Calibration of a subcutaneous amperometric glucose sensor. Part 1. Effect of measurement uncertainties on the determination of sensor sensitivity and background current

The calibration of a continuous glucose monitoring system, i.e. the transformation of the signal I(t) generated by the glucose sensor at time (t) into an estimation of glucose concentration G(t), represents a key issue. The two-point calibration procedure consists of the determination of a sensor sensitivity S and of a background current I(o) by plotting two values of the sensor signal versus the concomitant blood glucose concentrations. The estimation of G(t) is subsequently given by G(t) = (I(t)-I(o))/S. A glucose sensor was implanted in the subcutaneous tissue of nine type 1 diabetic patients during 3 (n = 2) and 7 days (n = 7). For each individual trial, S and I(o) were determined by taking into account the values of two sets of sensor output and blood glucose concentration distant by at least 1 h, the procedure being repeated for each consecutive set of values. S and I(o) were found to be negatively correlated, the value of I(o) being sometimes negative. Theoretical analysis demonstrates that this phenomenon can be explained by the effect of measurement uncertainties on the determination of capillary glucose concentration and of sensor output.     

Advances in plasmatic fractions used in blood transfusion.

I tried to give a bird's eye view of some of the intriguing recent advances in the production and clinical use of albumin, immunoglobulins, coagulation factors, protease inhibitors and plasma enzyme preparations. I aimed to put into the limelight of my lecture the basic differences between the reaction of plasma proteins in solutions in the test tube or on model surfaces in vitro as opposed to the well-balanced intricate protein-protein and cell membrane-plasma protein interactions in vivo. In the light of these recent advances we have to reassess the biological properties, structural and functional integrity of our plasma protein preparations. In spite of technical and regulatory difficulties new fractionation techniques have to break through. Especially gentle techniques which do not denature proteins are badly needed. Purified albumin with an adequate fatty acid content seems to be the safest and therapeutically most useful preparation among the albumin containing fractions. PPF and even albumin are, however, not so safe as we have thought. There is an urgent need to fight more intensively against their unjustified use. There is a growing need for specific immunoglobulins, coagulation factors, protease inhibitors and therapeutically useful enzyme preparations.     

Calibration of a subcutaneous amperometric glucose sensor implanted for 7 days in diabetic patients. Part 2. Superiority of the one-point calibration method

Calibration, i.e. the transformation in real time of the signal I(t) generated by the glucose sensor at time t into an estimation of glucose concentration G(t), represents a key issue for the development of a continuous glucose monitoring system. OBJECTIVE: To compare two calibration procedures. In the one-point calibration, which assumes that I(o) is negligible, S is simply determined as the ratio I/G, and G(t) = I(t)/S. The two-point calibration consists in the determination of a sensor sensitivity S and of a background current I(o) by plotting two values of the sensor signal versus the concomitant blood glucose concentrations. The subsequent estimation of G(t) is given by G(t) = (I(t)-I(o))/S. RESEARCH DESIGN AND METHODS: A glucose sensor was implanted in the abdominal subcutaneous tissue of nine type 1 diabetic patients during 3 (n = 2) and 7 days (n = 7). The one-point calibration was performed a posteriori either once per day before breakfast, or twice per day before breakfast and dinner, or three times per day before each meal. The two-point calibration was performed each morning during breakfast. RESULTS: The percentages of points present in zones A and B of the Clarke Error Grid were significantly higher when the system was calibrated using the one-point calibration. Use of two one-point calibrations per day before meals was virtually as accurate as three one-point calibrations. CONCLUSION: This study demonstrates the feasibility of a simple method for calibrating a continuous glucose monitoring system.     

A new plot for the kinetic analysis of dead-end enzyme inhibitors

A new procedure to characterize reversible dead-end inhibitors is presented. Preliminary identification of the inhibitor type is made by plotting vo/vi against the inhibitor concentration at different substrate concentrations. The inhibition constants for competitive, uncompetitive and mixed dead-end inhibitors are determined by secondary plots of l/(slope) vs [S], l/(slope) vs l/[S] and (slope)(Ks + [S] vs [S] respectively. These secondary plots render straight lines only for their corresponding type of inhibitor. For noncompetitive inhibitors all the secondary plots used yield straight lines. Therefore, the application of this plotting procedure leads to unambiguous diagnosis of the inhibitor type. An important feature of the procedure presented here is that the variable used (vo/vi) is independent on Vmax values. Therefore, experimental values obtained from enzyme preparations showing significant differences in their specific activities -i.e. enzyme coming from different purification steps- can be used.     

Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

The use of I50 (concentration of inhibitor required for 50% inhibition) for enzyme or drug studies has the disadvantage of not allowing easy comparison among data from different laboratories or under different substrate conditions. Modifications of the Michaelis-Menten equation for treatment of inhibitors can allow both the determination of the type of inhibition (competitive, noncompetitive, and uncompetitive) and the Ki for the inhibitor. For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs. For noncompetitive inhibitors the Ki = I50 and this relationship is valid with changing [S]. The equations developed require a single substrate, reversible-type inhibitors, and kinetics of the Michaelis-Menten type. Examples of the use of the equations are illustrated with experimental data from scientific publications.     

Congenital deficiency of factor VII in a canine family

Prolonged prothrombin time in the blood coagulation test was seen in some beagle dogs whose activated partial prothrombin times were distributed within the normal range. This phenomenon suggested possible abnormalities in coagulation factors II, V, VII, and/or X. Therefore, a revised cross-matching test was given and a determination of coagulation factors related to the extrinsic system was performed. We also determined whether or not factor VII inhibitor was present. The results were as follows: 1) In the revised cross-matching test, the prolonged prothrombin times were revised when normal canine serum was added to the plasma that showed prolongation of prothrombin time, but not when pooled normal canine plasma absorbed with BaSO4 was added to it. 2) The level of factor VII in the plasma with prolonged prothrombin time was 5 approximately 10% of the level in normal canine plasma. 3) Factor VII inhibitor was not detected in the plasma with prolonged prothrombin time or in normal plasma. Consequently, the prolongation of prothrombin time was attributed to a deficiency in factor VII. This abnormality was confirmed to be congenital.     

The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme''s active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin.     

An endogenous Ca2+-sensitive proteinase converts the hepatic alpha 1-adrenergic receptor to guanine nucleotide-insensitive forms

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.     

Neutrophil cathepsin G promotes prothrombinase and fibrin formation under flow conditions by activating fibrinogen-adherent platelets

Human neutrophil proteases cathepsin G and elastase can directly alter platelet function and/or participate in coagulation cascade reactions on the platelet or neutrophil surface to enhance fibrin formation. The clotting of recalcified platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (to shut down contact activation) was studied in well-plates or flow assays. Inhibitors of cathepsin G or elastase significantly delayed the burst time (t(50)) of thrombin generation in neutrophil-supplemented PRP from 49 min to 59 and 77 min, respectively, in well-plate assays as well as reduced neutrophil-promoted fibrin deposition on fibrinogen-adherent platelets under flow conditions. In flow assays, purified cathepsin G was a far more potent activator of platelet-dependent coagulation than elastase. Anti-tissue factor had no effect on neutrophil protease-enhanced thrombin formation in PRP. The addition of cathepsin G (425 nm) or convulxin (10 nm) to PRP dramatically reduced the t(50) of thrombin generation from 53 min to 17 or 23 min, respectively. In contrast, the addition of elastase to PRP left the t(50) unaltered. Whereas perfusion of PFP (gamma(w) = 62.5 s(-1)) over fibrinogen-adherent platelets did not result in fibrin formation until 50 min, massive fibrin could be observed on cathepsin G-treated platelets even at 35 min. Cathepsin G addition to corn trypsin inhibitor-treated PFP produced little thrombin unless anionic phospholipid was present. However, further activation inhibition studies indicated that cathepsin G enhances fibrin deposition under flow conditions by elevating the activation state of fibrinogen-adherent platelets rather than by cleaving coagulation factors.     

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.     

