Henry S. Kingdon

Use of Recombinant Antihemophilic Factor in the Treatment of Two Patients with Classic Hemophilia

COMMERCIAL concentrates of human antihemophilic factor (factor VIII) have been available for nearly 20 years and have resulted in dramatic changes in the treatment of classic hemophilia. Home thera...

Thrombogenic Materials in Prothrombin Complex Concentrates

Abstract Prothrombin complex concentrates are now available for use for treatment of bleeding complications associated with deficiencies of factors II, VII, IX, or X. The purpose of this report is ...

Inhibition of the heparin-antithrombin III/thrombin reaction by active site blocked-thrombin☆

Abstract Active site blocked-thrombin, prepared by reacting thrombin with valyl-isoleucyl-prolyl-arginine chloromethyl ketone, inhibits the heparin enhanced-antithrombin III/thrombin reaction. Since active site blocked-thrombin does not interact with antithrombin III it was concluded that active site blocked-thrombin was competing for heparin in the reaction system. The heparin concentration dependence for maximum enhancement of the antithrombin III/thrombin reaction in the presence and absence of active site blocked-thrombin indicated that heparin was binding to thrombin to enhance the reaction rate. A dissociation constant value of 6.4×10−9M was estimated for the heparin·thrombin complex which is similar to the value of 5.8×10−9M previously reported (Griffith M.J. (1979) J. Biol. Chem. in press). Antithrombin III·thrombin complexes were also found to bind heparin with an affinity equivalent to thrombin. The results were interpreted to indicate that heparin binds to thrombin as the first step in the mechanism of action of heparin in enhancing the antithrombin III/thrombin reaction.

An effect of predilution on potency assays of factor VIII concentrates

The effect of prediluent on potency assays for Factor VIII concentrates was evaluated systematically using a well defined one stage Factor VIII assay. It was demonstrated that potency values were higher when Factor VIII deficient plasma was used as the prediluent compared to identical assays performed when barbital buffered saline solution was used as prediluent. Prediluent is therefore an important variable to evaluate in establishing the validity of any proposed Factor VIII potency assay. Since the goal of Factor VIII potency measurement is to predict therapeutic effect, the use of Factor VIII deficient plasma is the more satisfactory alternative where a difference exists, since this diluent approximates best the conditions which occur when a Factor VIII concentrate is injected into the bloodstream of a patient with hemophilia A.

Fractionation of heparin by affinity chromatography on covalently-bound human α-thrombin☆

Abstract Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of N-α-tosyl-L-glycyl-L-prolyl-L-arginine- p -nitroanilide hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.

Effect of monovalent cations on the heparin-enhanced antithrombin III/thrombin reaction

Abstract The antithrombin III/thrombin reaction was enhanced by the monovalent cations lithium, sodium and potassium. In the presence of 0.1 M cation, the maximum relative rate enhancement by heparin was 2000-fold with lithium, 1,800-fold with sodium and 1,100-fold with potassium. In the absence of additional cation, heparin enhanced the antithrombin III/thrombin reaction rate 100-fold. The amount of heparin required to maximally enhance the antithrombin III/thrombin reaction rate increased as the concentration of cation was increased. A 10-fold higher concentration of heparin was required in the presence of 0.2 M lithium chloride, while a 100-fold and 150-fold higher concentration of heparin was necessary with 0.2 M sodium chloride and 0.2 M potassium chloride present, respectively. The binding of heparin to antithrombin III was not detectably affected by 0.13 M lithium chloride, suggesting that the heparin-antithrombin III interaction was not involved in the mechanism of action of heparin in this system. The results demonstrate the sensitivity of the heparin-antithrombin III/thrombin reaction rate to small increases in cation concentration and suggest that heparin activity assays using this system must be standardized to accomodate the contribution of cations present in the heparin samples being assayed.

A simple and inexpensive high pressure liquid chromatographie system as an adjunct to gas chromatography in identification of amino acid phenylthiohydantoins

Abstract A simple and inexpensive system has been developed in our laboratory for the identification and quantitation of amino acid phenylthiohydantoins by high pressure liquid chromatography. Isocratic methodology has been employed, so that expensive complex gradient makers can be avoided. The method is rapid, and retention times of 10 min or less are obtained in all but one case. This paper presents the rationale and details of the method, and demonstrates a comparison between gas-liquid chromatograms and high pressure liquid chromatograms of selected residues from actual automated sequence analyses of both sperm whale myoglobin and egg white lysozyme. The method is intended to complement rather than replace gas-liquid chromatography, as it cannot completely resolve all 20 phenylthiohydantoins, but rather identifies easily those residues which are difficult or impossible by gas chromatography.

Hydrolysis of N-α-benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide by human alpha thrombin in the presence of heparin

Abstract The effect of heparin on the hydrolysis of N-α-benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide by human alpha thrombin was studied. When the substrate concentration was low i.e. less than 4.0 × 10 −5 M, heparin enhanced the initial rate of substrate hydrolysis. At higher substrate concentrations heparin appeared to inhibit thrombin activity. The extent of inhibition was dependent on the order of mixing heparin with thrombin and substrate. When the substrate concentration was 1.1 × 10 −4 M, premixing heparin with substrate decreased the initial rate of substrate hydrolysis approximately 10%, whereas premixing heparin with thrombin decreased the initial rate approximately 75%. Substrate solutions appeared slightly turbid upon addition of heparin (10 −7 M). Turbidity was independent of the mixing regimen. After centrifugation pelleted material was found to contain heparin, substrate and enzyme. Kinetic data indicated that inhibition of the initial rate of substrate hydrolysis which was observed when heparin and substrate were premixed was due, primarily, to a decrease in the free substrate concentration caused by heparin-substrate aggregation. The inhibition observed when heparin and thrombin were premixed was due, primarily, to a decrease in the free enzyme concentration caused by the co-aggregation of thrombin with heparin and substrate. It was concluded that heparin does not have a direct inhibitory effect on the hydrolysis of this substrate by thrombin.

Purification and Partial Characterization of Deoxyribonuclease I from Bovine Parotid Gland

Deoxyribonuclease I has been purified from bovine parotid gland. The purification procedure utilizes an acid extraction of minced parotid gland, salt fractionation, gel filtration, and ion-exchange chromatography. The last step, chromatography on Sulfopropyl-Sephadex, resolves the enzymatic activity into several fractions. The major fraction, designated DNase A, was subjected to further investigation. This enzyme has, as expected, an alkaline pH optimum and an obligate requirement for divalent cations. The presence of calcium chloride protects DNase A from inactivation by proteolytic enzymes. Despite the previously described immunologic dissimilarity, there appears to be a large amount of homology between the parotid and pancreatic DNases.

Cellular activation of factor IX (christmas factor)

Abstract We have demonstrated Factor IX activation by sonicated polymorphonuclear leukocytes (PMNs). This activation reaction required the disrupted leukocytes, calcium chloride, and a small amount of normal human plasma. The requirement for normal plasma was met by plasma deficient in all of the known coagulation factors, and thus the substance present in normal plasma which facilitates this reaction was not identified. The fact that factor XI deficient plasma supported the reaction as well as normal plasma implied that factor XIa was not involved in this activation. Strontium ions could not substitute for calcium ions in the activation reaction, also implying that factor XIa was not involved. This factor IX activating principle in leukocytes could provide a mechanism for by-passing the contact factors of blood coagulation, thus providing an explanation for the discrepancy in clinical severity between deficiencies of the contact factors on the one hand and hemophilias A and B on the other.