Abstract: Methods are disclosed for producing thrombin. The protein is produced from host cells transformed or transfected with DNA construct(s) containing information necessary to direct the expression of thrombin precursors. The DNA constructs generally include the following operably linked elements: a transcriptional promoter, DNA sequence encoding a gla-domainless prothrombin, and a transcriptional terminator. Thrombin precursors produced from transformed or transfected host cells are activated either in vivo or in vitro.

Abstract: The present invention relates to a biological support for cell cultures formed by the coagulated mixture of a concentrate of plasma proteins and thrombin.The protein concentrate is obtained by precipitating fresh plasma with ethanol and contains balanced proportions of fibrinogen, Factor XIII and fibronectin. The thrombin concentration is adjusted to obtain the desired consistency of the support coagulated in the form of a film.The biological support is preferably used for preparing a culture of keratinocytes, recovering them in the form of a reconstituted tissue and transporting same. The reconstituted tissue is thus particularly suitable for use as a graft.

Abstract: A description is given of the possibility of using transglutaminases in a process for the preparation of an immunosuppressant.Additionally described is a pharmaceutical containing a transglutaminase and a plasminogen activator inhibitor.

Abstract: A protein having human plasmin inhibiting activity, substantially equivalent reactivity with human plasmin to a human .alpha..sub.2 -plasmin inhibitor derived from plasma, a binding ability to human fibrin about 1/4 to about 1/3 of that of the human .alpha..sub.2 -plasmin inhibitor derived from plasma, and a molecular weight of about 50,000 to about 77,000.

Abstract: A method for the recombinant production of forms of human protein C with higher activity is described. These forms differ from native protein C in their increased amidolytic and functional activities and novel carbohydrate structures. DNA compounds, vectors, and transformants useful in the method are also disclosed.

Abstract: The present invention relates to a method for preparing a high purity Factor IX concentrate. The starting material for the process is the supernatant fraction of a cryoprecipitated human plasma. A pre-purification step is performed by DEAE-Sephadex chromatography. The resulting Factor IX fraction has a specific activity of at least 0.5 IU/mg protein. The purification method of the invention comprises two successive chromatography separations. First, ion-exchange chromatography on DEAE-sepharose is conducted so that the Factor IX is eluted when the ionic force of the buffer is increased to 0.34-0.38M sodium chloride. Then, affinity chromatography is conducted on heparin-sepharose. The elution buffer is a citrate buffer at a pH of 7.4 adjusted with 0.45M sodium chloride and supplemented with arginine as a stabilizer for Factor IX activity. Lysine is added as a stabilizer before freeze-drying of the purified Factor IX.

Abstract: The present invention provides isolated DNA molecules comprising a DNA segment encoding novel human Kunitz-type inhibitors. Also provided are DNA constructs comprising a first DNA segment encoding a novel human Kunitz-type inhibitor wherein said first DNA segment is operably linked to additional DNA segments required for the expression for the first DNA segment, as well as host cells containing such DNA constructs and methods for producing proteins from the host cells.

Abstract: A process for the activation of t-PA or IgG after expression in prokaryotes is described. The process includes cell digestion, solubilization under denaturing and reducing conditions and activation under oxidizing conditions in the presence of GSH/GSSG.

Abstract: A method for at least partially separating vitamin K-dependent blood clotting factors from a mixture containing at least one such factor, e.g. a prothrombin complex concentrate, which comprises adsorption of said mixture on to a chelate of a polyvalent metal immobilized on an inert support, e.g. Cu.sup.2 + -primed Chelating Sepharose, followed by elution to yield one or more fractions enriched in respect of one of said factors.

Abstract: A dry reagent consisting essentially of (a) a protein having thrombin activity, (b) at least one additive selected from the group consisting of amino acid, a salt thereof and saccharide and optionally (c) magnetic particles, and a method of assaying fibrinogen in an assay sample using the above dry reagent. The dry reagent obviates the time required for preparing a reagent and warming an assay sample, and permits facile fibrinogen assay by only diluting an assay sample. The fibrinogen assay range being broad, the dry reagent substantially obviates the procedure of remeasuring plasma having a fibrinogen concentration outside the assay range of a liquid reagent. The assay result by the dry reagent and that by a liquid reagent well correlate with each other as compared with the result by any known thrombin-containing dry reagent, and the assay by the dry reagent can be performed with good reproducibility in achievement and reliability in measurement.

