Direct Anticoagulant Activity Research Articles

Eco-friendly preparation of Bi2O3, Ag-Bi2O3 and Ag-Bi2O3-rGO nanomaterials and their photocatalytic H2 evolution, dye degradation, nitrite sensing and biological applications

The current studies demonstrate the preparation of Bi2O3, Ag-Bi2O3, Ag-Bi2O3-rGO nanocomposite using a clear and concise, environmentally sustainable, low-cost green combustion method employing citrus limon as fuel. The synthesised materials were characterised through XRD, UV-DRS, FT-IR, TGA, TEM, SEM and Raman studies. The synthesised Ag-Bi2O3-rGO composite exhibited much improved photocatalytic H2 evolution activity (432.7 μmolg-1h−1) which is 5.5 times greater than bare-Bi2O3 (78.6 μmolg-1h−1) and 4.2 times greater than Ag-Bi2O3 (101.2 μmolg-1h−1) NPs. Further, Bi2O3, Ag-Bi2O3 and Ag-Bi2O3-rGO composite degraded the MB dye by 63.96, 74.9 and 100 % respectively in 90 min. Ag-Bi2O3-rGO exhibited superior electrochemical nitrite sensing property (with the LOD of 19.792 μM). Ag-Bi2O3-rGO nanocomposite exhibited potential antiradical property with IC50 of 900 μg/mL Ag-Bi2O3-rGO was found to have potent direct haemolytic, anticoagulant, antiplatelet and antibacterial activities. This study illustrates a simple, environmental friendly green synthetic technique for large-scale synthesis of multifunctional Ag-Bi2O3-rGO nanocomposite.

Muscle Mass and Direct Oral Anticoagulant Activity in Older Adults With Atrial Fibrillation.

Direct oral anticoagulants (DOAC) are hydrophilic drugs with plasma levels inversely proportional to lean body mass. Sarcopenic patients with low muscle mass may be at risk for supra-therapeutic DOAC levels and bleeding complications. We therefore sought to examine the influence of lean body mass on DOAC levels in older adults with atrial fibrillation (AF). A prospective cohort study was conducted with patients 65 years of age or more receiving rivaroxaban or apixaban for AF. Appendicular lean mass (ALM) was measured using a bioimpedance device and a dual X-ray absorptiometry scanner. DOAC levels were measured using a standardized anti-Xa assay 4 hours after (peak) and 1 hour before (trough) ingestion. The cohort consisted of 62 patients (47% female, 77.0 ± 6.1 years). The prescribed DOACs were apixaban 2.5 mg (21%), apixaban 5 mg (53%), and rivaroxaban 20 mg (26%). Overall, 16% had supra-therapeutic DOAC levels at trough and 25% at peak. In the multivariable logistic regression model, lower ALM was independently associated with supra-therapeutic DOAC levels at trough (odds ratio per ↓ 1-kg 1.23, 95% confidence interval 1.02 to 1.49) and peak (odds ratio per ↓ 1-kg 1.18, 95% confidence interval 1.02 to 1.37). Addition of ALM to a model consisting of age, total body weight, and renal function resulted in improved discrimination for supra-therapeutic DOAC levels. Our proof-of-concept study has identified an association between ALM and DOAC levels in older adults with AF. Further research is needed to determine the impact of ALM on bleeding complications and the potential role of ALM-guided dosing for sarcopenic patients.

Reversal agents for use with direct and indirect anticoagulants.

