Houria I. Hassouna

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

Abstract 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

MECHANISMS OF THROMBOSIS IN SPINAL CORD INJURY

Many studies failed to identify a hypercoagulable imbalance in the blood factors or decreased anticoagulant activity. On the other hand, fibrinolysis, a process unrelated to hypercoagulability but closely related to endothelial cell integrity, is predictably altered and contributes to the persistence of venous occlusion by thrombosis. There is considerable evidence that interruption of neurologic impulses and the ensuing paralysis cause metabolic changes in blood vessels and that blood vessel changes are accountable for venous thrombosis. Altered venous competence with complete spinal cord injury manifests by a decrease in venous distensibility and capacity and an increase in venous flow resistance. Vascular adaptations to inactivity and muscle atrophy, rather than the effect of a nonworking leg-muscle pump and sympathetic denervation, seem to lead to the thrombosis; indicating that thrombosis resulting from venous incompetence cannot be reversed by anticoagulation alone.

Effects of prothrombin fragments on thrombin, on thrombin formation, and separation from Ac-globulin (factor V)

Abstract Bovine prothrombin fragment 2 (profragment 2) and prethrombin 1 were found in normal oxalated bovine plasma after extensive adsorption with barium carbonate. This implies continuous physiological utilization of prothrombin. Ac-globulin-profragment 2 complex was isolated from plasma by the method of Dombrose and Seegers. This purified complex dissociated at pH 7.2 in a buffer consisting of 0.15 M NaCl in 0.15 M phosphate buffer. The two proteins were separated by ultrafiltration. The profragment 2 passed through the filter while the Ac-globulin (Factor V) was retained in pure form. Purified bovine profragment 2 enhanced esterase activity of thrombin and retarded the clotting function, thus producing a large esterase/coagulation ratio in prethrombin 1 activation mixtures. Inhibition of the thrombin-fibrinogen reaction by profragment 2 must be taken into account in some assays for prothrombin or prethrombin 1. If profragment 2 is not bound in the activation mixture of a prothrombin assay, a low thrombin titer may be recorded. Contrary to a previous conclusion reached in this laboratory, there is no enzyme with only esterase activity in the following degradation sequence: Prothrombin → Prethrombin 1 → Prethrombin 2 → Thrombin. The esterase enhancement of thrombin and its coagulation inhibition by profragment 2 were neutralized by additions of purified Ac-globulin or by specific antibodies to profragment 2. The procoagulant or anticoagulant properties of purified bovine prothrombin fragment 1 (profragment 1) were studied. For that purpose, purified bovine profragment 1 was incubated with purified Ac-globulin, purified autoprothrombin C (Factor Xa), crude “cephalin,” and calcium ions. Due to profragment 1, the procoagulant function of the mixture diminished when tested against purified prothrombin as substrate, but the procoagulant function increased when tested against purified prethrombin 1 as substrate. Thus, depending upon conditions, profragment 1 can function as a procoagulant or as an anticoagulant.

Thrombophilia and Hypercoagulability

This is a review of less well-known aspects of thrombophilia and hypercoagulability as they relate to thrombosis. Thrombosis is an abnormal fibrin clot that develops in circulating blood with clinical symptoms of one or more arterial and/or venous obstructions exclusively identified by imaging techniques. The terms thrombophilia and hypercoagulability are often used indiscriminately when they are in fact separate entities. Thrombophilia is an inherited or acquired clinical phenotype manifesting in selected individuals as a greater risk to develop recurrent thrombosis at a younger age than the general population, with considerable differences in the magnitude of risks among individuals in the same family with the same thrombophilic gene defect. Hypercoagulability is a laboratory phenotype whereby in vivo activation of clotting, fibrinolysis, endothelial cells and platelets is identified in vitro by specialized clotting techniques and by specific antibodies directed at biomarkers of clotting activation and damaged vasculature. Hypercoagulability may be provoked by drugs to treat bleeding in hemophilia, by sepsis, inflammation, surgery, blood stasis, atherosclerosis, and it manifests selectively in inherited and acquired thrombophilia. A chronology of the discovery of acquired and inherited thrombophilia puts in perspective the data analyzed in two representative large family studies that address whether venous and arterial thrombosis are a necessary outcome in thrombophilia, and the question, whether patients with inherited antithrombin, protein C and protein S deficiencies need to be treated after a first episode of thrombosis. The liberal use of case vignettes emphasizes a close relationship and the distinction between thrombosis, thrombophilia and hypercoagulability.

STATIN DRUGS AND DIETARY ISOPRENOIDS AS ANTITHROMBOTIC AGENTS

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis. Additional processes may enhance nitric oxide production and induce other processes. Downregulation of thrombin-stimulated events should promote hypothrombotic or quiescent conditions that reduce cardiovascular disease, thus contributing to longevity.

Immunological aspects of some vitamin K-dependent factors and preparation of depleted plasmas.

