A monoclonal antibody that inhibits activation of human Hageman factor (factor XII)

A monoclonal antibody to human Hageman factor (HF, factor XII) was derived from BALB/c mouse spleen cells fused with NS-1 mouse myeloma cells. This antibody, purified from ascites fluid, reacted with HF to inhibit the activation of HF, purified or in normal pooled plasma, as measured by a coagulation assay. The antibody did not inhibit the coagulant activity of activated HF. The antibody also inhibited the generation of amidolytic activity in HF-ellagic acid mixtures, but failed to inhibit the amidolytic properties of the carboxy-terminal fragment of HF (HFf). Amidolytic activity, absent in an HF-monoclonal antibody mixture, was generated upon treatment with insoluble trypsin. Monoclonal antibody, bound to CNBr Sepharose 4B gel (Pharmacia Fine Chemicals, Piscataway, NJ), reversibly bound HF in plasma or in buffer, without activating it. HF was then eluted with 4 mol/L guanidine HCI. The passage of 125I-labeled HF enzymatically cleaved by trypsin through a column of monoclonal antibody-CNBr Sepharose 4B gel resulted in flow- through of HFf with a molecular weight (mol wt) of 30,000 and HF fragments of mol wt 12,000. Elution with 4 mol/L guanidine HCI yielded several HF fragments (mol wt 80,000, 52,000, and 40,000) but not HFf. These data suggest that the single determinant recognized by the murine monoclonal antibody is not on HFf, but rather on the amino-terminal fragment thought to be involved in the binding activity of HF. The monoclonal anti-HF bound to CNBr-activated Sepharose 4B gel could be used to artificially deplete plasma samples of HF.     

A monoclonal anti-human platelet antibody: a new platelet aggregating substance

A monoclonal anti-human platelet antibody, TP82, is described, which caused irreversible aggregation of platelets in association with the release of adenosine triphosphate or [14C] serotonin, and which inhibited ristocetin-induced agglutination. Immunofluorescence assay showed that the antibody binds to platelets, megakaryocytes, and common acute lymphoblastic leukemia cells. The antibody (IgG1) immunoprecipitated a polypeptide of 23,000 daltons with an isoelectric point of about 7.0. The aggregation induced by the purified antibody and/or F(ab')2 fragments occurred in platelet-rich plasma and with washed platelets, but not with formalin-fixed washed platelets. TP82- induced aggregation was completely inhibited by disodium ethylendiaminotetraacetate, diltiazem, W-7, PGE1, and several metabolic inhibitors. At a concentration of apyrase or CP/CPK, which inhibited adenosine 5-diphosphate-induced aggregation. TP82-induced aggregation was only partially affected. Thrombin was not required for the antibody- mediated effects, since two thrombin inhibitors failed to block the reaction. The antibody, at least at a high concentration, induced platelet aggregation by a mechanism almost independent of thromboxane A2 formation, since cyclooxygenase inhibitors had little inhibitory effect on aggregation. TP82 monoclonal antibody is a new platelet- aggregating substance that interacts with a low-molecular-weight binding site on the platelet membrane.     

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.     

Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase.

Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor beta subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor beta subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.     

A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage

To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.     

Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody

A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.     

A monoclonal antibody to exfoliated surface vesicles that recognizes a membrane-associated erythroid burst-promoting activity

A monoclonal antibody (MoAb) recognizing a membrane-associated erythroid burst-promoting factor was prepared by immunizing BALB/c mice with plasma membrane-derived vesicles exfoliated from lymphocytes under serum-free conditions. Hybrids secreting antibody reactive with lymphocyte plasma membranes were formally cloned and IgG was purified from monoclonal supernatants or from BALB/c mouse ascites fluid. Two clones (D3-E4 and D3-G9) were found to suppress burst forming unit- erythroid (BFU-E) proliferation when added directly to serum-free human marrow culture. Inhibition to a level of 100% was observed in a dose- dependent fashion over a wide range of antibody concentrations (0-200 micrograms/mL). Neither antibody altered the proliferation of colony forming unit-granulocyte macrophage (CFU-GM) or colony forming unit- granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM) progenitor cells in human bone marrow culture. The D3-E4 clone was found to produce an IgG1 antibody which adsorbs an erythroid burst-promoting activity (BPA) from supernatants of, and octylglucoside extracts of shed vesicles present in, serum-free, lymphocyte conditioned medium (LCM), and which recognizes a vesicular protein of Mr approximate 30,000 on immunoblots of membrane proteins electrophoresed on sodium dodecyl sulfate (SDS)/polyacrylamide and transferred to nitrocellulose. In contrast, the D3-G9 clone was found to produce an IgG1 cytotoxic antibody. These antibodies will be important to the study of cell-cell and growth factor-cell interactions in vitro.     

