Separation of blood microsamples by exploiting sedimentation at the microscale.

Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. These results were confirmed by a clinical study scrutinising 8 blood markers in obese patients.

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis".

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.

Fixed dosage of low-molecular-weight heparins causes large individual variation in coagulability, only partly correlated to body weight.

BACKGROUND: Low-molecular-weight heparins (LMWHs) are routinely given without the control of their effect on coagulation. The endogenous thrombin potential (ETP) is a sensitive detector of the heparin effect. QUESTION: What is the interindividual variation in TG after a fixed dose of LMWH in normal volunteers, is it explained by variation in weight? METHODS: Subcutaneous (s.c.) injection, in 12 healthy volunteers, of 9000 aXa-units of unfractionated heparin (UFH) and of three heparins with narrow MW distribution around 10.5, 6.0 and 4.5 kD. Measurement of anti-thrombin (aIIa) and antifactor Xa (aXa)-activities and ETP at 11 time points over 24 h. RESULTS: The coefficient of variation (CV) of the AUCs of aXa- and aIIa-activities is 50% for UFH and 22-37% for LMWHs. Because of the hyperbolic form of the dose-response curve, the CV of the inhibition of the ETP is lower: 32% for UFH and 13-21% for the LMWHs. Fixed dosage of LMWH caused under-dosage in 10-13% of the samples and over-dosage in 5-11%. High or low response is an individual property independent of the type of heparin injected and only partially explained by variation in body weight. CONCLUSION: Optimized individual dosage of LMWH is possible through recognition of high and low responders, which requires one measurement of the heparin concentration or, preferably, the heparin effect on the ETP, 2-5 h after a first injection.

Free factor Xa is on the main pathway of thrombin generation in clotting plasma.

The effect of a synthetic pentasaccharide that specifically causes the inactivation of factor Xa on the development of prothrombinase activity in human plasma was monitored using four triggers of coagulation: (a) human brain thromboplastin; (b) contact activation; (c) factor X activating enzyme complex; (d) prothrombin activating enzyme complex. Inhibition was similar with the triggers a, b and c. With prothrombinase (d), the inhibition strongly decreased with increasing amounts of factor Va present. This indicates that only free factor Xa is inhibited. Because both the intrinsic pathway (b) and the extrinsic pathway (a) are inhibited by the pentasaccharide, we conclude that free factor Xa plays a rate-limiting role in the pathways, so that there is no reason to postulate the existence of 'supercomplexes' consisting of factors IXa, VIIIa, X(a), Va and prothrombin adsorbed on the same phospholipid particle (intrinsic system) or factor VII(a), X(a), Va and prothrombin adsorbed on tissue thromboplastin (extrinsic system).

Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule.

Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.

Draculin, the anticoagulant factor in vampire bat saliva, is a tight-binding, noncompetitive inhibitor of activated factor X.

The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K(i)(K(d)) = 14.8+/-1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M(-1)s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K(d) obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces <14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors.

Determination of glyphosate and aminomethylphosphonic acid residues in water by gas chromatography with tandem mass spectrometry after exchange ion resin purification and derivatization. Application on vegetable matrixes

An analytical method for the determination of glyphosate and its principal metabolite, aminomethylphosphonic acid (AMPA), in water of different hardnesses (5, 20, and 30 degrees DH, french hardness) has been developed. Samples were fortified at different levels (0.05, 0.1, 1, and 5 microg/L) and were purified by column chromatography on ion-exchange resins. After derivatization with TFAA/HFB mixture, the derivatives were quantified by using capillary gas chromatography with an ion-trap tandem mass spectrometric detector. Analytical conditions for MS/MS detection were optimized, and the quantification was carried out on the sum of areas of the three most representative ions: m/z 283, 223, and 181 for AMPA and m/z 440, 321, and 261 for glyphosate. The limit of quantification was demonstrated to be at 0.05 microg/L for each compound. The mean recovery value and the relative standard deviation (n = 65) were 93 and 12% for AMPA and 95 and 13% for glyphosate.