The properties of three oral anticoagulant-specific reversal agents are reviewed, and guidance is presented to assist pharmacists in planning for the agents' introduction to the market. Idarucizumab, which received Food and Drug Administration approval in October 2015, is a humanized monoclonal antibody fragment that immediately neutralizes the anticoagulant effect of dabigatran, as evidenced by reduced unbound dabigatran concentrations and normalized coagulation tests. Preliminary Phase III trial results demonstrated a median maximum reversal of 100%, a median time to bleeding cessation of 11.4 hours, and normal intraoperative hemostasis in 92% of patients requiring anticoagulation reversal before an urgent procedure. Andexanet alfa is a factor Xa (FXa) decoy that binds to direct and indirect FXa inhibitors. In Phase III trials in healthy volunteers, andexanet alfa reduced anti-FXa activity by more than 90%, reduced the concentration of unbound direct FXa inhibitor, and inhibited thrombin generation. Ciraparantag is a reversal agent under development for reversal of anticoagulation with direct and indirect FXa inhibitors and certain factor IIa inhibitors; it exerts its effect through hydrogen bonding. Concerns for thromboembolic events directly related to administration of idarucizumab, andexanet alfa, or ciraparantag have not arisen. Pharmacists need to begin preparing for the introduction of these specific reversal agents through protocol development and provider education; in addition, pharmacy departments need to plan for procurement and storage. The specific reversal agents should be incorporated into antithrombotic stewardship or other clinical pharmacy programs for surveillance. As agents that provide rapid reversal of direct oral anticoagulant activity become available, advance planning will help hospitals to optimize their use.

The Study of the Mechanism of Action of the Newly Synthesized Direct Anticoagulant of Thiazoline Ammonium -4-Chlorophenyl-2-Hydroxy-4-Oxo-2-Butenoate

The aim of this research is to study the mechanism of action of the newly synthesized substance - Thiazoline Ammonium 4-Chlorophenyl-2-Hydroxy-4-Oxo-2-Butenoate (FS-169), which possesses the direct anticoagulant activity. The research was conducted with the use of «АPG4-02-P» coagulometer. For the purposes of the study the rabbit plasma was used. The results show that FS-169 effectively changes Partial thromboplastin time and prothrombin time indicators. However, FS-169 has no influence on Thrombin time. The substance also does not affect the activity of II and V coagulation factors. FS-169 effectively changes the activity of the X coagulation factor.

Antithrombotic activity of protein S infused without activated protein C in a baboon thrombosis model

Protein S (ProS) is an essential plasma protein that enhances the anticoagulant activity of activated protein C (APC). In vitro , purified native human Zn2+-containing ProS also exerts direct anticoagulant activity by inhibiting prothrombinase and extrinsic FXase activities independently of APC. We investigated antithrombotic effects of ProS infused without APC in a baboon shunt model of thrombogenesis that employs a device consisting of arterial and venous shear flow segments. In in vitro experiments, the Zn2+-containing human ProS used for the studies displayed >10-fold higher prothrombinase inhibitory activity and anticoagulant activity in tissue factor-stimulated plasma, and four-fold higher inhibition of the intrinsic pathway than the Zn2+-deficient ProS used. In the thrombosis model, ProS (33 μg/minute for 1 hour) or saline was infused locally; platelet and fibrin deposition in the shunt were measured over 2 hours. During experiments performed at 50 ml/minute blood flow, Zn2+-containing ProS inhibited platelet deposition 73-96% in arterial-type flow segments and 90-99% in venous-type flow segments; Zn2+-deficient ProS inhibited platelet deposition 52% in arterial-type flow segments and 65-73% in venous-type flow segments. At 100 ml/min blood flow rate, Zn2+-containing ProS inhibited platelet deposition by 39% and 73% in the respective segments; Zn2+-deficient ProS inhibited platelet deposition by 5% and 0% in the respective segments. Zn2+-containing ProS suppressed fibrin deposition by 67-90%. Systemic APC-independent ProS activity was significantly increased and thrombin-antithrombin complex levels were significantly decreased after infusion of ProS. Thus, infused human Zn2+-containing ProS is antithrombotic in primates, and may have therapeutic potential even in protein C-deficient human patients.