Abstract: The Prothrombin Time (PT) is used as a screening test for blood coagulation factor deficiencies and for monitoring oral anti-coagulant therapy using coumadin. Thromboplastin reagents activate the extrinsic pathway of coagulation and are the basis for the Prothrombin Time (PT) test. This invention describes the use of barium sulfate and chaotropic agents, and nonionic detergents, for the extraction of sensitive thromboplastin reagents from tissue. Extraction with sodium thiocyanate alone also greatly enhances thromboplastin sensitivity. This invention should be useful for all thromboplastins and will improve their sensitivity for all PT-based tests and specific assays.

Abstract: A process for the preparation of a pasteurized factor VIII concentrate with high specific activity and stability is described, which comprises adsorbing impurities from the solution containing the factor VIII by at least two-fold adsorption with Al(OH).sub.3, an anion exchanger or Ca.sub.3 (PO.sub.4).sub.2, preferably with two different adsorbents from this group.

Abstract: Minactivin (also known as Plasminogen Activator Inhibitor-2 [PAI-2]), a protein inactivator of urokinase-type plasminogen activator, has been shown to be a natural inactivator of this plasminogen activator which is associated with invasive tumors, and is therefore indicated as a crucial element in the body's normal defense against tumor invasion and metastasis. It may be produced by the cultivation of minactivin-producing cells in vitro, and recovery of the cell culture supernatant. By controlling the culture conditions, the protein minactivin may be produced in a partially purified form which may be used for diagnosis and treatment of tumors. The specification discloses purification of biologically active native minactivin, as well as peptides derived from minactivin and their amino acid sequences.

Abstract: Cryoprecipitated mammalian plasma proteins with associated glycoproteins, polysaccharides, and numerous other macromolecular entities are transferred directly in the course of controlled thawing and centrifuging from native plasma phase across the boundary layer into a pre-prepared substrate transfer medium at sustained solidus--liquidus equilibrium regulated to residual icing from about 5 weight percent to about 95 weight percent to produce cryoprecipitates with enhanced productivity and enhanced qualifications for in vivo tissue bonding applications.

Abstract: A test article for determining coagulation capability in a blood sample comprises a porous membrane having a coagulation initiator and substrate impregnated therein. The pore dimensions and composition of the membrane are selected so that only blood plasma can pass into the interior of the membrane, where coagulation is initiated. The substrate is activated by a component of the coagulation pathway, typically thrombin, and produces a detectable signal upon activation. By utilizing membrane matrix materials which are substantially free from interference with the coagulation pathway, accurate results can be achieved.

Abstract: Test articles comprise a solid phase membrane having dry thromboplastin immobilized thereon or within. The thromboplastin is substantially free from substances which might cause aberrant functioning intermediate transition states as the thromboplastin is rehydrated with liquid sample. Coagulation neutral agents which facilitate rehydration of the dry thromboplastin are also provided on the solid phase membrane.

Abstract: Factor IX is selectively adsorbed by means of hydrophobic chromatography from an aqueous mixture containing at least one plasma zymogen or a vitamin-K dependent protein in addition to factor IX. By this method, the efficient enrichment of factor IX for the production of pharmaceutical preparations has become possible.

Abstract: The invention relates to a process for purifying human von Willebrand factor from a cryoprecipitated plasma fraction, which comprises a combination of three chromatographic separation steps. The first chromatographic separation step comprises contacting a cryoprecipitated fraction with a large-pore vinyl polymer resin having DEAE group. The effluent from this separation step is again contacted with a large pore vinyl polymer resin having DEAE groups in the second chromatographic step. In the third chromatographic separation step, the effluent from the second step is subjected to affinity chromatography by contacting with gelatin-Sepharose. The concentrate obtained has very high specific activity and a high percentage of high molecular weight multimers. The concentrate is intended, in particular, for therapeutic use.

Abstract: A reagent blood coagulation that causes an increase in turbidity changes when added to a plasma sample containing a substance activating coagulation factor activating such as tissue thromboplastin, phospholipid and thrombin, calcium ion, and molecular substance such as high molecular vinyl and a high molecular polysaccharide. By adding a molecular substance to the reagent, the turbidity change due to blood coagulation is increased, and hence the changing quantity of transmitted light or scattered light increases, thereby making it possible to achieve a more accurate detection. Besides, by adjusting the electric conductivity, pH and osmotic pressure to proper values, the turbidity change due to blood coagulation may be further amplified.

Abstract: An ultra-pure, clear thrombin solution having a high specific activity is described as well as a method of manufacture.

