gradient by plasma salts and proteins. In the standard assay the clot was formed by addition of thrombin at 4 degrees C then incubated at 37 degrees C. Retraction and lysis of these clots were inhibited by removal of platelets and by increasing concentrations of purified thrombin. Retraction and lysis were also inhibited by inactivation of any one of the following factors: gammaM globulin, complement components C4 and 3, and (in the case of lysis) plasminogen. Evidence that some of the above serum factors were adsorbed to the platelet membrane was obtained by aggregation of washed platelets by antisera to these factors (i.e. fibrinogen, gammaM, and C4 or C3). These platelets were not aggregated by antisera to other serum proteins (by albumin, transferrin, gammaG globulin). These and other studies suggested that platelets, thrombin, fibrinogen, gammaM globulin (cold agglutinin), complement components, and plasminogen influenced and facilitated retraction and lysis of clots. These studies also suggested that platelets and some of these factors were physically associated. Because of this physical association, and because of the fact that clot retraction is associated with aggregation and retraction of platelets, we extended the above observations to include a study of the effect of these same serum factors on serum-induced aggregation and retraction of washed platelets. (Other terms which have been in use in the past to describe serum-induced platelet aggregation and retraction have included those such as platelet "fusion" and "viscous metamorphosis," neither of which fully described the phenomena.)Platelet aggregation and retraction induced by serum was markedly accelerated by addition of increasing concentrations of thrombin and (or) cold agglutinin. Hirudin and antisera to gammaM globulin inhibited seruminduced aggregation and retraction of platelets. Reconstitution of inactivated serum with purified C4, 3, and 5 and thrombin restored its capacity to induce aggregation and retraction of platelets.Therefore, we postulated that platelet aggregation and retraction were necessary for clot retraction and that platelet aggregation and clot retraction facilitated clot lysis. More sp

Topics: Antibodies; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Buffers; Cell Membrane; Clot Retraction; Complement System Proteins; Fibrinogen; Fibrinolysin; Hemolysis; Hirudins; Humans; Hydrogen-Ion Concentration; Immunoglobulin M; Methods; Plasminogen; Platelet Adhesiveness; Thrombin; Time Factors; Venoms