Blood Coagulation Enzymes Research Articles

Active site-specific immunoassays

This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

Characterization of the Anticoagulant Activities from a Brazilian Arrow Poison

A recent report indicated that an arrow poison used by the native Indians of Rondonia, Brazil, to kill small animals was associated with profuse bleeding. The arrow poison was prepared from the bark of a tree, known locally as Tike-Uba. We have obtained bark and sap specimens from this tree and have characterized a potent anticoagulant activity in both the crude bark and sap samples as well as in more highly purified preparations. An aqueous extract of the bark significantly prolongs both prothrombin times and activated partial thromboplastin times in plasma based assays. Further fractionation of the bark extract and sap by molecular weight indicated that all of the anticoagulant activity could be isolated in a molecular weight fraction of equal to or greater than 30,000 daltons. The anticoagulant activity was also further purified by C-18 reverse phase chromatography. When highly purified preparations of the anticoagulant activity from the Tike-Uba tree were examined in specific blood coagulation enzyme assays utilizing chromogenic substrates, the highest inhibitory potency was found versus thrombin, followed by factor Xa. These studies establish the presence of a compound(s) in a Brazilian arrow poison, which potently disrupts mammalian blood clotting, and which may account for some of the observed toxicities associated with the arrows.

7 Factor V: A prototype pro-cofactor for vitamin K-dependent enzyme complexes in blood clotting

The relative abundance of factor V, factor X and prothrombin has enabled detailed analyses of the prothrombinase complex. Determination of the primary structure for factor V has provided the basis for examination of structure-function relationships. The imminent in vitro expression of recombinant factor V will provide the opportunity for site-specific mutagenesis and a verification of these structure-function relationships. A comparison of the physical properties and primary structures for factors V and VIII has revealed extensive similarities in these two cofactor proteins. This observation indicates that a direct application of the technology developed for the analysis of prothrombinase will lead to an equal understanding of the factor Xase complex. Whether similar relationships exist for other blood coagulation enzyme complexes remains to be determined.

Family with Factor X Variant Defective Only in Factor VII Activation (X + Riyadh)

This is the first report, to our knowledge, of a familial occurrence of the abnormal variant of factor X which is defective only in its reaction with factor VII in the extrinsic coagulation pathway...

Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells.

Tumor necrosis factor (TNF) dramatically alters the levels of various surface components of the blood vessel wall, such as blood coagulation enzyme receptors, leukocyte-adhesive receptors, and class 1 major histocompatibility complex antigens, which may have relevance to its effects in septic shock, angiogenesis, and tumor growth. However, the precise mechanism by which the cytokine is able to accomplish this remodeling of the endothelial cell surface has not been defined. We have demonstrated that exposure of bovine and human endothelial cells to TNF leads to suppression of the functional cell surface thrombin receptor, thrombomodulin (TM), and TM mRNA of virtually identical magnitude. The cytokine has no significant effect on the stability of TM mRNA or endothelial receptor turnover. Nuclear run-on studies reveal that the treatment of endothelial cells with TNF for short periods reduces TM gene transcription to as little as 3% of control values and that this inhibition does not require new protein synthesis.

Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells.

Tumor necrosis factor (TNF) dramatically alters the levels of various surface components of the blood vessel wall, such as blood coagulation enzyme receptors, leukocyte-adhesive receptors, and class 1 major histocompatibility complex antigens, which may have relevance to its effects in septic shock, angiogenesis, and tumor growth. However, the precise mechanism by which the cytokine is able to accomplish this remodeling of the endothelial cell surface has not been defined. We have demonstrated that exposure of bovine and human endothelial cells to TNF leads to suppression of the functional cell surface thrombin receptor, thrombomodulin (TM), and TM mRNA of virtually identical magnitude. The cytokine has no significant effect on the stability of TM mRNA or endothelial receptor turnover. Nuclear run-on studies reveal that the treatment of endothelial cells with TNF for short periods reduces TM gene transcription to as little as 3% of control values and that this inhibition does not require new protein synthesis.

