Eric Lian

Purification and characterization of l-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation

Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

Thrombocytopenic purpura and cardiomyopathy in pregnancy reversed by combined plasma exchange and infusion.

Abstract: Thrombocytopenia and hemolytic anemia have been seen with thrombotic thrombocytopenic purpura (TTP), HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), and hemolytic uremic syndrome (HUS). Differentiating between TTP, HUS, and HELLP syndrome is often difficult. Coexistence of TTP and HELLP is possible. Cardiomyopathy occurring in pregnancy can be idiopathic or associated with TTP. We describe a previously healthy woman who developed thrombocytopenia and hemolysis at 34 wk gestation. The patient underwent delivery after transfusion with platelets and RBCs. The suspicion of TTP was raised, and plasma exchange was begun by the third hospital day. On the seventh day of treatment, the patient developed shortness of breath, and an echocardiogram showed global hypokinesis with an ejection fraction of 25%. Plasma infusion, one unit q 4 h, was initiated in addition to the daily plasma exchange. The patient improved and her ejection fraction normalized. Plasma exchange and infusion and corticosteroids were gradually tapered off. von Willebrand factor (vWF) protease activity in the plasma upon transfer was completely deficient with the presence of inhibitor. This case illustrates that vWF protease assay and detection of inhibitor can be used for the diagnosis of TTP during pregnancy; and a severe cardiomyopathy in TTP can be reversed rapidly with combined plasma exchange and infusion.

Binding Properties and Inhibition of Platelet Aggregation by a Monoclonal Antibody to CD31 (PECAM-1)

CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1) is expressed on platelets, endothelial cells, myeloid cells, monocytes, and certain lymphocyte subsets. It has been shown that CD31 is involved in the homophilic and heterophilic cellular adhesion and leukocyte transendothelial migration, but little is known about the role of CD31 in platelet functions. Previously we have shown that monoclonal antibody (MAb) AAP2 produced in our laboratory bound to a 110-kD platelet antigen and gave an enhanced binding to activated platelet membrane. In this study we demonstrated that platelet lysate depleted of the antigen through adsorption by an AAP2-solidified affinity column was bound by MAbs against CD62 and CD42 but not by MAb 5.6E against CD31 or AAP2 on the immunoblot. Rabbit antibodies against CD31 completely inhibited the binding of AAP2 to platelets in the flow cytometry analysis. This indicates that AAP2 is specifically against CD31. 125I-labeled AAP2 bound to resting platelets with 5587 +/- 1765 sites/platelet and a Kd of 1 nmol/L and to thrombin-activated platelets with 17,625 +/- 4865 sites/platelet and a Kd of 0.24 nmol/L. Addition of 10 micrograms/mL AAP2 inhibited the aggregation induced by 4 mumol/L ADP by 78.6%, 6 mumol/L epinephrine by 79.4%, 1 microgram/mL collagen by 78.7%, and 0.25 U/mL thrombin by 29%. The platelet aggregation was completely restored when higher concentration of agonists were used. MAb 5.6E did not have any effect on platelet aggregation. These results suggest that AAP2 binds to a special epitope of CD31 on platelets and that CD31 is involved in platelet aggregation.

Plasma from a Patient with Thrombotic Thrombocytopenic Purpura Induces Endothelial Cell Apoptosis and Platelet Aggregation

1.1. Plasma Samples Thrombotic thrombocytopenic purpura (TTP) A TTP plasma sample was obtained from plasmais characterized pathologically by platelet pheresis during treatment through a blood bank. thrombi deposition and localized microvasThe diagnosis was based on clinical manifestations cular endothelial cell proliferation in arterioles and of thrombocytopenia, schizocytic hemolytic anecapillaries [1]. The pathogenesis of this syndrome mia, renal dysfunction, and fluctuating neurologic is still obscure. The disease process may be initiated symptoms. Plasma controls were obtained from by an abnormal interaction between plasma and healthy volunteers through venepuncture as well platelets and/or vascular endothelium, leading to as from two non-TTP patients undergoing plasmaplatelet thrombi formation with some fibrin in mipheresis. Informed consents were obtained and the crovessels [2]. The presence of factors in plasmas study approved by the Institutional Ethics Comfrom patients with TTP to induce platelet aggregamittee on Human Research. tion/agglutination has been reported [3–7]. Endothelial cell damage has also been speculated to be 1.2. Endothelial Cell Culture a crucial feature in some serial events that proceed to develop microvascular thrombotic lesions [8–10]. Human endothelial cell line ECV-304 from AmeriIn the present study, we demonstrate that subcan Type Culture Collection (Rockville, MD) is a stance(s) in the plasma from a patient with TTP spontaneously transformed immortal endothelial induces both endothelial cell apoptosis and platecell line established from the vein of an apparently let aggregation. normal human umbilical cord. Cells were maintained in M-199 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a huAbbreviations: dUTP, deoxyuridine triphosphate; HBSS, Hank’s balanced salt solution; TTP, thrombotic thrombocytopenic purmidified incubator at 378C with 5% CO2. pura; mAb monoclonal antibody; PI, propidium iodide; PE, R-Phycoerythrin; TdT, terminal deoxynucleotidyl transferase. 1.3. Cell Treatment This manuscript was presented at the International Society on Thrombosis and Hemostasis, Florence, Italy, June 1997. Corresponding author: Eric C.-Y. Lian, M.D., Sylvester Cancer 2310 viable cells in 1 ml were seeded in 6-well Center (D8-4), 1475 NW 12th Avenue, Miami, FL 33136. Tel: plates and grown to a confluence. Before use, cells 11 (305) 326 8041; Fax 11 (305) 324 3375; E-mail: ,elian@ mednet.med.miami.edu.. were rinsed with M-199 medium and then incu-

Sunitinib-induced microangiopathic hemolytic anemia with fatal outcome.

