Nucleic acid scavengers inhibit thrombosis without increasing bleeding

Development of effective, yet safe, antithrombotic agents has been challenging because such agents increase the propensity of patients to bleed. Recently, naturally occurring polyphosphates such as extracellular DNA, RNA, and inorganic polyphosphates have been shown to activate blood coagulation. In this report, we evaluate the anticoagulant and antithrombotic activity of nucleic acid-binding polymers in vitro and in vivo. Such polymers bind to DNA, RNA, and inorganic polyphosphate molecules with high affinity and inhibit RNA- and polyphosphate-induced clotting and the activation of the intrinsic pathway of coagulation in vitro. Moreover, [NH₂(CH₂)₂NH₂]∶(G = 3);dendri PAMAM(NH₂)₃₂ (PAMAM G-3) prevents thrombosis following carotid artery injury and pulmonary thromboembolism in mice without significantly increasing blood loss from surgically challenged animals. These studies indicate that nucleic acid-binding polymers are able to scavenge effectively prothrombotic nucleic acids and other polyphosphates in vivo and represent a new and potentially safer class of antithrombotic agents.     

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.     

Use of phage display for the generation of human antibodies that neutralize factor IXa function.

The use of libraries of phage-displayed human single-chain antibody fragments (scFv) has become a new, powerful tool in rapidly obtaining therapeutically useful antibodies. Here, we describe the generation of human scFv and F(ab')2 directed against the gamma-carboxyglutamic acid (Gla) domain of coagulation factor IX. A large library of human scFv, displayed either on M13 phage or expressed as soluble proteins, was screened for binding to human Gla-domain peptide (Tyr1-Lys43). Among a panel of scFv that bound to the factor IX-Gla domain, six scFv clones recognized full-length factor IX and exhibited strong inhibitory activity of factor IX in vitro. After reformatting as F(ab')2, the affinity for factor IX of three selected clones was determined: 10C12 Kd = 1.6 nmol/l, 13D1 Kd = 2.9 nmol/l, and 13H6 Kd = 0.46 nmol/l. The antibodies specifically bound to factor IX and not to other coagulation factors, as assessed by enzyme-linked immunosorbent-type and human plasma clotting assays. The complementarity determining region amino acid sequences of clones 10C12 and 13D1 only differed at a single residue, whereas 13H6 showed little homology, suggesting that 13H6 binds to a different epitope within the factor IX-Gla domain. Despite the slightly lower affinity of 10C12 F(ab')2 versus 13H6 F(ab')2, 10C12 was consistently more potent than 13H6 in prolonging the activated partial thromboplastin time (APTT), in inhibiting platelet-mediated plasma clotting, and in inhibiting factor X activation by the intrinsic Xase complex. Finally, 10C12 F(ab')2 also recognized and neutralized factor IX/factor IXa of different species, as demonstrated by the specific APTT prolongation of dog, mouse, baboon and rabbit plasma. In summary, the results validate the usefulness of scFv phage-displayed libraries to rapidly generate fully human antibodies as potential new therapeutics for thrombotic disorders.     

Mapping of genes that control the antibody response to human factor IX in mice

We tested the hypothesis that the antibody response to human factor IX in mice is controlled by genetic factors, especially histocompatibility antigens. Seven inbred mouse strains were immunized against human factor IX by adenoviral gene transfer or serial injections of human factor IX protein. A/J mice had the highest antibody response and 2 C57 mouse strains had the lowest response. We used the adenovirus vector to immunize 26 recombinant inbred mouse strains (AXB and BXA) derived from A/J and C57BL/6J mice and observed highly significant linkage (logarithmic odds [LOD] scores approximately 4.8) for the polymorphic D17Mit62 marker that is 1 centimorgan ( approximately 300 000 base pair [bp]) from the mouse major histocompatibility complex (MHC) locus (H-2). Experiments in mice with chimeric MHC genes indicated that class IaK or class II H-2 (or both) genes were critical, but other genes contributed to the antibody response. Polymorphic markers from chromosomes 1 and 10 that are near important immunoregulatory genes such as interleukin 10 and the interferon-gamma gene show suggestive linkage (LOD scores of approximately 2.3-2.6) to the factor IX antibody response. This study confirms the hypothesis that H-2 (and other) genes control factor IX antibody development in mice and suggests their potential importance for factor IX antibody development in humans with hemophilia B.     