Cofactor proteins in the assembly and expression of blood clotting enzyme complexes.

Cofactor proteins in the assembly and expression of blood clotting enzyme complexes.

Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants.

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.

Biosynthesis of factor XIII A and B subunits.

Factor XIIIa (FXIIIa), the blood coagulation enzyme, functions as a transglutaminase to covalently stabilize fibrin clots by introducing γ-glutamyl-e-lysyl peptide bonds between fibrin molecules (for detailed reviews, see references 1 and 2). The FXIII proenzyme exists in two zymogenic forms.3 Extracellular or plasma FXIII is composed of two subunits, A(Mr =80,500)4 and B(M =76,500),5 associated non-covalently as a tetrameric complex, A2B2.6,7 Intracellular FXIII is a dimer of only A subunits, A2.6 The A chains in both zymogens are identical and contain the active center sulfhydryl group.7,8 Upon activation by thrombin in the presence of calciurn ions, both zymogens give rise to the same enzymatic activity, FXIIIa.8,9

Heparin promotes the inactivation of antithrombin by neutrophil elastase.

Heparin is an acceleratory cofactor for antithrombin, a circulating inhibitor of blood coagulation enzymes. The presence of heparin on blood vessel walls is believed to contribute to the nonthrombogenic properties of those surfaces. In apparent opposition to this function, heparin was found to greatly accelerate the in vitro inactivation of antithrombin by neutrophil elastase. Inactivation rates in solution were potentiated several hundredfold by specific heparin fractions with anticoagulant activity. Although the data suggest that a heparin-antithrombin complex is essential for the inactivation by elastase to occur, the enzyme itself interacts tightly with heparin. These results suggest a mechanism which, if operating in vivo, could lead to a localized neutralization of the anticoagulant function of heparin at the endothelial surface.

Isolation and properties of blood clotting enzyme from the venom of Lachesis muta: F. Aragon-Ortiz, 1 F. Gubensek2 ( 1Department of Biochemistry, School of Medicine, University of Costa Rica, San Jose, Costa Rica, and 2Department of Biochemistry, J. Stefan Institute, Ljubljana, Yugoslavia)

Isolation and properties of blood clotting enzyme from the venom of Lachesis muta: F. Aragon-Ortiz, 1 F. Gubensek2 ( 1Department of Biochemistry, School of Medicine, University of Costa Rica, San Jose, Costa Rica, and 2Department of Biochemistry, J. Stefan Institute, Ljubljana, Yugoslavia)

Anticoagulant Effect of Sulphated Poly/Vinyl Alcohol-Acrylic Acid/Copolymers

Copolymers of poly/vinyl alcohol-acrylic acid/ with various content of sulphate and carboxyl groups have been synthetized and tested for their in vitro effect on blood coagulation. The results indicate that the sulphated copolymers display an inhibitory effect but there is a requirement in the charged groups of about 20% in the molecule to possess effective anticoagulation. The biochemical mechanism of their actions is complex, i.e. the inhibition of blood clotting is a consequence of both the accelerated inactivation rate of thrombin by antithrombin-III and a direct inhibitory effect on the thrombin-fibrinogen reaction. Moreover, additional effects may occur on other blood coagulation enzymes than thrombin, depending on the chemical composition of the copolymers.

“Clotspeed,” a mathematical simulation of the functional properties of prothrombinase.