An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.     

The immunodepletion of factor VII from human plasma using a monoclonal antibody

A murine hybridoma cell line which secretes monoclonal antibody to factor VII has been prepared to facilitate the immunodepletion of this clotting factor from plasma. Specific monoclonal antibody was purified from mouse ascites tumours by protein A-Sepharose chromatography and shown to be of the IgG1 immunoglobulin subclass. On immunoblotting, this antibody reacted with a single protein band identical to purified factor VII. The purified monoclonal antibody was coupled to Sepharose 4B and was used to immuno-deplete factor VII from pooled normal human plasma. The prothrombin time of plasma immunodepleted in this way was 35 s compared to 12 s for the starting plasma. Specific factor assays of the immunodepleted plasma showed factor VII activity to be less than 1% while the levels of the other clotting factors were unchanged. The immunodepleted plasma was equivalent to severe congenital factor VII deficient plasma as a substrate for factor VII assays. Bound factor VII could be eluted from the immunoaffinity column with citrate buffer, pH 6.0, with good recovery.     

Initiation of plasma prorenin activation by Hageman factor-dependent conversion of plasma prekallikrein to kallikrein.

Plasma prorenin is an inactive form of renin (EC 3.4.99.19) that can be converted to active renin in acid-treated plasma by an endogenous serine protease that is active at alkaline pH (alkaline phase activation). To identify this enzyme we first tested the ability of Hageman factor fragments, plasma kallikrein (EC 3.4.21.8), and plasmin (EC 3.4.21.7) to activate prorenin in acid-treated plasma. All three enzymes initiated prorenin activation; 50% activation was achieved with Hageman factor fragments at 1 microgram/ml, plasma kallikrein at 2-4 microgram/ml, or plasmin at 5-10 microgram/ml. We then showed that the alkaline phase of acid activation occurred normally in plasminogen-free plasma but was almost completely absent in plasmas deficient in either Hageman factor or prekallikrein; alkaline phase activation was restored to these latter plasmas when equal parts were mixed together. Therefore, both Hageman factor and prekallikrein were required for alkaline phase activation to occur. We then found that, although plasma kallikrein could activate prorenin in plasma deficient in either Hageman factor or prekallikrein, Hageman factor fragments were unable to activate prorenin in prekallikrein-deficient plasma. These studies demonstrate that alkaline phase prorenin activation is initiated by Hageman factor-dependent conversion of prekallikrein to kallikrein which, in turn, leads to activation of prorenin. In this fashion, we have revealed a possible link between the coagulation-kinin pathway and the renin-angiotensin system.     

Beta 2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: loss of inhibition by clipped beta 2-glycoprotein I

Activation of factor XI (FXI) by thrombin in vivo plays a role in coagulation by providing an important positive feedback mechanism for additional thrombin generation. FXI is activated in vitro by thrombin, or FXIIa in the presence of dextran sulfate. In this report, we investigated the effect of beta(2)-glycoprotein I (beta(2)GPI) on the activation of FXI. beta(2)GPI bound FXI in vitro and inhibited its activation to FXIa by thrombin and FXIIa. The affinity of the interaction between beta(2)GPI and FXI was equivalent to the interaction between FXI and high molecular weight kininogen. Inhibition of FXI activation occurred with lower concentrations of beta(2)GPI than found in human plasma. Proteolytic clipping of beta(2)GPI by plasmin abolished its inhibition of FXI activation. The results suggest a mechanism of regulation whereby physiological concentrations of beta(2)GPI may attenuate thrombin generation in vivo by inhibition of FXI activation. Plasmin cleavage of beta(2)GPI provides a negative feedback that counteracts its inhibition of FXI activation.     

