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SummaryArdeparin sodium (Normiflo®, Wyeth-Ayerst) is a low molecular weight heparin undergoing clinical evaluation as an antithrombotic agent. The objective of this study was to evaluate the absolute and comparative bioavailability of ardeparin following subcutaneous administration of three different formulations [two formulations of ardeparin at 10,000 anti-factor Xa (aXa) U/ml, but with different preservatives, and a 20,000 aXa U/ml formulation]. The study was conducted using a randomized 4-period crossover design (three subcutaneous treatments and one intravenous treatment) in 24 healthy subjects, and the pharmacokinetics of ardeparin were characterized by plasma anti-factor Ila (alia) and anti-factor Xa (aXa) activities. The mean absolute bioavailability of ardeparin based on alia activity ranged from 62% to 64% and the mean absolute bioavailability based on aXa activity ranged from 88% to 97%. Based on bioequivalence testing criteria, the three ardeparin formulations were bioequivalent.

SummaryIn this paper an inhibitor is described that is found in hemophilic plasma and serum different from any till now described inhibitor. The inhibitor only inhibits prothrombin activation in the “intrinsic clotting systems”. This inhibitor is probably not present in normal human plasma or serum. It is destroyed by ether and freeze drying, is labile to acid and storage at room temperature. It is stable upon dialysis and has not been adsorbed on barium sulfate, aluminum hydroxide or kaolin. It precipitates at 50% v/v saturation with alcohol. The nature of this inhibitor seems to be a protein or lipoprotein.Factor VIII was isolated from hemophilic plasma. The amount isolated was the same as from normal plasma and the activity properties were not different. Hemophiliacs have normal amounts of factor VIII.

SummaryTo elucidate the mechanisms triggering disseminated intravascular coagulation (DIC) in shock, acidosis was produced in dogs by intravenous infusions of lactic acid over a period of 4 h. As the pH of the blood dropped, the blood pressure increased and the heart rate fell. The platelet counts dropped and whole blood clotting times shortened. The partial thromboplastin times and prothrombin times decreased as did the r-times and maximal amplitudes of the thrombelastograms. Simultaneously, the levels of prothrombin, fibrinogen, factors V and VIII decreased with time in acidosis. There was no evidence of a systemic activation of the fibrinolytic system. The euglobulin lysis times increased as did the thrombin times and there was no lysis observed in the thrombelastograms.Thrombi in the lungs, liver, spleen and kidneys, together with the coagulation changes indicated that the infusion of acid produced DIC, with the changes being similar to those produced in shock. The glomeruli were especially affected and the pictures were identical to those described in kidneys of animals having experienced a generalized Sanarelli-Shwartzman phenomenon.A pH value of 7.20 or lower appeared to be a potent trigger of intravascular coagulation. The lower the pH, the more thrombi were found in the tissues. The consumption of clotting factors and thrombus formation thus corresponded to the degree of acidosis.A generalized fibrinolytic state was induced using Streptokinase, after a 4 h period of acidosis and 1 h of compensation with sodium bicarbonate. Streptokinase was infused along with human euglobulin fractions on the basis of the streptokinase tolerance test of each dog’s blood and plasma. Thrombolysis was maintained for 3-4 h and this was reflected in the reduction of fibrin deposits in the tissues. Of 100 glomeruli counted/dog’s kidney, the average containing fibrin was 44 ± 11% after acidosis and 0.4 ± 0.3% after thrombolysis.

SummaryAutoprothrombin I, autoprothrombin II, and autoprothrombin II-A have been derived from bovine prothrombin and concentrated.Autoprothrombin I is formed when thrombin is used to activate prothrombin which has been chromatographed on IRC-50 or diethylaminoethyl cellulose. The activation is inhibited by calcium ions, and goes more rapidly as the alkalinity is increased up to pH 9.7. During the activation the liberation of acidic groups predominates over that of the basic groups. In activation mixtures proline, alanine, threonine and valine were found as N-terminal amino acids. The activity was identified with a fraction that contained N-terminal valine, but an alteration can be made with IRC-50 so that the activity is all retained with a change in N-terminal amino acid. The activity is tentatively ascribed to a molecule that has a sedimentation constant of 3.8 in an ultracentrifuge. The autoprothrombin I was found to be soluble in ammonium sulfate solution at 50% of saturation and precipitated when the salt concentration is at 70% of saturation. There was very little loss of activity associated with drying from the frozen state at pH 8.0. Acetone precipitation destroyed the activity, and acidification predisposed to a loss in activity.All prothrombin preparations we have worked with develop autoprothrombin I activity in the presence of platelet factor 3 and calcium ions, but not, if the calcium ions are not there. The solubility of the protein is the same as when obtained by thrombin activation or by separation from serum.Autoprothrombin II is formed when purified thrombin is used to activate non-chromatographed prothrombin. The activation is inhibited with calcium ions, and there is a pH optimum between 7 and 8. During activation nonbuffered solutions become acidic. The activity is identified with a fraction in which the N-terminal amino acid is proline and the C-terminal amino acid is tyrosine. The activity was concentrated in a fraction that was practically homogenous upon ultracentrifuge analysis. The sedimentation constant is near S = 3.7 (tentative). The activity is easily precipitated at 50% of saturation with ammonium sulfate. No activity was lost upon dialysis or drying from the frozen state around pH 7 to 8. The concentrates with autoprothrombin I activity are precipitinogenic against antiprothrombin and anti-bovine serum. The autoprothrombin II of serum is also precipitated at 50% of saturation with ammonium sulfate.Autoprothrombin II-A is an anticoagulant that was found to be closely related to autoprothrombin II in physical chemical characteristics. The anticoagulant had arginine as N-terminal amino acid and tyrosine as C-terminal amino acid. After IRC-50 resin chromatography the autoprothrombin II-A protein was found to be changed and had autoprothrombin II activity, N-terminal proline and C-terminal tyrosine. The autoprothrombin II-A activity was retained during dialysis and during drying from the frozen state. The sedimentation constant found with the ultracentrifuge was 3.8. The activity was also found in concentrates from serum.Autoprothrombin II corrects the recalcified clotting time of PTC deficient plasma. Hemophilia A blood clots rapidly with autoprothrombin II. The recalcified clotting time of hemophilia A plasma is also brought to the normal range with concentrates of autoprothrombin II. In general the prothrombin utilization does not seem to be increased when the procoagulant is added to hemophilic blood or plasma.