Studies on the different modes of action of the anticoagulant protease inhibitors DX-9065a and Argatroban. I. Effects on thrombin generation

The present study began with mathematical modeling of how inhibitors of both factor Xa (fXa) and thrombin affect extrinsic pathway-triggered blood coagulation. Numerical simulation demonstrated a stronger inhibition of thrombin generation by a thrombin inhibitor than a fXa inhibitor, but both prolonged clot time to a similar extent when they were given an equal dissociation constant (30 nm) for interaction with their respective target enzymes. These differences were then tested by comparison with the real inhibitors DX-9065a and argatroban, specific competitive inhibitors of fXa and thrombin, respectively, with similar K(i) values. Comparisons were made in extrinsically triggered human citrated plasma, for which endogenous thrombin potential and clot formation were simultaneously measured with a Wallac multilabel counter equipped with both fluorometric and photometric detectors and a fluorogenic reporter substrate. The results demonstrated stronger inhibition of endogenous thrombin potential by argatroban than by DX-9065a, especially when coagulation was initiated at higher tissue factor concentrations, while argatroban appeared to be slightly less potent in its ability to prolong clot time. This study demonstrates differential inhibition of thrombin generation by fXa and thrombin inhibitors and has implications for the pharmacological regulation of blood coagulation by the anticoagulant protease inhibitors.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.     

Lysophosphatidylcholines containing polyunsaturated fatty acids were found as Na+, K+-ATPase inhibitors in acutely volume-expanded hog

Na+, K+-ATPase inhibitors possessing inhibitory activities against the specific binding of ouabain to Na+, K+-ATPase and 86Rb uptake into hog erythrocytes have been purified from the plasma of acutely saline-infused hog. The purifications were performed by a combination of Amberlite XAD-2 adsorption chromatography and four steps of high-performance liquid chromatography with four different types of columns. Fast atom bombardment (FAB) mass and proton NMR spectrometric studies identified the purified substances as gamma-arachidoyl- [LPCA(gamma), 34%], beta-arachidoyl- [LPCA(beta), 4%], gamma-linoleoyl- (LPCL, 33%), and gamma-oleoyl- (LPCO, 25%) lysophosphatidylcholine, expressed in molar ratio in the plasma. Small amounts of gamma-docosapentaenoyl-, gamma-eicosatrienoyl-, and gamma-palmitoyllysophosphatidylcholine were also detected by both FAB mass and 1H NMR spectrometric studies. Only gamma-acyl-LPC's showed inhibitory activities on Na+,K+-ATPase and ouabain-binding activities. These LPC's were effective at 100 microM levels in attaining 50% inhibition of the enzyme activity. The inhibition of Na+,K+-ATPase activity due to these compounds was always more sensitive than that of both ouabain-binding and 86Rb uptake activities. The ouabain-displacing activity in plasma due to these compounds increased with time during saline infusion. The maximal plasma level was approximately 10 times higher than that in the preinfusion plasma sample. Although these results suggest the gamma-acyl-LPC's with long-chain polyunsaturated fatty acids are not simple competitive inhibitors to Na+, K+ -ATPase, these compounds could be implicated in the pathogenesis of the circulation abnormality through the modulation of membrane enzyme.     

Aminoglycoside-3'-phosphotransferase I from aminoglycoside-polyresistant strain E. coli 182]

An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.     

Rise in background current over time in a subcutaneous glucose sensor in the rabbit: relevance to calibration and accuracy

In order to calibrate a continuous glucose monitor, accurate determination of the background current (I0) is necessary, in part because I0 could change over time. We compared two methods of I0 measurement: (1), extrapolation of sensor output data (as a function of glucose level) to the intercept at zero glucose and (2) direct measurement of the output of a blank anode with no enzyme coat. We implanted telemetric sensors subcutaneously in rabbits and measured their outputs during tri-level glucose clamps once per week for 5 weeks. The two methods yielded similar results. I0 rose substantially over time and this increase reached significance during week 3 by the direct method but not until week 5 by the extrapolation method. Using the direct method, I0 rose from 3.41 (0.60-8.48 nanoamperes (nA), median and range) during week 1 to 13.42 (9.1-14.3) during week 5. Using the extrapolation method, I0 rose from 0.57 (0-16.7) during week 1 to 15.3 (12.2-21.6) during week 5. We conclude that I0 can rise over time. If this rise went undetected and was assumed to be stable, a one-point calibration procedure would overestimate glycemia in the hypoglycemic range, i.e. fail to appreciate the severity of hypoglycemia. It is recommended that during validation of a chronic glucose sensor, I0 be measured sequentially over time.     

Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein

The interaction of dynein with ATP gamma S, a phosphorothioate analogue of ATP, has been investigated in depth. The hydrolyses of ATP gamma S and of ATP were shown to be mutually competitive. ATP gamma S induced complete dissociation of the microtubule-dynein complex such that the time course of dissociation monitored by stopped-flow light-scattering methods followed a single exponential. The ATP gamma S concentration dependence of the rate of dissociation was hyperbolic, indicating that the dissociation is at least a two-step process: M.D + ATP gamma S in equilibrium M.D.ATP gamma S----M + D.ATP gamma S. The fit to the hyperbola gives an apparent Kd = 0.5 mM for the binding of ATP gamma S to the microtubule-dynein complex, and the maximal rate of 45 s-1 defines the rate of dissociation of the ternary M.D.ATP gamma S complex. Rapid quench-flow experiments demonstrated that the hydrolysis of ATP gamma S by dynein exhibited an initial burst of product formation. The size of the burst was 1.2 mol/10(6) g of dynein, comparable to that in the case of ATP hydrolysis. The steady-state rate of ATP gamma S turnover by dynein was activated by MAP-free microtubules. Because the rate of ATP gamma S turnover is severalfold (4-8) slower than ATP turnover, the rate-limiting step must be release of thiophosphate, not ADP. Thus, microtubules can activate the rate of thiophosphate release. The stereochemical course of phosphoric residue transfer was determined by using ATP gamma S stereospecifically labeled in the gamma position with 18O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of thrombin inhibitors on positive feedback in the coagulation cascade

The coagulation cascade is a series of sequential reactions of limited proteolysis of protein factors resulting in generation of thrombin. Thrombin mediates both positive and negative feedback in regulating this cascade by taking part in activation of several factors. Some thrombin inhibitors, by affecting positive feedback, inhibit generation of thrombin itself. In the current study, we used two thrombin inhibitors: argatroban, a low molecular weight reversible competitive inhibitor that binds to the active site, and bivalirudin, a bivalent oligopeptide that blocks the active site and binding center of protein substrates (exosite I). Appearance rate and total amount of thrombin were measured in a thrombin generation assay (TGA) using a fluorescent substrate. We found that argatroban slows the appearance of thrombin and lowers its amount. Bivalirudin also slows appearance of thrombin, but it does not decrease its amount, perhaps because the region being bound to the active site undergoes hydrolysis so that the inhibitor stops binding to thrombin. Many reactions of the coagulation cascade proceed on the surface of phospholipid micelles (PLMs). In the case of argatroban, PLMs do not affect the results of the TGA, whereas for bivalirudin they lower its inhibitory activity. It seems that PLMs stabilize protein complexes (wherein thrombin exosite I is hindered) mediating positive feedback in the coagulation cascade, e.g. complexes of thrombin with factor V and VIII.     

Design of constructs for the expression of biologically active recombinant human factors X and Xa. Kinetic analysis of the expressed proteins

Activation of vitamin K-dependent plasma proteases occurs by specific interaction with components of the blood coagulation cascade. In this report, we describe the direct expression and enzymatic characterization of the human coagulation zymogen factor X and its activated form, factor Xa, from transformed Chinese hamster ovary fibroblast cell lines. Expression was achieved using either a full-length factor X cDNA or a unique mutant factor Xa cDNA. The functional factor Xa precursor contained a novel tripeptide bridge in place of the native 52-amino acid activation peptide. This mutation allowed for intracellular processing and secretion of the activated form of factor X. Secreted recombinant factors X (rX) and Xa (rXa) were purified by sequential anion-exchange and immunoaffinity chromatography. The enzymatic activities of factors rX and rXa were compared with those of plasma factors X and Xa in three independent assay systems. In comparison to human plasma factor X, the amidolytic, prothrombinase complex, and plasma clotting activities of factor rX were 50, 85, and 43%, respectively. The corresponding comparative activities for factor rXa were 32, 64, and 48%, respectively. The ability to directly express mutant forms of biologically active human factor X will facilitate the structure/function analysis of this important blood coagulation protein and may lead to the development of novel coagulation inhibitors.