Increased sensitivity of factor IX to phenprocoumon as a cause of bleeding in a patient with antiphospholipid antibody associated thrombosis

Harbrecht U, Oldenburg J, Klein P, Weber D, Rockstroh J, Hanfland P (University of Bonn, Bonn; University of Würzburg, Würzburg; and Evangelische Diakonissen Anstalt Bremen, Bremen, Germany). Increased sensitivity of factor IX to phenprocoumon as a cause of bleeding in a patient with antiphospholipid antibody associated thrombosis. J Intern Med 1998; 243 : 73–77.
We report one patient who presented with a spontaneous bleeding complication under phenprocoumon therapy. Oral anticoagulation was initiated due to deep-vein thrombosis which was attributed to an antiphospholipid antibody syndrome. Coagulation analysis revealed a strong and selective reduction of factor IX (F IX) activity to 1%, whereas the other vitamin K-dependent factors (II, VII, X), the prothrombin time and International Normalized Ratio (INR) were within the therapeutic range. After withdrawal of phenprocoumon, all vitamin K-dependent factors including F IX normalized. Because the patient suffered from a recurrence of thrombotic events, he was re-exposed to phenprocoumon and the disproportionate decline of F IX was observed again. These findings indicate an increased sensitivitiy of F IX to vitamin K antagonists, representing an uncommon mechanism associated with bleeding complications under oral anticoagulant treatment.     

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.     

Guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis

Background  The phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation. Methods and results  Guggulsterone inhibited TNF-α-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-α-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-α-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner. Conclusions  Guggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.     

Immunochemical characterization of a polyclonal human antibody to factor IX.

Inhibitors of clotting factors occuring in humans are often antibody molecules synthesized in response to exogeneous proteins used in replacement therapy. Extensive studies of inhibitors to factor VIII indicate such antibodies may be monoclonal or polyclonal in nature. To date, only one factor IX inhibitor has been subjected to detailed immunochemical analysis and it appears to be a monoclonal IgGA lambda antibody. We have discovered a second inhibitor of factor IX in a patient with severe hemophilia B and have subjected it to immunochemical analysis. Studies on this second inhibitor have been carried out before and after an anamnestic response. Column chromatography, preparative zone electrophoresis, and specific inhibitor neutralization assays using monospecific heterologous antisera to human immunoglobulin classes, subclasses, and light-chain types indicate that the antibody is of the IgG class and contains both kappa and lambda light chains and probably all four IgG subclasses. Thus, the inhibitor appears to be polyclonal by immunochemical and structural criteria. In addition, preparative isoelectric focusing of pre- and postanamnestic inhibitor samples indicates that recruitment of new clones of IgG antibody occurs as a result of anamnesis. It is conceivable that an antibody initially restricted in immunoglobulin subclass became polyclonal following an anamnestic response.     

Monoclonal antibody that specifically inhibits a human Mr 52,000 plasminogen-activating enzyme

Monoclonal antibodies against a human plasminogen activator of M_r ≈52,000 (HPA52) were derived by immunization of mice with an impure preparation of the enzyme (urokinase), subsequent hybridization of spleen cells with NSI-Ag4/1 myeloma cells, and cloning of the hybridomas. Selection of mice for hybridization and screening of hybridomas were based solely on direct inhibition of an enzymatic assay of the plasminogen activator with the impure enzyme preparation. A cloned hybridoma produced IgG1 antibodies that bound to and inhibited the enzymatic activity of HPA52 irrespective of whether the HPA52 was derived from urokinase or from human glioblastoma cells, whereas there was no inhibition of or binding to a plasminogen activator of M_r ≈70,000 from human melanoma cells or a plasminogen activator of M_r ≈36,000 that is a degradation product of HPA52 and present in urokinase. Nor did the anti-HPA52 IgG1 inhibit a murine plasminogen activator of M_r ≈48,000 derived from sarcoma virus-transformed cells. By using affinity chromatography with columns of anti-HPA52 IgG1 bound to Sepharose, HPA52 was purified from urokinase to homogeneity as evaluated by NaDodSO₄/polyacrylamide gel electrophoresis. This study demonstrates that inhibitory monoclonal antibodies against enzymes can be derived with the sole use of impure enzyme preparations and shows how such antibodies subsequently can be used for enzyme purification.     

JE-2147: A dipeptide protease inhibitor (PI) that potently inhibits multi-PI-resistant HIV-1

We designed, synthesized, and identified JE-2147, an allophenylnorstatine-containing dipeptide HIV protease inhibitor (PI), which is potent against a wide spectrum of HIV-1, HIV-2, simian immunodeficiency virus, and various clinical HIV-1 strains in vitro. Drug-resistant clinical HIV-1 strains, isolated from seven patients who had failed 9-11 different anti-HIV therapeutics after 32-83 months, had a variety of drug-resistance-related amino acid substitutions and were highly and invariably resistant to all of the currently available anti-HIV agents. JE-2147 was, however, extremely potent against all such drug-resistant strains, with IC(50) values ranging from 13-41 nM (<2-fold changes in IC(50) compared with that of wild-type HIV-1). The emergence of JE-2147-resistant HIV-1 variants in vitro was substantially delayed compared with that of HIV-1 resistant to another allophenylnorstatine-containing compound, KNI-272, and other related PIs. Structural analysis revealed that the presence of a flexible P2' moiety is important for the potency of JE-2147 toward wild-type and mutant viruses. These data suggest that the use of flexible components may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1. Further development of JE-2147 for treating patients harboring multi-PI-resistant HIV-1 is warranted.     