Endothelial cells produce a substance that inhibits contact activation of coagulation by blocking the activation of Hageman factor.

Human umbilical vein endothelial cells (HUVECs) produce a property that impairs the generation of coagulant and amidolytic activity initiated when normal human plasma is exposed to glass. This inhibitory property blocks the adsorption of Hageman factor (factor XII) to glass, thereby preventing the activation of Hageman factor, but does not impair the coagulant or amidolytic activity of already activated Hageman factor (factor XIIa). This property in HUVEC lysates could be neutralized by a purified preparation of Hageman factor but not by purified prekallikrein or high molecular mass kininogen. A partially purified inhibitory fraction from cell lysates exhibited a single homogeneous band in SDS/PAGE of approximately 22.5 kDa. Inhibitory activity was also found in concentrates of conditioned media from HUVECs, which also impaired the binding of Hageman factor to a surface; it may not be identical with that found in cell lysates.     

Measurement of plasma total renin by the anti-human renin monoclonal antibodies.

The authors evaluated the assay performances and clinical usefulness of a newly developed solid phase radioimmunoassay (RIA) for total renin concentration (TRC) in human plasma. The direct total renin RIA was performed by a sandwich technique with a pair of anti-human renin monoclonal antibodies. Renin activation with trypsin did not change TRC. The RIA showed satisfactory assay performances and demonstrated full compatibility with a direct RIA-kit for active renin concentration (ARC) in human plasma. The values of TRC were 105.3 +/- 8.6 pg/mL in normal subjects and 136.5 +/- 14.6 pg/mL in patients with essential hypertension. The values of TRC and the ratios of ARC to TRC were high in patients with renovascular hypertension and were low in patients with primary aldosteronism. Although the TRC value in diabetic patients was 134.4 +/- 14.8 pg/mL, the ratio of ARC to TRC was low. The RIA procedure was simple since prior purification or activation of renin was not required. These results suggest that the total renin RIA and its combined use with the active renin RIA may be helpful in understanding the renin-angiotensin system in human plasma.     

A human antibody that inhibits factor IX/IXa function potently inhibits arterial thrombosis without increasing bleeding

10C12, a human antibody F(ab')2, which specifically binds to the gamma-carboxyglutamic acid domain of factor IX/factor IXa (F.IX/IXa), interferes with all known coagulation processes in which F.IX/IXa is involved. In a rabbit model of carotid artery injury, intravenous administration of 10C12 or heparin decreased thrombosis dose dependently. The dose that resulted in a 90% reduction of thrombus mass (ED90) was a 30-microg/kg bolus of 10C12 or a 100-U/kg bolus plus 1.0 U x kg(-1) x min(-1) infusion of heparin. Heparin, at and below the ED90, significantly prolonged coagulation times and cuticle bleeding times. In contrast, 10C12 had no effect on coagulation or bleeding times at doses up to 4 times the ED90. To further evaluate the effect of 10C12 on bleeding, it was compared with heparin in a novel model of blood loss. At the ED90 of heparin, blood loss induced by a standardized injury to the vasculature of the rabbit tibia increased to more than 2 times that of saline controls. In contrast, the dose of 10C12 required to produce a similar increase in blood loss was more than 30 times the ED90. The antithrombotic potency and relative safety of this fully human antibody suggests that it may have therapeutic value for treatment of thrombotic disorders.     

A monoclonal antibody reactive with a subset of human plasma cells

A mouse monoclonal antibody (R1-3) raised against a human plasmacytoma xenograft and reactive with human plasma cells is described. The antibody reacts with a subset of plasma cells and lymphocytes, but is nonreactive with granulocytes, normoblasts, thymus, spleen or T lymphocytes. Flow cytometer double labelling experiments showed that approximately 50% of R1-3 positive bone marrow cells expressed surface immunoglobulin, but no R1-3 positive cells also expressed B1 or Leu 4. In a myeloma patient the R1-3 reactive cells were found to have a higher rate of DNA proliferation and were aneuploid as determined by flow cytometry. The R1-3 antigen is expressed on late B lymphocytes and early plasma cells.     

Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding

Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulation factor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ～ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.     

Identification of a surface antigen on Theileria parva sporozoites by monoclonal antibody.

A mouse monoclonal antibody (mAbD1) that neutralizes sporozoites of different stocks of the protozoan parasite Theileria parva has been used to localize and identify a sporozoite antigen. Protein A-colloidal gold was used to localize bound mAbD1 in immunoelectron microscopic studies. mAbD1 bound to sporozoite antigen, which was evenly spread over the surface of all sporozoites. Immune complexes were obtained by incubation of sporozoite suspensions with mAbD1 followed by Zwittergent 3-14 extraction and precipitation with protein A-Sepharose. One- and two-dimensional NaDodSO4/polyacrylamide gel electrophoretic analyses were performed on these complexes, and a major protein with a molecular size of 68 kDa was identified. Other related components of 52 kDa, 47 kDa, and 28 kDa were also detected. Since antibody to this antigen(s) neutralizes T. parva sporozoites from different stocks, the results could be of relevance to the development of a broad spectrum vaccine against the cattle disease East Coast fever, which is caused by T. parva.     

A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody.

A single amino acid substitution, Asp-63 to Asn-63, was detected in the hemagglutinin of an antigenic variant of the 1968 Hong Kong (H3) influenza virus that was selected by growth of the wild-type virus in the presence of a monoclonal antibody. The mutation generates an oligosaccharide attachment site, Asn-Cys-Thr at residues 63-65, that is glycosylated. Immunoprecipitation experiments with extracts from variant virus-infected cells prepared in the presence or absence of tunicamycin, which inhibits glycosylation, demonstrate that addition of the new oligosaccharide side chain is required to prevent reaction with the monoclonal antibody. Similar experiments with the virus of the 1969 Hong Kong influenza epidemic, A/England/878/69, which also contains a hemagglutinin glycosylated at residue 63, support this conclusion and provide evidence for the epidemiological significance of carbohydrate-mediated modifications of hemagglutinin antigenicity.     

Myeloid progenitor surface antigen identified by monoclonal antibody

A mouse monoclonal antibody, WM-15, has been developed which reacts with a human myeloid lineage-restricted cell surface antigen. WM-15, an IgG1 antibody, reacts with a mean of 3.8% of normal bone marrow mononuclear cells, identifying predominantly promyelocytes and myeloblasts, and in fluorescence-activated cell sorting experiments produces enrichment of bone marrow granulocyte-macrophage progenitor cells. Normal mature monocytes and granulocytes are weakly labelled by WM-15, but other haemopoietic cells including erythroblasts and all lymphoid cells are unreactive. The myeloid specificity of this antibody is highlighted by its reactivity with myeloid leukaemia cell lines and 65% of cases of acute myeloid leukaemia, while lymphoid cell lines and leukaemias are WM-15 negative. WM-15 appears to be a useful reagent for further investigation of normal and abnormal myelopoiesis.     

抗人DR5单克隆抗体诱导肝癌细胞株SMMC-7721凋亡实验研究

目的探讨抗人DR5单克隆抗体对肝癌细胞株SMMC-7721致凋亡作用。方法常规培养肝癌细胞SMMC-7721,通过MTT法检测细胞抑制作用,流式细胞术定量分析凋亡细胞率。结果抗人DR5单抗能够诱导肝癌SMMC-7721细胞凋亡,在抗人DR5单抗浓度2μg/ml作用48h,对肝癌SMMC-7721细胞的杀伤率可达50.10%,增加抗人DR5单抗浓度细胞凋亡作用无明显增加。结论抗人DR5单抗能够诱导肝癌细胞SMMC-7721凋亡,以死亡受体为靶点的抗体制剂为肝癌治疗提供新途径。