Protein C anticoagulant system—anti-inflammatory effects

Activated protein C (APC) plays active roles in preventing progression of a number of disease processes. These include thrombosis due to its direct anticoagulant activity which is likely augmented by its cytoprotective activity, thereby limiting exposure of procoagulant cellular membrane surfaces on cells. Beyond that, the pathway signals the cells to prevent apoptosis, to dampen inflammation, to increase endothelial barrier function, and to selectively downregulate some genes implicated in disease progression. Most of these functions are manifested to APC binding to endothelial protein C receptor (EPCR) allowing PAR1 activation, but activation of other PARS is also implicated in some cases. In addition to EPCR orchestrating these changes, CD11b is also capable of supporting APC signaling. Selective control of these pathways offers potential in new therapeutic approaches to disease.

Zn²(+) -containing protein S inhibits extrinsic factor X-activating complex independently of tissue factor pathway inhibitor.

Protein S (PS) has direct anticoagulant activity, independently of activated protein C (APC). The mechanisms underlying this activity remain unclear, because PS preparations differ in activity, giving rise to conflicting results. Some purification procedures result in loss of intramolecular Zn²(+) , which is essential for inhibition of prothrombinase. To investigate the inhibition of extrinsic factor (F)Xase by Zn²(+) -containing PS. Purified component extrinsic FXase assays were used to determine FXa generation in the presence and absence of PS and/or tissue factor pathway inhibitor (TFPI). Binding assays, immunoblots and thrombin generation assays in plasma supported the FXase data. Zn²(+) -containing PS potently inhibited extrinsic FXase in the presence of saturating phospholipids, independently of TFPI, whereas inhibition of extrinsic FXase by Zn²(+) -deficient PS required TFPI. Immunoblots for FXa and functional assays showed that Zn²(+) -containing PS inhibited primarily the quantity of FXa formed by tissue factor (TF)-FVIIa, rather than FXa amidolytic activity. Zn²(+) -containing PS, but not Zn²(+) -deficient PS, bound to TF with high affinity (K(dapp) = 41 nm) and targeted TF function. Binding of PS to FVIIa was negligible, whereas PS showed appreciable binding to FX. Increasing FX concentrations 10-fold reduced PS inhibition five-fold, suggesting that PS inhibition of FXase is FX-dependent. PS also exhibited TFPI-independent and APC-independent anticoagulant activity during TF-initiated thrombin generation in plasma. PS that retains native Zn²(+) also retains anticoagulant functions independently of APC and TFPI. Inhibition of extrinsic FXase by PS at saturating levels of phospholipids depends on PS retention of intramolecular Zn²(+) , interaction with FX, and, particularly, interaction with TF.

Les déficits congénitaux en protéine S ; difficultés diagnostiques

Protein S is a physiologic inhibitor of coagulation acting as a cofactor of activated protein C (APC) that inhibits factor Va and VIII. Approximately 60% of PS is bound to C4bBP, a protein of the complement system and only the free PS has a cofactor PCa role. Congenital PS deficiencies are diagnosed by immunologic dosage of free and total PS and functional assay evaluating APC cofactor activity. However, it has been demonstrated a direct anticoagulant activity of free PS, non-dependant of APC on the cascade coagulation and even PS bound to C4bBP seems to have anticoagulant properties. So, it appears that functional assays available estimate only a part of PS anticoagulant activities and, in addition, many interferences are reported with these tests (lupus anticoagulant, factor V Leiden, factor VIII excess…). Immunologic dosages are more reliable in spite of rare qualitative PS deficiencies that could be non-diagnosed. PS deficiencies are often difficult to diagnose because of an overlapping between normal and pathological values. Familial studies are necessary to prove the hereditary origin because there are several causes of acquired and sometimes persistent PS deficiencies (liver insufficiency, vitamin K absence, hormonal therapy in women, PS auto immune deficiency). About 200 different mutations were retrieved and, therefore, molecular studies are not of current practice. It is recommended currently to measure in first intention the free PS, if possible in association with PCa cofactor activity.