A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein- HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (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.     

An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates.

Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8-11D12 and TF9-9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9-9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8-11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8-11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8-11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.     

Reduced bleeding events with subcutaneous administration of recombinant human factor IX in immune-tolerant hemophilia B dogs

Intravenous administration of recombinant human factor IX (rhFIX) acutely corrects the coagulopathy in hemophilia B dogs. To date, 20 of 20 dogs developed inhibitory antibodies to the xenoprotein, making it impossible to determine if new human FIX products, formulations, or methods of chronic administration can reduce bleeding frequency. Our goal was to determine whether hemophilia B dogs rendered tolerant to rhFIX would have reduced bleeding episodes while on sustained prophylactic rhFIX administered subcutaneously. Reproducible methods were developed for inducing tolerance to rhFIX in this strain of hemophilia B dogs, resulting in a significant reduction in the development of inhibitors relative to historical controls (5 of 12 versus 20 or 20, P <.001). The 7 of 12 tolerized hemophilia B dogs exhibited shortened whole blood clotting times (WBCTs), sustained detectable FIX antigen, undetectable Bethesda inhibitors, transient or no detectable antihuman FIX antibody titers by enzyme-linked immunosorbent assay (ELISA), and normal clearance of infused rhFIX. Tolerized hemophilia B dogs had 69% reduction in bleeding frequency in year 1 compared with nontolerized hemophilia B dogs (P =.0007). If proven safe in human clinical trials, subcutaneous rhFIX may provide an alternate approach to prophylactic therapy in selected patients with hemophilia B.     

Asialo-von Willebrand factor inhibits platelet adherence to human arterial subendothelium: discrepancy between ristocetin cofactor activity and primary hemostatic function

Platelet adherence at high wall shear rates requires plasma von Willebrand factor (vWF). Clinically, the ristocetin cofactor (RCof) activity is the only widely available assay for vWF function. When purified vWF is treated with neuraminidase to yield asialo-vWF (AS- vWF), its RCof activity is increased by 20% to 40%. AS-vWF binds to normal human platelets independently of ristocetin and induces platelet aggregation in the presence of fibrinogen. To determine whether AS-vWF also shows an enhanced capacity to support platelet adherence to subendothelium, we used the Baumgartner technique. Intact vWF, AS-vWF, or AS-vWF treated with beta-galactosidase (asialo, agalacto-vWF; AS,AG- vWF) was added to normal citrated whole blood before perfusion over human umbilical artery segments (wall shear rate, 2,600 sec-1). Four micrograms per milliliter AS-vWF caused a 69% reduction in total platelet adherence compared with citrated whole blood (P less than .001), and 4 micrograms/mL AS,AG-vWF led to a 48% reduction (P less than .005). With 4 micrograms/mL intact vWF, the platelet adherence values were not significantly different from the controls. No significant differences in subendothelial platelet thrombi or postperfusion platelet counts were evident among any of the groups. In reconstituted afibrinogenemic perfusates, 4 micrograms/mL AS-vWF caused a 42% reduction in platelet adherence (P less than .05). Thus, AS-vWF is a potent inhibitor of platelet adherence, despite its enhanced RCof specific activity. Abnormalities in vWF carbohydrate may play a role in impaired primary hemostasis in some patients with von Willebrand's disease.     

A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor

The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4kappa antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)     

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.     

A monoclonal antibody against human thrombospondin inhibits platelet aggregation.

A monoclonal antibody (C6.7) has been generated against the calcium-replete form of human platelet thrombospondin (TSP). C6.7 is specific for TSP as determined by both competitive radioimmunoassay and immunoprecipitation. This antibody inhibits both thrombin- and A23187-induced aggregation of gel-filtered platelets in a concentration-dependent manner without affecting the secretion of serotonin. The epitope on TSP recognized by C6.7 has been localized to an 18-kDa fragment that is present in mild chymotryptic digests of TSP. This fragment is disulfide-linked to a 120- to 140-kDa fragment in unreduced digests, and both reduction and denaturation are required to separate the 18-kDa peptide from the larger fragments. A 25-kDa heparin binding domain is also present in the chymotryptic digest. However, the 18-kDa peptide is distinct from the heparin binding domain. The amino acid sequence at the NH2 terminus of the 18-kDa fragment is Asp-Thr-Asn-Pro-Thr-Arg-Ala-Gln-Gly-Tyr-.