Abstract: The present invention relates to a process of making a concentrate of coagulation proteins starting with whole human or animal plasma. This concentrate is used as a biological adhesive when extemporaneously mixed to thrombin. The concentrated proteins include mostly fibrinogen, fibrin stabilizing factor (factor XIII) and fibronectin. The claimed process has the advantage of being short of execution while providing an excellent yield of coagulable proteins. No protease inhibitor has to be added during the process. The process involves steps of separation by "salting-out" in presence of amino-6 hexanoic acid which prevents co-precipitation of plasminogen with the desired coagulable proteins. The proteins so obtained are very stable after reconstitution in water for at least 24 hours at room or body temperature. After mixing with thrombin and calcium, the adhesive shows excellent strength and biocompatibility.

Abstract: Use of human blood coagulation factor XIII for the treatment of ulceration colitisThe use of human blood coagulation factor XIII for the treatment of ulcerative colitis is demonstrated in 4 representative cases. Treatment with factor XIII leads to rapid and total disappearence of the chief symptoms realizing transition to the remission stage.

Abstract: A pure Factor I protein essentially free of infectious virus. Factor B and C3. The protein is derived from plasma and pasteurized by heating to a temperature of 50.degree. to 65.degree. C. for 0.5 to 100 hours in the presence of one or more stabilizers for Factor 1. Preparations containing the protein are useful in the treatment of Factor I deficiency and autoimmune diseases.

Abstract: In the method for purifying factor VIII from cryoprecipitate, which is dissolved and then treated with alumina gel, the extract is diluted to a protein concentration not exceeding approximately 5 g/l and subjected to viral inactivation with solvent/detergent, the inactivated extract containing the solvent/detergent is then subjected to chromatography on a weak anion exchange column which is hydrophilic in nature and factor VIII is then eluted with a dissociating buffer.

Abstract: A purified therapeutic grade thrombin is described which is essentially free of lipid envelope viruses, has a specific activity of about 2200 NIH units per milligram of protein to about 3200 NIH units per milligram of protein, is essentially homogeneous and may be produced on a commercial-scale. The thrombin is acceptable from human administration.

Abstract: Thrombin-binding substances capable of promoting anti-thrombin III activity and inhibiting platelet aggregation, and by themselves possessing anti-thrombin activity are disclosed. The thrombin-binding substances are useful as an effective component of anticoagulant agents, and can be produced inexpensively on a large scale.

Abstract: A tissue factor protein mutant capable of neutralizing the ability of endogenous tissue factor to induce coagulation is provided. A representative tissue factor mutant designated K165A, K166A TF is useful in a method for inhibiting thrombin-induced platelet aggregation in a mammal, either separately or in combination with a thrombolytic agent, an anticoagulant, or a GPII.sub.b III.sub.a inhibitor.

Abstract: The invention relates to a process for the manufacture of a high-purity activated factor VII concentrate.This process comprises the use of a plasma free of cryoprecipitate, preferably of human origin, as well as at least one purification step involving chromatography at least once on an ion exchange resin, and a factor VII activation step, wherein the first stage is direct activation of the factor VII in the crude supernatant of plasma free of cryoprecipitate, without the addition of exogenous proteins.By virtue of the invention, the high-purity activated factor VII is essentially free of vitamin K-dependent factors and factors VIIIC and VIIICAg and has a factor VIIa/factor VII ratio greater than 5 with a specific activity of the activated factor VII greater than 200 IU/mg of proteins.

Abstract: An apparatus for resisting coagulation of blood, includes a blood container having disposed therein antibody to blood coagulation factors for resisting coagulation of blood. The apparatus further includes a needle and a holder for the needle which are both operatively associated with the blood container. The blood coagulation factor antibody inhibits coagulation of the blood drawn by the needle into the blood container so that an uncoagulated blood sample is obtained.

Abstract: Recombinant DNA expression vectors encoding a peptide which inhibits binding of von Willebrand factor to platelet membrane glycoprotein Ib, said vector including a nucleotide sequence encoding the amino acid sequence from HIS.sup.1 to LEU.sup.610, inclusive, of the amino terminal region of platelet membrane glycoprotein Ib.alpha.

Abstract: A Ca.sup.2+ dependent monoclonal antibody that specifically binds to a specific twelve peptide sequence (E D Q V D P R L I D G K) in the activation region of the Protein C. The antibody does not bind to Activated Protein C and can be used to inhibit activation of Protein C by thrombin-thrombomodulin. The antibody can be isolated from cell culture or ascites fluid in large quantities by affinity chromatography with mild conditions using the peptide bound to an immobilized substrate.The antibody has a number of specific uses in isolation and characterization of Protein C and as a model for the design of Ca.sup.2+ dependent antibodies for the isolation of other proteins, as a diagnostic, and as a therapeutic to prevent activation of Protein C. The Protein C can be naturally produced or produced by expression of the recombinant gene. Advantages of the antibody in purification of Protein C include the specificity for Protein C and not Activated Protein C, and the unique Ca.sup.