Prothrombinase is a Ca2+-dependent, 1:1, enzymatic complex of Factor Xa and Factor Va that assembles on the surface of negatively charged phospholipid vesicles or platelets. It catalyzes the proteolytic conversion of prothrombin to the blood-clotting enzyme thrombin. Experimentally determined kinetic parameters, plus Kd and n values for the interaction of substrate, cofactor (Factor Va), and serine protease (Factor Xa) for both phospholipid and each other, were used to develop a model that simulates the functional properties of the enzymatic complex. Through the use of a desk-top computer and a program designated "Clotspeed," the distribution of enzymatic components and substrate between the bulk fluid and phospholipid is determined for a given set of initial concentrations of reaction components. Simulated reaction rates are then calculated from the calculated distributions, fractional binding, and local and bulk concentration of reactants. Predicted behavior includes formal Michaelis-Mentenlike properties for the reaction, increasing apparent Km with increased levels of phospholipid, and apparent inhibition by excess substrate, enzyme, and phospholipid. Inhibition by excess enzyme and phospholipid was demonstrated experimentally in quantitative agreement with predicted results. The model is useful in that it rationalizes well the seemingly unusual properties of prothrombinase in straightforward physical terms, provides a means of rationally choosing experimental conditions to both further test and refine the model, and explores the properties not only of prothrombinase but also other blood-clotting or surface-bound enzymatic complexes.

Computer-generated models of blood coagulation factor Xa, factor IXa, and thrombin based upon structural homology with other serine proteases.

Computer-generated molecular models of the trypsin-like domains of blood coagulation factor IXa, Factor Xa, and thrombin have been prepared. These hypothetical models are based upon the sequence homology of the blood coagulation enzymes with the pancreatic serine proteases and the known three-dimensional structure of the pancreatic serine proteases. The internal structures and active sites of these enzymes are highly conserved. The high degree of substrate specificity which characterizes the blood coagulation enzymes appears to be defined not entirely by the active site, but by the unique molecular surface surrounding the active site of each enzyme. Several regions which demonstrate high sequence variability among these enzymes likely participate in forming the putative extended substrate binding sites.

Blood coagulation changes during effective thrombolysis using urokinase and heparin

The efficacy and the safeness of a new urokinase dosage regimen (loading dose 250,000 IU, initial maintenance dosage 2,000 IU/kg/h in combination with heparin) was studied in ten cases of deep vein thrombosis with regard to the changes of the blood coagulation and fibrinolytic enzyme system. The coagulation analyses demonstrated a pronounced activation of the fibrinolytic system with a statistically significant (p < 0.05) shortening of the euglobulin clot lysis time and increase of the FDP. The fibrinogen concentration ranged from 50 – 100 mg/dl already after 12 – 36 hours. Plasminogen was reduced by 63%, α 2-macroglobulin by 32% and factor VIII:C by 42% (p < 0.05 each). The decrease of fibrinogen (Clauss method) related well to the method of Ratnoff and Menzie, the reduction of plasminogen and the shortening of the euglobulin clot lysis time. According to our data, a sufficient plasma fibrinogenolytic activity may permit on its own an assessment of an adequate therapy with urokinase and the requirements of dose adjustment. The dosage regimen applied here proved effective and readily controllable. Relevant side reactions were not observed.

The lipids of red cell membranes. Compositional, structural and functional aspects.

The structural matrix of the erythrocyte membrane is provided by a lipid bilayer containing more than one hundred different molecular species of lipid. The phospho- and glycolipids are distributed over the two halves of the bilayer in a highly asymmetric fashion; the outer monolayer consisting of all the glycolipids and the greater part of the two choline-containing phospholipids, whereas the amino-containing phospholipids dominate the inner monolayer. Functional implications of this arrangement are still poorly understood, but some of them may be found in blood coagulation and lipid-requiring membrane-bound enzymes. The bilayer exhibits a high degree of stability as is illustrated by 31P-NMR studies of membranes treated with phospholipases. However, selective modification of the molecular species of lecithin in the outer monolayer may lead to such destabilizations that the structural integrity of the membrane is lost and the cells haemolyse.