The ionic contrast medium ioxaglate interferes with thrombin-mediated feedback activation of factor V, factor VIII and platelets.

Clinical observation shows that radiographic contrast media (CM) may influence thrombus formation. In the search for the underlying mechanism, we have shown that the ionic CM ioxaglate is a potent inhibitor of thrombin generation in platelet-poor and platelet-rich plasma, whereas the influence of the non-ionic contrast medium iodixanol is minimal. Ioxaglate boosts the inhibitory effect of the platelet GPIIb/IIIa antagonist abciximab and the effects of ioxaglate and heparin are additive. Ioxaglate inhibits the clotting of fibrinogen and the activation of factors V and VIII, and of platelets by thrombin. It does not inhibit hydrolysis of small chromogenic thrombin substrates, nor does it influence the heparin-catalyzed inactivation of thrombin by antithrombin. We assume therefore that ioxaglate interferes with the binding of macromolecular substrates to the anionic exosite I of thrombin. The biological correlation to the observed antithrombotic effect of ioxaglate is then to be found in the inhibition of thrombin generation via inhibition of thrombin-mediated feedback activations.

Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time.

Heparin can be quantified with antifactor Xa and IIa tests (aXa, aIIa) but the anticoagulant power of heparin depends upon plasma properties as well as upon heparin concentrations and thus differs between subjects. Measuring the effect, as with the activated partial thromboplastin time (APTT) therefore is clinically more relevant. Here we investigate the use of the endogenous thrombin potential (ETP) for this purpose. In 12 volunteers 9000 IU of four heparins of different mol. wt distributions were injected. Samples were taken at 11 time points between 0 and 24 h. With the exception of the 0 and 24-h time points, heparin could be demonstrated by its aIIa and aXa activity in virtually all samples. The APTT showed the effect of this heparin in 34% of the samples; the ETP in 80%. This is partly due to the wide margins of the normal values, caused by large interindividual variation [coefficient of variation (CV) approximately 12% for the APTT, approximately 17% for the ETP]. The intraindividual variation is much smaller (CV approximately 4% for the APTT, approximately 5% for the ETP). Relative to the baseline value of the individual, the heparin effect was recognized by the APTT in 55% of the cases and by the ETP in 98%. There were no large differences between the different types of heparin.

Fibrin polymerization is crucial for thrombin generation in platelet-rich plasma in a VWF-GPIb-dependent process, defective in Bernard-Soulier syndrome.

Defective prothrombin consumption has been reported in the proband case of Bernard-Soulier syndrome (BSS). There is no consensus, however, on whether the formation of platelet procoagulant activity (PPA) is impaired in BSS and, if so, whether this is due to the lack of GPIb-V-IX-dependent binding of thrombin or of von Willebrand factor (VWF). We show thrombin generation (TG) in platelet-rich plasma of BSS (BSS-PRP) to be defective provided that fibrin remains present in the reaction mixture and that the giant platelets are not damaged by frequent subsampling. In BSS-PRP addition of (thrombin-free) fibrin did not increase TG as in normal PRP, supporting our previous hypothesis that the interaction of fibrin, VWF and GPIb triggers PPA development. Fibrin formed during the lag phase of TG by a snake venom enzyme which only removed fibrinopeptide A induced an immediate burst of TG, that was inhibited by a monoclonal antibody against GPIb (6D1) that abolishes ristocetin-induced binding of VWF to platelets. Inversely, inhibition of polymerization decreased TG and the residual activity was insensitive to 6D1. We conclude that polymerizing fibrin interacts with VWF so as to activate GPIb.

The inhibition of blood coagulation by heparins of different molecular weight is caused by a common functional motif--the C-domain.

