Autoprothrombin II-A Research Articles

Assessment of Antithrombin III and Protein C in Saudi Myocardial Infarction Patients.

Myocardial infarction (MI) or acute myocardial infarction (AMI), commonly referred to as a heart attack, happens when the blood flow to part of the heart stops, causing damage to the heart muscle. Chest pain or discomfort that may flow into the shoulder, arm, back, neck, or jaw is the most common symptom. Most MIs occur due to coronary artery disease. High blood pressure, smoking, diabetes, lack of exercise, obesity, high blood pressure, poor diet, excessive alcohol use, etc. are risk factors. Antithrombin III (AT III) is a glycoprotein produced by the liver and consists of 432 amino acids. Protein C, also referred to as autoprothrombin IIA and factor XIV of blood coagulation, is a zymogen. In regulating anticoagulation, inflammation, cell death, and maintaining the permeability of blood vessel walls in humans and other animals, the activated form of protein C plays an important role. A case control study was conducted in Saudi Arabia to determine the levels of AT III and protein C in Saudi MI patients. Samples (n = 150) from MI patients as well as healthy controls (n = 50) were collected (2.5 mL of venous blood for sandwich ELISA). This study showed that the mean AT III and protein C levels were within normal levels in patients (86 ± 19.63 and 76.20 ± 30.64, respectively). A comparison of mean AT III and protein C levels in patient and control groups showed no significant difference (p-value = 0.26, 0.2, and 0.19, respectively). The results also showed that some of the samples had low levels of AT III (8.7%) and protein C (11.3%). A deficiency of AT III and protein C were not strong significant risk factors for myocardial infarction.

Autoprothrombin ii-a, thrombin, and epinephrine: interrelated effects on platelet aggregation.

Platelets aggregate with thrombin-free autoprothrombin II-A. Aggregation was dependent on an intact release mechanism since inhibition of aggregation occurred with adenosine, colchicine, or EDTA. Autoprothrombin II-A reduced the sensitivity of platelets to aggregate with thrombin, but enhanced epinephrine-mediated aggregation. Autoprothrombin II-A directly competes for membrane sites sensitive to thrombin. It allows complexes to from with epinephrine. There was no absolute requirement for autoprothrombin II-A since epinephrine aggregated dog platelets at the identical optimum concentration following Coumadin treatment.

Activated protein C.

Protein C is a vitamin K-dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to down-regulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor-1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APC's direct effects on various cell types, it is clear that APC's molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells.

Effects of Autoprothrombin II-A on Epinephrine-Induced Platelet Aggregation of Normal- and Coumadin-Treated Dogs

Platelet-rich plasma from dogs and from coumadin-treated dogs aggregated at the same optimum concentration of epinephrine. Neither protein C nor its active form called autoprothrombin II-A was necessary for aggregation of dog platelets with epinephrine. For platelet aggregation, suboptimal concentrations of epinephrine were potentiated by addition of purified autoprothrombin II-A. The latter, by itself, induced platelet aggregation in high concentration.

Some properties of autoprothrombin II-A anticoagulant.

Autoprothrombin II-A anticoagulant was isolated from bovine prothrombin. Purified prothrombin was applied to DEAE-cellulose chromatography after incubation with thrombin. Four protein peaks were obtained where the third peak corresponded to the anti-coagulant effect. The fractions under the third peak were pooled together and the anticoagulant effect was evaluated with different methods. From 25,470 +/- 2,800 U of prothrombin, 5,800 +/- 1,400 U of inhibitor were obtained. The inhibitor was found to be most effective at pH 7.2--7.8. In vitro, the inhibitor inhibited the thrombin time and the plasma clotting time highly significantly but had no effect on euglobulin lysis time and fibrin plates. In vivo, when injected into rabbits, the inhibitor effect was also significant on the same tests. The autoprothombin II-A anticoagulant had a protective effect on DIC formation with rabbit brain thromboplastin administration. This protective effect was found to be statistically significant.

Relationship of “new” vitamin K-dependent protein C and “old” autoprothrombin II-A

Vitamin K-dependent Protein C recently isolated by Stenflo ( J. Biol. Chem. 251, 355, 1976) was found to be related to autoprothrombin II-A by comparing amino acid analysis, molecular weight, immunochemical properties, and inhibitory activity in blood coagulation. Our purified Protein C converted to autoprothrombin II-A with purified thrombin in association with loss of a small peptide(s). Protein C or autoprothrombin II-A served as cofactors for epinephrine induced platelet aggregation in platelet rich plasma taken from dogs heavily dosed with warfarin. It is suggested that the designation Factor XIV be assigned to Protein C and Factor XIVa to autoprothrombin II-A with the following meaning: Factor XIV Thrombin Factor XIVa

Improved procedures for the purification of selected vitamin K-dependent proteins.

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4,340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8,200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III thrombin leads to Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin, and furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was also not converted to the active enzymes when the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The "activation peptide" released by RVV-X from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same "actid of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.

The ultrastructure of bovine autoprothrombin II-A

Abstract The morphology of bovine autoprothrombin II-A was investigated, before and after treatment with 8M urea, using transmission electron microscopy in combination with the negative staining procedure. Individual molecules were easily observed and many were composed of 5 subunits. The molecules of autoprothrombin II-A had a mean diameter of 80 A with 79% of the population in the range from 70 to 80 A. The average diameter of these molecules was increased to 100 A when samples of the bovine autoprothrombin II-A were spread on grids pretreated with 4% sodium phosphotungstate. Following urea treatment, the mean diameter of the modified bovine autoprothrombin II-A molecules was reduced to 74 A with 78% of the molecular population in the range from 50 to 89 A. The subunit arrangement was retained post urea.

