Immunopurification of human plasma kininogens

Human plasma kininogens were purified by immunoadsorption on Sepharose columns using two different approaches, either removing protein impurities with the respective immunospecific polymers or applying an anti-kininogen-specific immunoadsorbent column. An anti-kininogen serum developed and investigated in this laboratory in earlier studies was used. This antiserum recognizes the native conformational determinants in the kininogen heavy chain, the common denominator in plasma kininogens, and reacts with three heterogeneous molecular forms of high mol. wt kininogen (mol. wts 103,000, 92,000 and 90,000) as well as with low mol. wt kininogen. Heterogeneity of kininogens was shown by SDS gel electrophoresis and immunoelectrophoresis. With the antibody-specific polymers the yield was 80-100% compared to 75% or lower when several consecutive immunoadsorption steps were applied to remove impurities. Both methods serve the purpose of preparing immunologically pure kininogens suitable for immunization.     

Effect of the novel direct factor Xa inhibitor DX-9065a on thrombin generation and inhibition among patients with stable atherosclerotic coronary artery disease

INTRODUCTION: Thrombin, a pluripotential effector enzyme with prothrombotic, proinflammatory, and mitogenic properties, plays a pivotal role in the pathobiology and clinical expression of atherothrombotic coronary artery disease. Existing anticoagulant drugs have not been shown to attenuate thrombin generation or activity consistently. We sought to investigate the effect of DX-9065a on thrombin generation and inhibition in patients with stable CAD. DX-9065a is a small-molecule, synthetic, direct inhibitor of factor Xa. MATERIALS AND METHODS: Peripheral venous blood samples were collected serially during and after administration of either placebo or 1 of 4 weight-adjusted regimens of DX-9065a, in 73 patients with stable CAD participating in the XaNADU-1B study. RESULTS AND CONCLUSIONS: At baseline, the median (25th, 75th) prothrombin activation fragment 1.2 (F1.2) level was 2.56 (2.05, 3.20) nmol/L, and the median d-dimer level was 0.26 (0.19, 0.38) mug FEU/L. There were significant relationships between measured plasma DX-9065a concentrations and both F1.2 (4.9% decrease for each doubling of DX-9065a) (P<0.0001) and d-dimer (5.5% decrease for each doubling of DX-9065a) (P=0.001). F1.2 was suppressed (below baseline) at 96 h after administration of DX-9065a. Coronary thrombotic events did not occur during or after study drug administration. DX-9065a, the first in a class of small-molecule, direct, selective and reversible factor Xa inhibitors, reduces thrombin generation and fibrin formation among patients with stable CAD. The effect is concentration-dependent and persists for at least 96 h following drug cessation, without biochemical or clinical evidence of rebound.     

Methods for studying fibrinolytic pathway components in human plasma

Methods have been developed to quantitatively measure the major plasma components of the human fibrinolytic system. Plasminogen is measured functionally with a 9M excess of streptokinase and immunochemically by rocket immunoelectrophoresis; the normal range was found to be 16.7-23.8 mg/dl and 17.4-21.6 mg/dl, respectively. Alpha 2-plasmin inhibitor is measured functionally and immunochemically; the normal range for the major plasma plasmin inhibitor was found to be 5.30-6.60 mg/dl by both methods. Plasminogen activator concentrations, as well as, free, and complexed, protease activities are measured along with plasmin generation rates by spectrophotometric assays with chromogenic substrates. Both activator and free protease activities are zero in plasma samples from normal human subjects. Plasmin generation rates are 0.25-0.47% with urokinase and 5.30-9.70% with streptokinase; these values are the percentages of the respective initial velocities of activation in purified systems.     

A Mr 90 000 surface polypeptide of Trypanosoma cruzi as a candidate for a Chagas' disease diagnostic antigen

Trypanosoma cruzi (Peru strain) trypomastigotes and epimastigotes were biosynthetically labeled with [35S]methionine, and the proteins were analyzed by two dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE analysis of the trypomastigotes showed a complex array of polypeptides with distinct clusters at Mr 88 000-92 000, isoelectric point (pI) 5.6-6.0, and Mr 72 000-76 000, pI 5.6-5.8. 2D-PAGE analysis of the epimastigotes did not show the cluster of polypeptides at Mr 90 000. When the trypomastigote lysate was reacted with sera from either mice or humans chronically infected with T. cruzi, 10-50 polypeptides were immunoprecipitated. Five of these polypeptides were recognized by all sera tested. However, of these polypeptides, only three, two of Mr 90 000 and one of Mr 150 000, can be identified by immunoreaction of [35S]methionine-labeled live parasites as surface proteins of T. cruzi trypomastigotes. 125I-iminobiotinylated surface proteins isolated from T. cruzi trypomastigotes were immunoprecipitated with the same series of sera as described above. Chagasic sera immunoprecipitated an antigen of Mr 90 000. The [35S]methionine and 125I-labeled Mr 90 000 polypeptides were not immunoprecipitated with sera from individuals infected with Leishmania donovani, Leishmania braziliensis, Leishmania tropica or Leishmania mexicana. These data indicate that a surface polypeptide of Mr 90000, pI 5.8-5.9 is a viable candidate for a Chagas' disease diagnostic antigen.