BACKGROUND: Heparins in clinical use differ considerably as to mode of preparation, molecular weight distribution and pharmacodynamic properties. OBJECTIVES: Find a common basis for their anticoagulant action. METHODS: In 50 fractions of virtually single molecular weight (Mr), prepared from unfractionated heparin (UFH) and four low-molecular-weight heparins (LMWH), we determined: (i) the molar concentration of material (HAM) containing the antithrombin binding pentasaccharide (A-domain); (ii) the specific catalytic activity in thrombin and factor Xa inactivation; (iii) the capacity to inhibit thrombin generation (TG) and prolong the activated partial thromboplastin time (APTT). We also calculated the molar concentration of A-domain with 12 sugar units at its non-reducing end, i.e. the structure that carries antithrombin activity (C-domain). RESULTS: The antithrombin activity and the effects on TG and APTT are primarily determined by the concentration of C-domain and independent of the source material (UFH or LMWH) or Mr. High Mr fractions (>15 000) are less active, probably through interaction with non-antithrombin plasma proteins. Anti-factor Xa activity is proportional to the concentration of A-domain, it is Ca2+- and Mr-dependent and does not determine the effect on TG and APTT. CONCLUSION: For any type of heparin, the capacity to inhibit the coagulation process in plasma is primarily determined by the concentration of C-domain, i.e. the AT-binding pentasaccharide with 12 or more sugar units at its non-reducing end.

Chromatic adaptation in a mutant of Fremyella diplosiphon incapable of phycoerythrin synthesis.

A mutant of the chromatically adapting cyanobacterium Fremyella diplosiphon, incapable of phycoerythrin synthesis but responding to wavelength modulation of its biliprotein content, was isolated. The biliprotein composition of the mutant and of the wild type were identical after growth in red light, but green light induced, in the mutant, the synthesis of a biliviolin-type chromophore bound to some of the alpha subunits of its phycocyanin. Implications of the results on the regulation and possible pathways of biliprotein biosynthesis are discussed.

Standard and method independent units for heparin anticoagulant activities.

It is discussed why the current USP unit of heparin anticoagulant activity necessarily will render inaccurately the anticoagulant activities of low molecular weight heparins. It is shown that the outcome is bound to vary with the method used for comparison of the sample and the standard and with the nature of the standard used. As an alternative we define a unit of heparin in terms of anti-factor Xa- and antithrombin-activity that is independent of the heparin standard and of the assay method, but that is based upon a quantitative description of the catalytic effect of heparin on AT III mediated thrombin- and factor Xa breakdown. Expression of the results of existing anti-factor Xa- and antithrombin tests in terms of these units will allow to express heparin levels in plasma in terms of concentrations of active anticoagulant material. This approach makes it possible to separate heparin pharmacodynamics from heparin pharmacokinetics. Introduction of this unit does not require adaptation of current laboratory practice but changes the way in which the results obtained are expressed.

Thrombin generation in plasma: its assessment via the endogenous thrombin potential.

Thrombin generation is a pivotal function of plasma in haemostasis and thrombosis. Its mechanism is essentially the classical cascade, the velocity of which is governed by the availability of factors Va and VIIIa and that is confined to the surface of the procoagulant membranes which appear at the site of injury. There is no routine test that quantitatively renders the thrombin forming capacity of a plasma sample. Clotting times (PT-prothrombin time, APTT-activated partial tromboplastin time) do not reflect the over all thrombin generation and are insensitive to hypercoagulative states. The endogenous thrombin potential (ETP), i.e. the area under the thrombin generation curve, better represents this function. We developed a method to assess the ETP in the routine laboratory. The first results suggest that it is a sensitive indicator for every form of anticoagulation. It is increased in hypercoagulable states thus far studied, both congenital and acquired and can be designed to indicate deficiencies in protein C and S and APC (activated protein C) resistance.

A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes.

A method is described that, on the basis of the time course of amidolytic activity after the triggering of thrombin generation in normal plasma, allows the calculation of the velocity of prothrombin conversion independent of thrombin inactivating processes. It is shown how the reaction constants for the alpha 2M-dependent and the alpha 2M-independent thrombin inactivation processes can be obtained in a sample of whole plasma. The method is verified by demonstrating that the experimentally observed time courses of residual prothrombin and of alpha 2M-thrombin complex coincide with those calculated from the time course of amidolytic activity, and by showing that the course of prothrombin conversion in plasma without alpha 2-macroglobulin or AT III is adequately described if the alpha 2M or AT III-dependent breakdown constants are taken zero in the calculations. It appears that the inactivation of thrombin, endogenously generated in whole plasma, is about half as fast as that of exogenous thrombin added to the plasma. A computer program is presented that carries out the relevant calculations.