Protein S Is Antithrombotic In Vivo Independent of APC.

Protein S in plasma has APC-independent, direct anticoagulant activity. Preparations of purified protein S vary in this activity, depending on purification methods. Plasma and purified protein S form multimers that do not appear to differ in ability to inhibit prothrombinase activity (Heeb et al J Thromb Haemost 2006) except in the case of protein S purified using quarternary amine resins. We sought to determine if protein S purified by different methods has direct antithrombotic activity in vivo using a well-characterized baboon thrombosis model. A silicon rubber graft was placed between the femoral vein and artery and included a thrombogenic dacron segment that simulated thrombus formed under arterial flow conditions, followed by a teflon chamber of larger diameter that simulated thrombus formed under venous flow, then a region in which a propagating tail thrombus formed. 111In-labeled autologous platelets and 125I-labeled fibrinogen were infused prior to opening the graft. Protein S (2 mg/hr) or saline was infused 1 cm upstream circumferentially along the wall of the shunt for 1 hr after opening the graft. Deposition of platelets and fibrin was monitored for 2 hr. Eleven experiments were performed, using four different baboons. During the 1 hr infusion, affinity-purified recombinant and plasma-derived protein S inhibited platelet deposition by up to 62% in the arterial segment, up to 89% in the venous segment, and up to 100% in the propagating tail thrombus. Recombinant protein S purified by a different method using quaternary resins had weaker activity, consistent with in vitro clotting assay data. Protein S was more effective in experiments performed at flow rates of 40 ml/hr than in experiments performed at flow rates of 100 ml/hr. In other experiments, plasma-derived protein S was infused in the absence and presence of systemically administered antibody that blocked protein C activation (HPC4). Ex vivo blood samples showed that the ability of protein C to be activated was blocked by 91% or more by HPC4. During the 1 hr infusion, protein S suppressed at least 95% of platelet deposition in the absence of HPC4. In presence of HPC4, protein S suppressed platelet deposition by 79% in the dacron segment, and by 97% in the other segments. After protein S infusion was discontinued, a prothrombotic effect due to HPC4 was observed, in that platelet deposition occurred in the graft somewhat faster than in the saline control without protein S and HPC4, and the shunt occluded 20 min after cessation of protein S. Final fibrin deposition 2 hr after the beginning of the experiments was suppressed by protein S by 34 and 67% in the dacron segments and by 83 and 90% in the chambers, in the presence and absence of HPC4, respectively. Plasma from blood taken downstream of the shunt had modest but significant elevation of direct anticoagulant protein S activity, measured in endogenous thrombin potential assays, after local protein S infusion in the presence and absence of HPC4. APC cofactor activity was not significantly changed in any samples, but there was a trend to higher values after local protein S infusion. These experiments are the first to demonstrate that purified protein S has APC-independent, direct antithrombotic activity in vivo in primates, and suggest therapeutic potential for protein S as an antithrombotic agent.

Plasma contains protein S monomers and multimers with similar direct anticoagulant activity.

Protein S (PS) has activated protein C-independent, direct anticoagulant activity (PS-direct). We reported that both multimers and monomers of affinity-purified PS have PS-direct similar to that in plasma, in contrast to another report. We extended our studies to establish the molecular forms and activity of plasma PS. Novel ELISAs were developed that could detect only multimeric, not monomeric, PS because they employed the same monoclonal antibody for capture and detection. PS forms were also examined on native PAGE immunoblots. A new activity assay for PS-direct was applied to plasma and gel-filtered plasma fractions. Plasma PS multimers were clearly demonstrated using the ELISAs; 30-60% of free plasma PS appeared to be multimeric, a proportion similar to that of affinity-purified PS. On immunoblots, plasma PS multimers were more easily detected after gel filtration; plasma PS monomers and several apparent multimers comigrated with respective forms of affinity-purified PS. Antigen elution profiles after gel filtration of plasma revealed at least one major peak of apparent PS multimers (40-55% of free PS appeared multimeric). Biotin-factor Xa could bind to both plasma PS monomers and multimers. Strong plasma PS-direct was demonstrated, and plasma PS monomers, multimers, and PS-C4b-binding protein complexes each reconstituted PS-depleted plasma to similar levels of PS-direct. Our data are in disagreement with a report that monomeric purified PS has little PS-direct and that only monomeric PS exists in plasma. We find that both affinity-purified and plasma PS exist as monomers and multimers with similar PS-direct.

