-
Biochimica Et Biophysica Acta Nov 1995The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did...
The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by alpha v beta 3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C.
The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6-10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 microM), and the morphologic alteration was also less pronounced with BAPTA(15 microM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [3H]IP1 of HUVECs preloaded with [3H]myoinositol. This IP1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against alpha v beta 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin alpha v beta 3 on HUVECs, causing the subsequent activation of the endogenous G-protein coupled phospholipase C.
Topics: Ancrod; Calcium; Cell Adhesion; Endothelium, Vascular; Fibrin; GTP-Binding Proteins; Humans; Inositol Phosphates; L-Lactate Dehydrogenase; Morphogenesis; Pertussis Toxin; Phosphatidylinositols; Receptors, Vitronectin; Signal Transduction; Type C Phospholipases; Virulence Factors, Bordetella
PubMed: 7488643
DOI: 10.1016/0167-4889(95)00099-e -
Texas Heart Institute Journal 1995Anticoagulation in the form of intravenous heparin is used after coronary angioplasty to prevent thrombosis. Ancrod, a rapid-acting defibrinogenating agent, has been...
Anticoagulation in the form of intravenous heparin is used after coronary angioplasty to prevent thrombosis. Ancrod, a rapid-acting defibrinogenating agent, has been used in various clinical settings that require anticoagulation. We present the use of ancrod after percutaneous transluminal coronary angioplasty in a patients with heparin-induced thrombopathia.
Topics: Aged; Ancrod; Angioplasty, Balloon, Coronary; Anticoagulants; Blood Coagulation Disorders; Female; Heparin; Humans
PubMed: 8605439
DOI: No ID Found -
Anesthesiology Mar 1994
Normal activated clotting time despite adequate anticoagulation with ancrod in a patient with heparin-associated thrombocytopenia and thrombosis undergoing cardiopulmonary bypass.
Topics: Aged; Ancrod; Blood Coagulation; Cardiopulmonary Bypass; Fibrin; Heparin; Humans; Male; Thrombocytopenia; Thrombophlebitis; Time Factors; Whole Blood Coagulation Time
PubMed: 8141466
DOI: 10.1097/00000542-199403000-00029 -
Blood Dec 1993The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well...
The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.
Topics: Ancrod; Animals; Endotoxins; Fibrin; Humans; Infusions, Intravenous; Kinetics; Male; Plasminogen Activator Inhibitor 1; Rabbits; Recombinant Proteins; Thrombin; Time Factors; Tissue Plasminogen Activator
PubMed: 8260701
DOI: No ID Found -
Blood Dec 1993A new type of A alpha Glu-11 to Gly substitution has been identified in a congenitally abnormal fibrinogen, fibrinogen Mitaka II, derived from a 14-year-old female...
A new type of A alpha Glu-11 to Gly substitution has been identified in a congenitally abnormal fibrinogen, fibrinogen Mitaka II, derived from a 14-year-old female suffering from easy bruising since childhood. Plasma of the patient and fibrinogen purified therefrom were found to clot slowly by thrombin but in a normal fashion by ancrod, a thrombin-like snake venom enzyme. The ancrod-clotted fibrin gels were normally solid and turbid, whereas the thrombin-clotted gels were initially fragile and transparent but became gradually normalized during further incubation. On reverse-phase high-performance liquid chromatography, there was an additional peptide group eluted distinctly later than the corresponding normal fibrinopeptide A in the clot-liquor of the patient's samples. Sequence analysis of these aberrant peptides and isolated A alpha chains of the patient's fibrinogen showed that Glu at position 11 of the abnormal A alpha chain had been replaced by Gly. Studies using 125I-labeled thrombin showed that the binding with thrombin was evidently reduced for her fibrinogen and the aberrant fibrinopeptide A as compared with that for the normal controls, indicating that A alpha Glu-11 may be critical for the fibrinogen-thrombin interaction. Indeed, A alpha Glu-11 of fibrinogen has recently been proposed to stabilize the local conformation, including the beta-turn, and to form a salt bridge between its side-chain carboxyl group and the guanidino group of Arg-173 of thrombin based on crystallographic analyses using analogs of fibrinopeptide A complexed with thrombin (Stubb et al, Eur J Biochem 206:187, 1992 and Martin et al, J Biol Chem 267:7911, 1992).
Topics: Adolescent; Amino Acid Sequence; Ancrod; Female; Fibrinogens, Abnormal; Fibrinopeptide A; Glutamates; Glutamic Acid; Glycine; Humans; Kinetics; Male; Molecular Sequence Data; Point Mutation; Thrombin
PubMed: 7903170
DOI: No ID Found -
The Journal of Experimental Medicine Dec 1993Although "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and...
Although "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and chronic inflammatory responses. Early interactions between implants and inflammatory cells are probably mediated by a layer of host proteins on the material surface. To evaluate the importance of this protein layer, we studied acute inflammatory responses of mice to samples of polyester terephthalate film (PET) that were implanted intraperitoneally for short periods. Material preincubated with albumin is "passivated," accumulating very few adherent neutrophils or macrophages, whereas uncoated or plasma-coated PET attracts large numbers of phagocytes. Neither IgG adsorption nor surface complement activation is necessary for this acute inflammation; phagocyte accumulation on uncoated implants is normal in hypogammaglobulinemic mice and in severely hypocomplementemic mice. Rather, spontaneous adsorption of fibrinogen appears to be critical: (a) PET coated with serum or hypofibrinogenemic plasma attracts as few phagocytes as does albumin-coated material; (b) in contrast, PET preincubated with serum or hypofibrinogenemic plasma containing physiologic amounts of fibrinogen elicits "normal" phagocyte recruitment; (c) most importantly, hypofibrinogenemic mice do not mount an inflammatory response to implanted PET unless the material is coated with fibrinogen or the animals are injected with fibrinogen before implantation. Thus, spontaneous adsorption of fibrinogen appears to initiate the acute inflammatory response to an implanted polymer, suggesting an interesting nexus between two major iatrogenic effects of biomaterials: clotting and inflammation.
Topics: Ancrod; Animals; Biocompatible Materials; Cell Adhesion; Chemotaxis, Leukocyte; Fibrin; Fibrinogen; Humans; In Vitro Techniques; Inflammation; Mice; Mice, Inbred BALB C; Phagocytes; Polyethylene Terephthalates
PubMed: 8245787
DOI: 10.1084/jem.178.6.2147 -
The Biochemical Journal Sep 1993The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma....
The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.
Topics: Amino Acid Sequence; Ancrod; Base Sequence; Cloning, Molecular; Crotalid Venoms; DNA; Molecular Sequence Data; Polymerase Chain Reaction; Restriction Mapping; Sequence Analysis; Sequence Analysis, DNA
PubMed: 8373353
DOI: 10.1042/bj2940387 -
BMJ (Clinical Research Ed.) May 1993
Topics: Ancrod; Heparin; Humans; Thrombocytopenia
PubMed: 8518620
DOI: 10.1136/bmj.306.6889.1410-a -
Blood Sep 1992
Topics: Ancrod; Clinical Trials as Topic; Humans; Plasminogen Inactivators; Thrombosis
PubMed: 1520890
DOI: No ID Found -
Blood May 1992
Topics: Ancrod; Humans; Thrombosis
PubMed: 1571563
DOI: No ID Found