Abstract The specificity of the immune response elicited in rabbits to purified bovine prothrombin, autoprothrombin III (Factor X), Factor IX, and Protein C was studied. The immunizing proteins were homogeneous on polyacrylamide gel electrophoresis. By immunodiffusion analysis antigenic similarities were not detected between them in spite of known stretches of amino acid sequence homology. Immunoelectrophoresis of bovine plasma and each purified protein gave identical single precipitin lines with the homologous antisera. This experiment established that the antibodies prepared against each one of the purified vitamin K-dependent factors studied reacted with a single component in bovine plasma. Immunologic crossreactions were found between human, bovine, dog, rat and chicken prothrombin, Protein C, Factor X, and Factor IX. They were not species specific. Purified bovine prothrombin has at least four antigenic determinant sites. Incubation of the purified proteins with suitable proteolytic enzymes yielded fragments and intermediate products which were purified and tested for their potential capacity to react with antibodies in the native antisera. In immunoelectrophoresis tests both prothrombin fragments 1 and 2 and the intermediate product prethrombin 1 gave immunoprecipitates which were separately identified by their differing mobilities, while the enzyme portion, thrombin, did not react with antithrombin serum. In addition to prothrombin, bovine plasma contained prethrombin 1, prothrombin fragments 1 and 2, as well as prethrombin 2 and/or thrombin. Autoprothrombin III m (Factor Xβ) and autoprothrombin C (Factor Xa) crossreacted with anti-autoprothrombin III and the active form of Protein C. Gamma globulins of antisera to prothrombin, to autoprothrombin III, to Factor IX, and to Protein C were fractionated by using saturation ammonium sulfate solution and coupled to activated Agarose A-15m beads. The insolubilized antibodies were used to absorb the antigen from the plasma. Each depleted plasma served as test material for a specific clotting factor. The prolonged prothrombin time of the prothrombin depleted plasma was normalized by additions of purified prothrombin, but not by purified prethrombin 1. The prothrombin time of autoprothrombin III depleted plasma was shortened by fresh plasma, purified autoprothrombin III, and by purified autoprothrombin III m (Factor Xβ). In the case of Factor IX depleted plasma, the partial thromboplastin time was used. The depleted bovine plasmas are suitable for assay purposes. They serve the same role as the corresponding deficient plasma in bioassay coagulation tests.

Bovine antithrombin-III: Miscellaneous observations

Abstract Bovine antithrombin-III was purified by two procedures. With the ultracentrifuge we found S 20,w = 4.18 − 0.015X. It was found to have an NH 2 -terminal histidine residue, neutral sugars, sialic acid and disulfide bonds. It formed a complex with purified bovine thrombin in a 1:1 molar proportion with residual quantities of either component remaining. It formed a 1:1 molar complex with purified Factor Xa. In the ultracentrifuge this complex sedimented as a single component with S 20,w = 4.65 − 0.034X. Antibodies were used to deplete plasma of antithrombin-III. The depleted plasma progressively inactivated purified thrombin.

EDITORIAL COMMENT: Blood Stasis, Thrombosis and Fibrinolysis

Blood stasis is a very common occurrence associated with many of our daily activities. Prophylaxis against development of thrombosis in high-risk conditions is well established. In this issue of Hematology/Oncology Clinics of North America, international experts present new and provocative information on the basic mechanisms, diagnosis, and management of blood stasis, thrombosis, and fibrinolysis. Reviews of pertinent topics by three physicians in training also are included. I hope that the information provided will expand our understanding of this interesting subject.

Contents Vol. 18, 2009

493 Abstracts of Award-Winning Posters, 14th Annual Health Sciences Poster Conference, Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait, April 21–23, 2009 498 Abstracts of Theses Approved for the MSc and PhD Degrees at the Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait 504 Announcement 505 List of Reviewers 507 Author Index Vol. 18, 2009 510 Subject Index Vol. 18, 2009

Assessment of specific risks for the recurrence of deep vein thrombosis: a case report

IntroductionVenous thromboembolism is a multifactorial disease defined by multiple interactions between genetic and environmental components. It is managed by oral anticoagulation with warfarin sodium (Coumadin), a drug that targets the vitamin K epoxide reductase to prevent the recycling of vitamin K epoxide to the reduced form of vitamin K. The reduced form of vitamin K is an essential cofactor in the formation of active clotting factors II, VII, IX, X and regulatory factors protein C, and cofactor protein S through gamma-glutamyl carboxylation. The duration of Coumadin treatment, three to six months or life-long, should be based on the individual risk for recurrent deep vein thrombosis and on the associated increased risk for bleeding complications.Case PresentationA previously healthy 50-year-old white male developed a deep vein thrombosis consequent to surgical placement of a titanium rod to correct a fracture of the femur and he was maintained for over a year on daily oral doses of Coumadin 9 mg and aspirin 325 mg. When he began to bruise spontaneously with multiple large hematomas appearing without provocation, he requested that his primary care physician reconsider the anticoagulation. Because of his age, sex, and the possibility of an inherited or acquired anticoagulant protein deficiency he was maintained on Coumadin and a thrombophilia work up was ordered. Test results were interpreted as deficiencies in both protein C and protein S and he was instructed that life-long therapy with Coumadin was necessary. Is this a correct evaluation by his primary care physician?ConclusionThis case illustrates that Coumadin, a vitamin K agonist, was exerting a therapeutically acceptable negative influence on plasma activity levels of vitamin K-dependent protein C and protein S. Relying on the outcome of a thrombophilia work-up for a decision to maintain or cease Coumadin treatment of patients at risk for recurrent deep vein thrombosis has pitfalls that can be avoided. The use of real-time B-mode venous ultrasonography to verify complete restoration of venous flow before ceasing Coumadin treatment is not always considered in the long-term management of a patient with a first thrombosis, despite the well documented significant risk of deep vein thrombosis recurrence associated with an unresolved thrombosis.