Ximelagatran, direct thrombin inhibitor, oral anticoagulants, thromboprophylaxis

Warfarin sodium is an effective oral anticoagulant drug. However, warfarin has a narrow therapeutic window with significant risks of hemorrhage at therapeutic concentrations. Dosing is difficult and requires frequent monitoring. New oral anticoagulant agents are required to improve current anticoagulant therapy. Furthermore, while warfarin is effective in venous disease, it does not provide more than 60% risk reduction compared with placebo in venous thrombosis prophylaxis and considerably lower risk reduction in terms of arterial thrombosis. Ximelagatran is an oral pro-drug of melagatran, a synthetic small peptidomimetic with direct thrombin inhibitory actions and anticoagulant activity. As an oral agent, ximelagatran has a number of desirable properties including a rapid onset of action, fixed dosing, stable absorption, apparent low potential for medication interactions, and no requirement for monitoring of drug levels or dose adjustment. It has a short plasma elimination half-life of about 4 hours in cases of unexpected hemorrhage or need for reversal. Its main toxicity relates to the development of abnormal liver biochemistry and/or liver dysfunction with "long-term" use of the drug. This usually occurs within the first 6 months of commencing therapy, with a small percentage of patients developing jaundice. The biochemical abnormality usually resolves despite continuation of the drug. The cause of this toxicity remains unknown. Clinical studies to date have shown that ximelagatran is noninferior to warfarin in stroke prevention in patients with nonvalvular atrial fibrillation, noninferior to standard therapy as acute and extended therapy of deep vein thrombosis (DVT), and superior to warfarin for the prevention of venous thromboembolism post-major orthopedic surgery. It has also been shown to be more effective than aspirin alone for prevention of recurrent major cardiovascular events in patients with recent myocardial infarction.

Direct anticoagulant activity of protein S‐C4b binding protein complex in Heerlen heterozygotes and normals

Plasma protein S normally circulates free (40%) or complexed with C4b-binding protein (PS-C4BP); only free protein S is a cofactor for activated protein C during factor (F) Va inactivation. Protein S-Heerlen lacks a carbohydrate group, leading to low plasma free protein S levels, but normal levels of PS-C4BP. Because protein S-Heerlen is not associated with thrombosis, we investigated whether PS-C4BP is directly anticoagulant in plasma and whether PS-Heerlen-C4BP has enhanced direct anticoagulant activity. An assay for protein S direct activity was applied to Heerlen-heterozygous plasmas. Free and complexed protein S were repeatedly isolated from normal and Heerlen-heterozygous plasmas and tested for direct anticoagulant activity in prothrombinase assays and in plasma. Heerlen-heterozygous plasmas were deficient in free and total protein S antigen but had normal to high protein S direct anticoagulant activity. Purified Heerlen-heterozygous PS-C4BP was 7-fold more potent than normal PS-C4BP in inhibiting full prothrombinase activity, and 22-fold more potent in inhibiting prothrombin activation in the absence of FVa; it also specifically prolonged plasma clotting times 14-fold more than normal PS-C4BP. Heerlen-heterozygous PS-C4BP did not compete for limiting phospholipids any better than normal PS-C4BP. However, ligand blots and surface plasmon resonance studies showed that Heerlen-heterozygous PS-C4BP bound more avidly to FXa than did normal PS-C4BP (apparent Kd = 4.3 nm vs. 82 nm). Plasma-derived PS-C4BP has direct anticoagulant activity in plasma and in purified systems. Enhanced direct activity of PS-Heerlen-C4BP may compensate for low free protein S levels and low cofactor activity in individuals with protein S-Heerlen.

