Senichiro Hashimoto

BOVINE PLASMA COLD-INSOLUBLE GLOBULIN: GROSS STRUCTURE AND FUNCTION*

Bovine plasma CIg, like human CIg, is a glycoprotein with a molecular weight of approximately 450,000 daltons and consists of two homologous subunits, the alpha and beta chains. These subunits are covalently linked through disulfide bridges in their carboxyl terminal domains. The carboxyl terminal regions are presumed to contain the fibrin-reactive transamidation site. The covalent incorporation of CIg into fibrin has been conclusively demonstrated by isolation of the S-carboxymethyl derivative of the CIg-fibrin-alpha chain complex and by determination of its terminal amino acid sequences. Cold-insoluble globulin has been shown to exert a stimulatory effect on the urokinase-mediated activation of bovine plasminogen to plasmin.

Isolation and characterization of thrombomodulin from bovine lung.

Bovine thrombomodulin was isolated from the lung by Triton X extraction, affinity chromatography on diisopropyl phosphate-thrombin-agarose, and gel filtration on Ultrogel AcA-44. The final preparation was purified 6000-fold from the membrane extract with a yield of 21%. It showed apparent Mr of 78,000 and 105,000, before and after reduction, respectively, on polyacrylamide gel electrophoresis in SDS. The activity of the thrombomodulin was stable under the conditions of 1% SDS, 8 M urea, pH 2 and 10, and heat treatment at 60 degrees C for 30 min, but was unstable against treatment with 2-mercaptoethanol. Activation of protein C by thrombin in the presence of the thrombomodulin depended on Ca2+, and an equimolar complex formation between thrombin and thrombomodulin was required for the maximum rate activation. The rate of protein C activation by thrombin was increased 900-fold by thrombomodulin. Thrombomodulin inhibited the thrombin-induced fibrinogen clotting and platelet activation. However, it did not affect the inhibition of thrombin by antithrombin III with or without heparin, a protein C inhibitor or several synthetic inhibitors. These properties of bovine thrombomodulin were similar to those of rabbit thrombomodulin reported earlier.

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate, and its mechanism.

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VIII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content of dextran sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.

Synthesis and secretion of protein C inhibitor by the human hepatoma-derived cell line, Hep G2

The site of synthesis of protein C inhibitor, a recently identified human plasma inhibitor against activated protein C, is not known. We have studied the production and secretion of protein C inhibitor by an established human liver cell line derived from hepatocellular carcinoma (Hep G2). The concentration of protein C inhibitor, as measured by a specific radioimmunoassay, increased in the medium of Hep G2 cells with time. There was no evidence for a significant intracellular pool of this protein. Protein C inhibitor secreted from Hep G2 cells (G2 protein C inhibitor) inhibited the activity of purified activated protein C in a functional assay. De novo synthesis of protein C inhibitor was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 h of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by SDS-polyacrylamide gel electrophoresis showed a peak of radioactivity corresponding to Mr 57 000. These results indicate that the liver is a site of protein C inhibitor production.

Identical binding site on human platelets for von Willebrand factor and bovine platelet aggregating factor

Abstract Identity of the binding site on human platelets for von Willebrand factor (vWF) and bovine platelet aggregating factor (PAF) was investigated. Although the 125I labelled bovine PAF alone bound to human platelets fixed with paraformaldehyde, the binding of 1251 labelled vWF was dependent on the presence of ristocetin. The binding of 125I labelled bovine PAF was not inhibited by the presence of human plasma with ristocetin (at a final concentration of 1.5 mg/ml) or vWF alone, but was inhibited by vWF with ristocetin (at a final concentration of 4 mg/ml). Moreover, the binding of 125I labelled vWF with ristocetin was inhibited by the presence of bovine PAF. These results suggest that the binding sites on human platelets for vWF and bovine PAF are identical.

The influence of 2-mercaptoethanol on von willebrand factor and bovine platelet aggregating factor

Abstract The influence of a reducing agent, 2-mercaptoethanol (2-ME), on the activity of von Willebrand factor (vWF) and bovine platelet aggregating factor (PAF) was studied. 2-ME was found to inhibit the human platelet agglutination induced by vWF plus ristocetin or by PAF alone. However, binding ability of vWF in the presence of ristocetin or PAF to the platelets was not affected by the addition of 2-ME at a high concentration. Both vWF and PAF were found to be dissociated into subunits in the presence of 2-ME at concentrations inducing the inhibition of platelet agglutination. These findings indicate that although each subunit of vWF or PAF is capable of binding to platelets, a macromolecular constitution of vWF or PAF subunits, linked with disulfide bonds, is required for inducing the platelet agglutination.

A simple technique for purification of fibrinogen from plasma by affinity chromatography on ristocetin-agarose

Abstract Ristocetin-agarose adsorbs fibrinogen and other proteins from plasma. After incubation of ristocetin-agarose with plasma, the agarose packed in a column was washed with buffer containing 1M NaCl and treated with 8M urea. Fibrinogen eluted in urea fraction has a clottability of 95–97% and is not contaminated with cold-insoluble globulin, plasminogen, Factor VIII, Factor XIII, immunoglobulin G and other plasma proteins. Ristocetin-agarose is a suitable adsorbent for the purification of fibrinogen from plasma by affinity chromatography.

Vancomycin as well as ristocetin facilitates von Willebrand factor binding to platelets

A glycopcptidc antibiotic, ristocetin, induces platelet agglutination in the presence of von Wlllebrand factor (vWF), a part of Factor VIII complex (1) The role of ristocetin in the platelet agglutination has not yet been fully clarified. Recently, ristocctin was found to facilitate vWF binding to platelets in a concentration-dependent manner (2-6). Vancomycin, which is a structural analog of ristocetin, does not agglutinate as ristocatin does, but inhibits specificaliy ristocetin/vWF-induced platelet agglutination (7). As to the inhibltion mechanism of vancomycin, several researchers have proposed that vancomycin competes with ristocetin for binding to platelet membrane (8, 9). However, if vancomycin could interfere with the binding of vWF to platelets, then ristocetin/vWF-induced platelet agglutination might be inhibited. In the present study, we have examined the effect of vancomycin on the interaction of vWF with fixed and washed platelets in the absence or presence of ristocetln.