Autoprothrombin II-A: Thrombin removal and mechanism of induction of fibrinolysis

Traces of thrombin were removed from purified preparations of autoprothrombin II-A. This thrombin-free protein, when infused intravenously into a dog, retarded blood coagulation, stopped autoprothrombin III (Factor X) utilization, and induced plasmin activity. There were no associated changes in heart rate, respiratory rate or blood pressure. In experiments with purified components, it was found that autoprothrombin II-A possessed only weak ability to directly activate either bovine or canine plasminogen to plasmin, but did depress the function of plasmin inhibitors. Autoprothrombin II-A thus induced fibrinolysis by an indirect mechanism. There is clearly a pathway from the coagulation system to fibrinolysis. Autoprothrombin II-A retarded the activity of soybean trypsin inhibitor.

Purification and some properties of autoprothrombin II-A: An anticoagulant perhaps also related to fibrinolysis

Methods were developed for the assay and isolation of autoprothrombin II-A. The purified protein retarded blood coagulation in vitro as well as in vivo and induced fibrinolysis. It is tentatively concluded that the protein originated from prothrombin and possessed inhibitor-activator qualities respectively as anticoagulant and fibrinolytic agent. To obtain Auto II-A, concentrated prothrombin complex was digested with purified thrombin. Chromatography on a DEAE-cellulose column separated prethrombin, Auto II-A, and autoprothrombin III (Factor X). The inhibitor-activator was then purified by using a Sephadex G-100 or G-100 superfine column. A single component was indicated by polyacrylamide gel electrophoresis and by ultracentrifugation. The equation for S 20,w 0 was 3.9 + 0.0114X. Isoelectric point = pH 3.9. It is a competitive inhibitor of autoprothrombin C (Factor X a). N-terminal amino acids were Ile and Gly. An amino acid analysis was compared with a new amino acid analysis of purified prothrombin. The latter had 518 amino acid residues per mole. The protein contained sialic acid, neutral sugars, and amino sugars. Serum from rabbits immunized with purified prothrombin crossreacted with purified Auto II-A. A working theory is proposed as follows: Thrombin functions in a negative feedback system which retards thrombin formation due to the production of prethrombin and Auto II-A. Platelet factor 3 also produced prethrombin and Auto II-A. This feedback system could be the molecular basis of latent coagulation and fibrinolysis. In accelerated coagulation, the latent coagulation sequences are by-passed with massive formation of fibrin.

Prothrombin activation: Ac-Globulin, lipid, platelet membrane, and autoprothrombin C (factor X a) requirements

Thrombin formation was studied with the use of purified prothrombin, prothrombin complex, prethrombin, Ac-globulin, platelet factor 3, autoprothrombin C, autoprothrombin III (Factor X), and thrombin, With Auto-C alone, there was only a fractional yield of the potential thrombin from purified prothrombin complex or purified prothrombin. This low yield of thrombin was partly due to the simultaneous formation of prethrombin and autoprothrombin II-A, which is a competetitive inhibitor of Auto-C. Echis carinatus venom converted prothrombin completely to thrombin. In a standard reaction mixture consisting of prothrombin, Auto-C, Ac-G, calcium ions and crude “cephalin”, thrombin formation was rapid and complete. By progressively reducing the concentration of any of the five components, there was a corresponding reduction in yield and rate of thrombin formation. From the thrombin yield, the concentration of the limited reactant was determinable. The basic molecular biology, which applies to several clinical conditions, is outlined. With purified prothrombin as a substrate, Auto-C functioned most effectively in thrombin production in the presence of calcium ions, Ac-G, and lipids. The Km' was 9.50 × 10 −6 M prothrombin. The maximum rate of conversion of prothrombin to thrombin by Auto-C was an order of magnitude 120 times greater than that for prethrombin. Ac-G was regarded as a determiner protein becaue it modified the specificity of Auto-C. The lipid requirement was fulfilled equally well by platelet factor 3, cephalin, thromboplastin, and platelet membranes. One of the three basic reactions of blood coagulation may thus be related to the function of membranes. In a mixture consisting of prothrombin, Auto-III, thromboplastin, Ac-G and calcium ions, all of the prothrombin was converted to thrombin. No Factor VII was required.

Antithrombin II Anticoagulant (Autoprothrombin II-A)

SummaryAutoprothrombin II-A activity frequently develops when the autoprothrombin II fraction of prothrombin is dried from the frozen state. When the anticoagulant activity develops, the N-terminal amino acid arginine is found, but not when the anticoagulant activity does not develop. The anticoagulant autoprothrombin II-A retarded the conversion of prothrombin to thrombin when three different procoagulants were used; namely, platelet cofactor I, autoprothrombin II, and lung extract. The procoagulant power of the platelet cofactor I is most easily depressed. It is postulated that autoprothrombin II-A activity develops when prothrombin activates and that platelet cofactor I is a receptor of the anticoagulant. In that way the other procoagulants are not ordinarily inhibited by the autoprothrombin II-A. As a further extension of this speculation, attention is brought to the idea that autoprothrombin II-A could be present in certain hemorrhagic diseases. In the PTA patient there is no autoprothrombin II and most likely no autoprothrombin II-A. The close chemical relationship of these two prothrombin derivatives thus may account for the multiple deficiency.

Activation of Purified Prothrombin to Autoprothrombin I or Autoprothrombin II (Platelet Cofactor II or Autoprothrombin II-A)

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.