Activated Protein C-Dependent and –Independent Anticoagulant Activities of Protein S Have Different Structural Requirements

ABSTRACT Plasma protein S exhibits multiple anticoagulant activities. About 20% of protein S normally circulates in a form that is cleaved in its thrombin-sensitive region (TSR, residues 47–72) and this cleaved protein S is inactive as a cofactor for activated protein C (APC). To clarify whether the same cleavage(s) in the TSR neutralizes both APC-cofactor and APC-independent direct anticoagulant activities, protein S was treated with several proteases, and activities and cleavages were monitored. Thrombin cleaved protein S first at Arg49, which abolished protein S APC-cofactor activity, but not APC-independent activity. A slower second thrombin cleavage at Arg70 abolished the direct prothrombinase inhibitory activity of protein S and its ability to bind phospholipids. Factor Xa cleaved protein S only at Arg60 and abolished APC-cofactor activity but not APC-independent anticoagulant activity. The snake venom enzyme Protac C efficiently cleaved protein S at two sites in the TSR, which impaired both types of protein S anticoagulant activity in the presence of phospholipids. Protac C-cleaved protein S did not compete with Factor Xa for limiting phospholipid surfaces but could still inhibit prothrombinase activity in the absence of phospholipids. Thus, the APC-cofactor activity protein S is significantly more sensitive to structural changes in the TSR than is the APC-independent activity of protein S.

Purification and characterization of three acidic, cytotoxic phospholipases A 2 from Indian cobra ( Naja naja naja) venom

Three acidic phospholipases A 2 (NN-I 2c-PLA 2, NN-I 2d-PLA 2 and NN-I 2e-PLA 2) were purified by successive chromatography of Indian cobra ( Naja naja naja) venom on CM–Sephadex C-25, Sephadex G-50 and QAE–Sephadex A-25 columns. They have molecular weights of 13 000–14 500 by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. They showed tryptophan specific fluorescence emission spectra (∼345 nm). All the three phospholipases A 2 were enzymatically highly active with specific activities 9–17 μmol min −1 mg −1. They were non-lethal to mice when injected intraperitoneally in doses up to 10 mg kg −1 body weight. They induced edema in mouse foot pads and were cytotoxic to Ehrlich ascites tumour cells. They did not exhibit direct haemolytic and anticoagulant activities.

The Interaction of Protein S with the Phospholipid Surface Is Essential for the Activated Protein C-independent Activity of Protein S

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in plasma and in purified systems. This anticoagulant effect of protein S can be explained either by a direct interaction of protein S with one of the components of the complexes and/or by the interference with the binding of these components to phospholipid surfaces. During our investigation we noted that protein S preparations purified in different ways and derived from different sources, expressed discrepant APC cofactor and direct anticoagulant activity. In order to elucidate these differences and to study the mechanism of the APC-independent activity of protein S, we compared the protein S preparations in phospholipid-binding properties and anticoagulant activity. The dissociation constant for the binding of protein S to immobilized phospholipids ranged from 7 to 74 nM for the different protein S preparations. APC-independent inhibition of both prothrombinase and tenase activity performed on phospholipid vesicles and in plasma showed a strong correlation with the affinity for phospholipids. The APC-independent activity could be abolished by monoclonal antibodies that were either calcium-dependent and/or directed against epitopes in the Gla-region of protein S, suggesting that the protein S-phospholipid interaction is crucial for the APC-independent anticoagulant function of protein S. Protein S preparations with a low APC-independent activity expressed a high APC-cofactor activity suggesting that the affinity of protein S for phospholipids is of less importance in the expression of APC-cofactor activity of protein S. We conclude that high affinity interactions of protein S with the membrane surface are essential for the direct anticoagulant activity of protein S and we suggest that inhibition of the prothrombinase and the tenase complex by protein S is a consequence of the occupation of the phospholipid surface by protein S molecules.