Abstract: The subject invention concerns novel methods and compositions for thrombolytic therapy. More specifically, a receptor with high affinity for plasmin has been characterized, purified, cloned, and expressed. This receptor can be used in combination therapies where it is administered prior to, concurrently with, or after a plasminogen activator. Also, this receptor can be bound to plasmin and administered to humans or animals in need of fibrinolytic activity. Additionally, the invention pertains to a novel immobilized form of plasmin which advantageously accumulates at the point where antifibrinolytic activity is needed.

Abstract: Fibrin binding peptides disclosed include (a) peptides having the amino acid sequence of a human Blood Coagulation Factor XIIIA fragment (i.e., NKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGKWGAKIVMREDR SVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFNPWCEDDAVYLDNEKEREEYVLNDIGVIFY GEVNDIKTRSWSYGQF-R', where R, is --CONH.sub.2 or --NH.sub.2); (b) peptides which are fragments of the foregoing Factor XIIIA fragment and which retain the capability thereof of binding to fibrin; and (c) peptides which bind to fibrin, which have the amino acid sequence of any of the foregoing peptides, and which have additional amino acid residues attached to the N-terminal end and/or the C-terminal end thereof.The peptides are useful for localizing blood clots in vivo, inhibiting fibrin stabilization, and promoting thrombolysis.

Abstract: In a method of recovering proteins, an antibody raised against a peptide comprising at least a fragment of the protein is immobilized on a solid support, a crude preparation of the protein is contacted with the antibody under conditions resulting in a partial, reversible unfolding of the protein to obtain binding of the protein to the antibody, and the protein is eluted from the solid support under non-denaturing conditions to recover the protein in a form in which it is refolded into its native conformation.

Abstract: There is disclosed a pharmaceutical preparation based on Lys-plasminogen and available in the lyophilized form. This preparation contains a serine-protease inhibitor and optionally an inhibitor co-factor, preferably in an amount of 0.5 to 30 nmol per mg Lys-plasminogen. This preparation is free of side-effects and may be used for the treatment and prophylaxis of plasminogen deficiency syndromes and thromboses as well as for the production of thromboresistant artificial organs.

Abstract: Process for preparing a human thrombin concentrate from the PPSB fraction of plasma that does not involve any addition of factors of animal origin to induce activation of the prothrombin to produce thrombin, and which includes a viral inactivation step using solvent-detergent and purification by cation exchange chromatography. The choice of a protective medium ensures the high specific activity of the final product. The thrombin concentrate obtained using this process is intended for therapeutic use, either alone, to serve as a local hemostatic agent, or in combination with a fibrinogen concentrate to form biological glue.

Abstract: The present invention provides a fail-safe combination of chemical and physical means for rendering a blood product which comprises a labile blood protein free of viruses without incurring protein denaturation. The blood product is contacted with an effective amount of a selected chemical disinfectant, preferably, sodium thiocyanate in combination with a physical process, preferably, ultrafiltration. The blood product may be plasma, serum, plasma concentrate, cryoprecipitate, cryosupernatant, plasma fractionation precipitate or plasma fractionation supernatant containing viruses such as hepatitis or human immunodeficiency virus.

Abstract: A method is disclosed to make any protein in a form that can be isolated rapidly from a solution using a specific monoclonal antibody designated "HPC-4". It has now been determined that it is possible to form a fusion protein of the epitope with a protein to be isolated, and isolate the protein using HPC-4-based affinity chromatography. In the preferred embodiment, a specific protease cleavage site is inserted between the epitope and the protein so that the epitope can be easily removed from the isolated protein. In an example, a functionally active soluble tissue factor including the twelve amino acid epitope recognized in combination with calcium by HPC-4 and a factor Xa cleavage site was expressed from a vector inserted into a procaryotic expression system. The recombinant tissue factor can be rapidly isolated in a single chromatographic step using the HPC-4 monoclonal antibody immobilized on a suitable substrate.

Abstract: The subject invention provides purified polypeptide encoded by naturally-occurring wild-type platelet glycoprotein Ib alpha having a mutation which renders the polypeptide less reactive with von Willebrand factor. Preferably, the mutation is in the leucine rich region of GPIb.alpha., such as the substitution of phenylalanine for leucine at residue 57. DNA encoding the mutant polypeptides, as well as expression systems for the production of the mutant polypeptides, are also provided. Methods and compositions using the mutant polypeptides and DNA oligomers complementary to the mutant polypeptides are further provided.