SummaryThe method for the assay for platelet cofactors has been overhauled to make allowance for an adequate and uniform supply of Ac-globulin. Thrombin is added at zero time to reduce variability of response in the activation of prothrombin. The potency of purified platelet cofactor I is about twice that of platelet cofactor II (autoprothrombin II). The total mass of platelet factor 3 plus platelet cofactor I or platelet cofactor II is much greater in the conversion of a given amount of purified prothrombin to thrombin than when brain thromboplastin is used.In plasma and serum from the human being barium carbonate adsorbs platelet cofactor II, and ether extraction frees activity that is not adsorbed on barium carbonate before ether extraction. The extract contains an inhibitor(s) of prothrombin activation. The inhibition is strong in the intrinsic as well as the extrinsic prothrombin activation mechanisms. In the case of bovine plasma barium carbonate adsorption leaves the plasma richer in platelet cofactor activity and in that respect it differs from the plasma from human beings. Dog plasma and serum is comparable to that from man than the bovine species.The lipids extracted from plasma or serum with ether are predominantly anticoagulant. Since platelet cofactor I activity disappears during the clotting of blood, we think that a function of platelet cofactor I is to be the receptor for an anticoagulant. Then as autoprothrombin II arises from prothrombin it serves as a procoagulant and is not retarded by the anticoagulant neutralized by platelet cofactor I.

SummaryPurified bovine platelet cofactor I substituted for adsorbed plasma in the thromboplastin generation test when Ac-globulin was also supplied. Only 70 gamma/ml were needed. Purified bovine autoprothrombin II substituted for serum in the test. Only 9 gamma were equivalent to serum. Platelet cofactor I thus takes the place of the antihemophilic factor and autoprothrombin II substitutes for PTC and Stuart-Prower factor. We regard the autoprothrombin II preparation used as a single component derived from prothrombin.

SummaryBy using glass wool absorption followed by elution in strong salt solution and various precipitation methods and DEAE cellulose column chromatography, Hageman factor was purified from bovine plasma. Physico-chemical properties were studied in native protein and in 6 M urea (denatured) which was used in order to prevent monomer - polymer conversion. S°20,w was 7.07 in 0.1 M KC1 and 5.84 in 6 M urea. The intrinsic viscosity by Kragh’s method was 0.0540 ml/g inNaCl-phosphate buffer and 0.0815 ml/g in 6 M urea. Partial specific volume (v) by method based on amino acid residues in undenatured protein, was 0.724 cc/g. By using differential sedimentation equilibrium in 6 M urea-H20 and 6 M urea - D20 solution, v was determined as 0.740 cc/g. In urea, molecular weight (MW) was 142,000 as determined by meniscus depletion method. Similar values were obtained for nondenatured and denatured protein by Sephadex gel filtration, from amino acid composition using Brand’s method and Schachman’s equation. For undenatured and denatured Hageman factor, molar frictional ratios (f/fo) were 1.39 and 1.58, axial ratios were 5.0 and 7.0 and estimation of diffusion constant (D°20, w) based on S-rate, MW and v, gave values of 4.45 x 10-7 and 3.88 x 10-7 cm2/sec respectively. Our data indicate that the molecule was unfolded in 6 M urea solution. Two N-terminal groups, glycine and valine, were demonstrated by Edman procedure. Carbohydrate analysis revealed the following: hexosamine 0.8%, protein- bound hexose 1.9%, fucose 0.19% and sialic acid 0.35%. Isoelectric point was determined as 7.9 by electrofocussing technique. This protein migrated with gamma globulin on electrophoresis. Hageman factor was found to be a powerful immunogen. Anti Hageman factor reacted with both a plasma and a serum protein but not with purified IgG.

SummaryIn the handling of thrombin activity is lost by freeze drying, and by dialysis of dilute solutions. Acetone drying can be done without loss of activity. Acetylated thrombin does not lose activity during freeze drying. Prothrombin activity is retained during freeze drying and during dialysis, but is lost with acetone drying and when dilute solutions are placed in ordinary glassware that is not lined with paraffin.