Thrombin, thrombomodulin and the control of hemostasis

Thrombin, thrombomodulin and the control of hemostasis John C. Giddings, Austin, 1994, RG Landes, 186 pages, $89.95. It is becoming increasingly apparent that modulation of hemostasis is important in clinical vascular surgery and that the endothelium plays a pivotal role in this process. Thrombin, Thrombomodulin, and the Control of Hemostasis is a detailed overview of the hemostatic process, with particular attention to the interactions between thrombin, thrombomodulin, associated coagulation factors, and the vascular endothelium. This volume is extremely well written and provides a thorough, up-to-date discussion of the topic. Although there are several authors, Giddings is the editor and primary author, and there is little overlap between chapters in this tightly organized book. Each subject is introduced with a review of the pertinent basic science, which leads in logical progression to extensive discussion of the relevant molecular biology, molecular genetics, and cellular biology. Thus, although the book is directed primarily at researchers and scholars, its information is accessible to those at all levels of understanding. The book begins by introducing the nature of the relationships between thrombin, thrombomodulin, and the vascular endothelium that serve to maintain the balance between hemostasis and fibrinolysis. Concise summaries of the processes of coagulation and fibrinolysis and the naturally occurring inhibitors of these processes follow and provide the basis for subsequent discussion of the interactions between the titular elements. Much of the clinically relevant material in this work is included in the chapter on inhibitors. The mechanism of action of fibrinolytic agents is explained, as is the molecular basis of the prothrombotic states associated with protein C and S deficiencies and antithrombin III deficiency. The book would assume greater value to the clinician with expansion of these sections. The discussion of protein C and S deficiencies was particularly limited. The main focus of the book, the molecular biology of the thrombin-thrombomodulin interaction, and the role of the vascular endothelium in this process, is the topic of subsequent chapters. The structure, function, and regulation of these and related entities is covered in detail, including ongoing research and controversies. Thrombin exerts its effects on the hemostatic process in a myriad of ways, including activation of factor V, fibrinogen, and platelets, as well as initiating anticoagulant and fibrinolytic pathways. Thrombomodulin activity takes three forms. Thrombomodulin cofactor activity refers to the enhancement of thrombin's activation of protein C; direct anticoagulant activity refers to the inhibition of activation of factor V, fibrinogen, and platelets by thrombomodulin; antithrombin III – dependent activity refers to thrombomodulin's ability to enhance antithrombin III – mediated thrombin inhibition. The vascular endothelium is an important participant in the hemostatic process, affording both a surface on which reactions can take place, as well as providing important components of the process itself. Substances synthesized and associated with vascular endothelium include von Willebrand factor, fibronectin, tissue factor, thrombomodulin, and plasminogen activator inhibitor 1. This book provides a complete and well-researched discussion of a complex topic. It will be of great value to researchers in the field of thrombosis and hemostasis and will provide an understandable discussion of the issues to the interested but uninitiated clinician.

Purification of a basic phospholipase A 2 from Indian saw-scaled viper ( Echis carinatus) venom: characterization of antigenic, catalytic and pharmacological properties

A major basic phospholipase A 2 was purified from the Indian saw-scaled viper ( Echis carinatus) venom by the combination of column chromatography and electrophoresis. The purified phospholipase A 2 (EC-IV-PLA 2) has a mol. wt of 14,000 by SDS-PAGE. It is a basic protein with a p I value between 7.2 and 7.6, and has a fluorescence emission maxima at 340 nm. It induces neurotoxicity and oedema in mice with an i.p. ld 50 of 5 mg/kg body weight. It is devoid of direct haemolytic, myotoxic, cytotoxic and anticoagulant activities. Rabbit polyclonal antibodies prepared against EC-IV-PLA 2 inhibited the in vitro enzymatic activity dose dependently, but did not neutralize the toxic effects of EC-IV-PLA 2 in experimental animals.