Sunitinib, a new vascular endothelial growth factor receptor inhibitor, has demonstrated activity in renal cell carcinoma and is now widely used in the palliative treatment of patients with metastatic renal cell carcinoma. It is generally well tolerated but has been associated with a low incidence of grade 3 and 4 toxicities including fatigue, diarrhea, anorexia, mucositis, skin toxicity, immune thrombocytopenic purpura, hypertension, hypothyroidism, cytopenias, and decreased cardiac ejection fraction. Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) is a rare condition that is severe and may be fatal. Several medications have been implicated in causing TTP-HUS including clopidogrel, mitomycin C, cisplatin. In this report, we describe a case of atypical HUS-microangiopathic hemolytic anemia during treatment with sunitinib in a patient with metastatic renal cell carcinoma. To our knowledge, this is the fourth case of microangiopathic hemolytic anemia associated with sunitinib described in the literature and the first case with fatal outcome despite treatment with plasmapheresis, dialysis, and withdrawal of sunitinib.

Warm-Antibody Autoimmune Hemolytic Anemia Developing after Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) and warm-antibody autoimmune hemolytic anemia (AIHA) are uncommon diseases. Although TTP has been increasingly described in association with autoimmune antibodies, there are very few reports of the association with autoimmune hematological conditions, including idiopathic thrombocytopenic purpura and AIHA. Here we describe a patient with classic manifestations of TTP, who was successfully treated with plasma exchange. A few weeks later, she developed warm-antibody AIHA, which responded promptly to prednisone.

A case of severe thrombotic thrombocytopenic purpura with concomitant Legionella pneumonia: review of pathogenesis and treatment.

Thrombotic thrombocytopenia purpura (TTP) is a severe multisystem disorder characterized by fever, microangiopathic hemolytic anemia, thrombocytopenia, neurologic symptoms, and impaired renal function. Platelet counts are usually diminished, whereas the bone marrow shows a large number of megakaryocytes indicating peripheral destruction and consumption of platelets. Coagulation studies in patients with TTP are normal or slightly elevated, which helps differentiate this entity from disseminated intravascular coagulation. The peripheral smear shows an abundance of schistocytes, reticulocytes, and, at times, nucleated red blood cells. Serum lactate dehydrogenase and indirect bilirubin are elevated as a result of mechanical destruction of red blood cells. Legionella pneumophila has been identified as a relatively common cause of both community-acquired and hospital-acquired pneumonia. An association between Legionella and TTP has only been cited once in the literature. Here we present a case of severe TTP with concurrent Legionella infection. Our patient presented with the classic clinical findings of TTP and an ADAMTS13 level of less than 5% associated with an inhibitor. After a 3-week treatment course with plasma exchange, steroids, and antibiotics, he had complete clinical recovery and his ADAMTS13 level increased to greater than 75%.

Purification and some properties of a protein obtained from normal human plasma which inhibits the platelet aggregation induced by thrombotic thrombocytopenic purpura plasma

Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of washed human platelets, which was inhibited by preincubation with normal plasma. Using salt fractionation, ion exchange chromatography, and preparative agarose gel electrophoresis, we purified a protein from normal plasma which inhibited the platelet aggregation caused by TTP plasma. On SDS polyacrylamide gel, the purified inhibitor gave a single band with a M.W. of 150,000. The antiserum against the purified protein neutralized the activity of the inhibitor and formed an identical precipitin line against normal and TTP plasma.

Electron microscopic study of platelet agglutination induced by thrombotic thrombocytopenic purpura plasma containing 37-KDa platelet agglutinating protein

It has been demonstrated that plasma from a patient with thrombotic thrombocytopenic purpura (TTP) and 37-KDa platelet-agglutinating protein (PAP p37) purified from the same plasma caused the agglutination of platelets from normal subjects as well as from the same patient after recovery without the requirement of extracellular Ca++ and fibrinogen. Experiments were designed to study the morphologic changes of platelets as a result of agglutination and the distribution of platelet receptors for PAP p37 under transmission electron microscope. Following incubation with TTP plasma or PAP p37 with stirring, platelets showed shape change, pseudopod formation, variable degrees of degranulation, dilatation of open canalicular systems and formation of agglutinates composed of a few to several hundred platelets. After platelets were incubated with TTP plasma or PAP p37 they were washed and further incubated with rabbit anti-PAP p37 serum without stirring followed by immuno-staining. Abundant electron dense reaction products were bound directly and randomly to the outer surface of the membrane of solitary platelets. When the reaction mixture was stirred, electron dense particles were also present between the platelet membranes in the agglutinates. No staining was observed in control experiments using normal plasma or non-immune rabbit serum. These results indicate that the TTP plasma containing PAP p37 causes agglutination, shape change, and variable degrees degranulation in platelets and that PAP p37 binds randomly to the outer surface of platelet membrane.