Abstract: Pharmaceutical compositions which consist essentially of a vascular anticoagulant annexine, Ca.sup.2+, Zn.sup.2+, and a pharmaceutically acceptable carrier are disclosed. Also disclosed are processes for preparing such pharmaceutical compositions. Also disclosed are methods of preventing the coagulation of blood by mixing the blood with a vascular anticoagulant annexine, Ca.sup.2+ and Zn.sup.2+.

Abstract: An intraocular anticoagulant including antithrombin III is applicable as an injection into the ocular chamber of the eye at the time of cataract surgery, intraocular lens implant surgery, and other ocular surgeries. The anticoagulant prevents fibrin deposits from forming on the intraocular lens surface after surgery and avoids postoperative viscoelastic material inducing transient elevation in intraocular pressure.

Abstract: Processes for stabilizing active PAI-1 protein comprising combining active PAI-1 protein with an aqueous buffer having an ionic strength of at least about 5 millisiemens and a sugar selected from the group consisting of monosaccharides and disaccharides, and/or subjecting the active PAI-1 protein to lyophilization, are disclosed. Also described are compositions, including pharmaceutical compositions and kits, which include the stabilized active PAI-1 protein, and therapeutic processes employing the same in the treatment of fibrinolysis.

Abstract: The present invention relates to a process of making a concentrate of coagulation proteins starting with whole human or animal plasma. This concentrate is used as a biological adhesive when extemporaneously mixed to thrombin. The concentrated proteins include mostly fibrinogen, fibrin stabilizing factor (factor XIII) and fibronectin. The claimed process has the advantage of being short of execution while providing an excellent yield of coagulable proteins. No protease inhibitor has to be added during the process. The process involves steps of acidic precipitation in presence of amino-6 hexanoic acid which prevents co-precipitation of plasminogen with the desired coagulable proteins. The proteins so obtained are very stable after reconstitution in water for at least 24 hours at room or body temperature. After mixing with thrombin, the adhesive shows excellent strength and biocompatibility.

Abstract: The present invention relates to a process for purifying Factor IX from an impure protein fraction containing Factor IX. The purification process comprises the steps of adding a solvent and a detergent to an impure protein fraction and incubating the solvent/detergent protein solution to inactivate any viral contaminants. Factor IX is purified from the solvent/detergent protein solution by chromatography on a sulfated polysaccharide resin without first removing the solvent and detergent prior to the purification on the sulfated polysaccharide resin. The Factor IX, purified by the process has a specific activity of at least 85 units/mg.

Abstract: Factor IX is selectively adsorbed by means of hydrophobic chromatography from an aqueous mixture containing at least one plasma zymogen or a vitamin-K dependent protein in addition to factor IX. By this method, the efficient enrichment of factor IX for the production of pharmaceutical preparations has become possible.

Abstract: The present invention describes APC polypeptides and anti-peptide antibodies capable of inhibiting activated Protein C anticoagulant activity. The polypeptide and antibody are useful in diagnostic methods and systems for measuring APC in vascular fluid samples. In addition, the polypeptide and anti-peptide antibody are useful in therapeutic methods for inhibiting APC in a human patient.

Abstract: Recombinant thrombin-binding substances, derived from thrombomodulin by modification of the c-terminal glycosaminoglycan (GAG) binding site and capable of promoting anti-thrombin III activity and inhibiting platelet aggregation, and by themselves possessing anti-thrombin activity are disclosed. The thrombin-binding substances are useful as an effective component of anticoagulant agents, and can be produced inexpensively in a large scale.

Abstract: The Prothrombin Time (PT) is used as a screening test for blood coagulation factor deficiencies and for monitoring oral anti-coagulant therapy using coumadin. Thromboplastin reagents activate the extrinsic pathway of coagulation and are the basis for the Prothrombin Time (PT) test. This invention describes the use of barium sulfate and chaotropic agents, and nonionic detergents, for the extraction of sensitive thromboplastin reagents from tissue. Extraction with sodium thiocyanate alone also greatly enhances thromboplastin sensitivity. This invention should be useful for all thromboplastins and will improve their sensitivity for all PT-based tests and specific assays.

Abstract: A method for the recombinant production of zymogen forms of human protein C is described. These zymogen forms differ from native zymogen protein C in their increased sensitivity to activation by thrombin and thrombin/thrombomodulin. DNA compounds, vectors, and transformants useful in the method are also disclosed.

Abstract: A process is provided for preparing fibronectin and antihemophilic factor (Factor VIII) concentrates from a blood plasma cryoprecipitate.