Effect of rabbit thrombomodulin on thrombin inhibition by antithrombin in the presence of heparin

Thrombomodulin acts as a cofactor for protein C activation by thrombin (PC activation cofactor activity) and inhibits thrombin-induced fibrinogen clotting (direct anticoagulant activity). In addition, rabbit thrombomodulin has been shown to promote thrombin inactivation by antithrombin (AT-dependent anticoagulant activity). However, a non-acidic form (i.e non-retarded on ion-exchange chromatography) of thrombomodulin generated by limited proteolysis retained only the PC activation cofactor activity. The acidic form (retarded on ion-exchange chromatography) of thrombomodulin is now shown to prevent the rapid inactivation of thrombin by antithrombin in the presence of heparin, presumably by preventing the formation of the ternary thrombin-AT-heparin complex. This effect was not observed with non-acidic thrombomodulin. When submitted to chondroitinase digestion, thrombomodulin was converted into an essentially non-acidic form that lacked both the AT-dependent and the direct anticoagulant activities but showed a PC activation cofactor function indistinguishable from that of native thrombomodulin. This chondroitinase-digested form did not prevent the catalytic effect of heparin on the inhibition of thrombin by AT. It is concluded that the acidic domain of rabbit thrombomodulin, a chondroitin (dermatan) sulfate glycosaminoglycan, interacts with a site of the thrombin molecule that is not involved in the protein C activation cofactor function, but is essential to the cleavage of fibrinogen or binding of heparin.

A basic phospholipase A from the venom of the australian king brown snake ( Pseudechis australis) showing diverse activities against membranes

1. 1. A basic phospholipase A (MSPA) was isolated from the venom of the Australian king brown snake, Pseudechis australis. 2. 2. MSPA had an approximate M r of 13,000 and consisted of a single polypeptide chain of 119 amino acid residues cross-linked by seven disulphide bridges. 3. 3. MSPA exhibited direct haemolytic, anticoagulant and myotoxic activities. 4. 4. Treatment of MSPA with p-bromophenacyl bromide modified a single histidine residue, resulting in complete loss of enzyme activity.

Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin.

Rabbit thrombomodulin displays three distinct blood anticoagulant activities: it promotes the activation of protein C by thrombin (protein C activation cofactor activity); it promotes the inactivation of thrombin by thrombin (direct anticoagulant activity). The effects on these activities of mouse anti-thrombomodulin monoclonal antibodies and of the heparin-neutralizing proteins, platelet factor 4, histidine-rich glycoprotein, and S-protein, were investigated. One of the antibodies, which did not influence the functional properties of thrombomodulin, was used as an immunoaffinity ligand for purification of the protein. Two other antibodies, which were found to abrogate the protein C activation cofactor activity of the purified thrombomodulin, also abolished the antithrombin-dependent and the direct anticoagulant activities. The heparin-neutralizing proteins all inhibited the two latter activities, albeit to a varying extent, but did not appreciably affect the activation of protein C. These results are interpreted in relation to our previous finding that rabbit thrombomodulin contains an acidic domain, tentatively identified as a sulfated glycosaminoglycan (Bourin, M.-C., Boffa, M.-C., Björk, I., and Lindahl, U. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5924-5928). It is proposed that the acidic domain interacts with thrombin at the protein C activation site and that this interaction is a prerequisite to the expression of direct as well as antithrombin-dependent anticoagulant activity. The interaction is not essential to, but compatible with, the activation of protein C. Experiments involving treatment of thrombomodulin with various glycanases or with nitrous acid, followed by measurement of anticoagulant activities, indicated that the acidic domain is constituted by a sulfated galactosaminoglycan and not by a heparin-related polysaccharide as previously suggested.