Chromogenic substrate assays: Present and potential applications

Chromogenic substrates are now available for the study of many aspects of blood coagulation and related enzyme reactions in blood. These substrates have opened up new vistas, including ease of control of routine anticoagulant therapy. The overall impact on routine haemostatic testing will probably be immense since automated systems may be used as an initial screen for patients with haemostasis problems. This applies not only to the coagulation but also for related systems such as the fibrinolytic pathway. The major point one must remember in their use is that activity directed against these substrates may not be identical with the biological activity one wishes to examine. This must make one cautious in their interpretation in the same way as one would be cautious in interpreting the results employing antibodies for antithrombin III levels. A great advantage of chromogen substrate techniques is their accuracy and reproducibility. Some of these points will be illustrated with investigations in our laboratory with the chromogenic substrate S2238. This substrate can assay thrombin formation in a thrombin generation system consisting of recalcified platelet rich plasma. Early in these studies it was shown that S2238 recognized thrombin activity long after the biological activity of the enzyme had disappeared against fibrinogen. The reason for this is because thrombin is inactivated in plasma initially by binding α₂ microglobulin, and that thrombin so bound can still attack S2238. This is a good example of the possible lack of correlation between biologic and substrate activity in certain circumstances. Such property however, would not exclude these agents from being very useful in a general automated screen of clotting problems in large numbers of patients coming through teaching hospitals. One could identify patients with haemostatic problems which might then be investigated by standard coagulation techniques.

Stability differences between high coagulant (alpha) and noncoagulant (gamma) human thrombins. Denaturation.

Human alpha-thrombin, the two (covalently linked)-chain, highly coagulant blood-clotting enzyme was compared with its noncoagulant, yet estero/amidolytically active derivative, gamma-thrombin, a three (noncovalently associated)-domain enzyme which results from two proteolytic cleavages of the coagulant a form. Studies of their denaturation behavior by Tos-Arg-OMe esterase activity, by intrinsic fluorescence, by fluorescence of active serine-directed dansyl labels, and by monitoring the ESR of a fluorosulfonylphenyl spin-labeled inhibitor clearly demonstrated the reduced stability of the noncovalently associated gamma-thrombin form. At pH 6.5, 0.75 M NaCl, gamma-thrombin unfolds in approximately 2. 1 M urea while the more stable a form denatures at approximately 4 M urea. By monitoring active serine probes (spin label or fluorescent labels), these transitions were slightly lower, 1.0 +/- 0.1 and 2.8 +/- 0.2 M urea for spin-labeled gamma- and alpha-thrombins, respectively. Similar behavior was found for the same spin-labeled derivatives in guanidine HCl with unfolding transitions of 0.4 M and 1.0 M for spin-labeled gamma- and alpha-thrombin, respectively. These differences in structural stabilization serve as a good physical diagnostic for the two thrombin species.

The influence of pregnancy upon blood coagulation and plasma fibrinolytic enzyme function

Fibrinogen catabolism has been studied in 27 patients from early pregnancy through delivery and up to 4 months post partum using the noninvasive technique of plasma fibrinogen chromatography. This technique quantifies in plasma the percentage and concentration of high-molecular weight fibrinogen or fibrinogen/fibrin complexes (HMWFC), which reflect the rate of fibrin formation in vivo, native fibrinogen, and derivatives of fibrinogen smaller than the native molecule resulting from the actions of the enzyme plasmin on fibrinogen, an assay reflecting the rate of fibrinogenolysis. Plasma fibrinogen chromatographic findings were similar to control findings during the first gestational month but during the second gestational month plasma HMWFC percentage and concentration were elevated twofold to threefold over control values and thereafter increased with gestational age, being fivefold control values prior to delivery. Plasma fibrinogen increased by the third month and its increase was significantly (p < 0.01) correlated with increase of plasma HMWFC. Fibrinogen first-derivative concentration was mildly elevated during pregnancy. These results demonstrate that intravascular fibrin deposition, presumably involving the uteroplacental circulation, is the earliest and most persistent alteration in blood coagulation function noted during pregnancy. Further, the fivefold increase in fibrin deposition during pregnancy over the physiologic, nonpregnant state is accompanied by a significant increase in compensatory fibrinolysis.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

It is shown that in a system where thrombin acts on fibrinogen, the clotting time can be used to assess coagulation velocity because clotting time and clotting velocity are inversely proportional.