The relative importance of the factors II, VII, IX and X for the prothrombinase activity in plasma of orally anticoagulated patients.

The individual importance of each of the four vitamin K-dependent clotting factors on the generation of prothrombinase activity in the plasma of orally anticoagulated patients has been investigated. Addition of purified factors VII, IX or X to plasma from deeply anticoagulated patients (International Normalized Ratio 2.8-4.8) did not influence the amount of prothrombinase activity or the amount of thrombin formed. Only the prothrombin level in the plasma determines the course of thrombin generation. Addition of increasing amounts of purified factor II, VII, IX or X to plasmas deficient in respectively factor II, VII, IX or X showed that the prothrombinase activity increases linearly with the concentration of factor II added and that the concentration below which the factors VII, IX and X start to have a measurable effect on prothrombinase activity are 5%, 20%, and 30%, respectively. Half maximal prothrombinase activity was found at about 1% factor VII, 5% factor IX and 8% factor X respectively. From these observations we conclude that primarily the variation in factor II level determines thrombin generation and hence presumably the antithrombotic effect of oral anticoagulant therapy. It therefore seems likely that, for the control of oral anticoagulant therapy, tests that reflect factor II activity would be suitable.

Factor IX a inhibition contributes to the heparin effect.

We investigated whether the inactivation of factor IXa contributes to the partial inhibition of thrombin formation that is observed at therapeutic concentrations of heparin. The action of standard unfractionated heparin (0.05 U/ml) on thrombin formation in the intrinsic system was compared to that of a mixture of dermatan sulfate (DS) and a synthetic pentasaccharide (PS). DS enhances the action of heparin cofactor II which inhibits thrombin only. PS specifically enhances the anti-factor Xa activity of antithrombin III (AT III). The concentrations of DS and PS were chosen so as to obtain equal anti-thrombin and anti-factor Xa activities as in 0.05 U/ml heparin. An extra inhibitory effect of heparin over the mixture is observed in situations where free factor IXa, not bound to factor VIIIa and phospholipid, limits the rate of thrombin formation, notably in contact activated plasma. We conclude that the inactivation of free factor IXa by heparin contributes importantly to the inhibition of thrombin formation in the intrinsic system such as e.g. measured in the activated partial thromboplastin time.

The effect of fibrin clots and clot-bound thrombin on the development of platelet procoagulant activity.

We tested different types of clot for their ability to provoke procoagulant activity in platelets: normal clots from platelet poor plasma (des AABB- or fibrin II clots), similar clots in which the adsorbed thrombin has been inhibited by hirudin, and clots obtained by the action of two snake venom enzymes that release only fibrinopeptide A (des AA- or fibrin I clots). Analogous clots from fibrinogen solutions were also tested. In platelet rich plasma (PRP), where platelet coagulant phospholipids (PCP) are rate limiting for thrombin generation, the addition of any type of clot enhances the generation of thrombin thus it induces the appearance of PCP. Clots containing active adsorbed thrombin are the most potent ones in this respect. Lactate dehydrogenase (LDH) levels do not increase in the course of the thrombin generation so the platelets are not damaged in the process. Non-centrifugable PCP could be demonstrated to appear during the process, so the production of procoagulant phospholipid microparticles must be part of the mechanism. Membrane transbilayer phosphatidyl serine movement (flip-flop) can not be demonstrated in PRP as the activated platelets are caught in the emerging clot. In order to demonstrate flip-flop, we tried to investigate the influence of clots on washed platelets. However, contrary to platelets in a plasma milieu, isolated platelets are damaged by fibrin clots, especially in the presence of thrombin, as can be judged from the appearance of LDH. We conclude that, in PRP, clots induce the appearance of PCP from platelets by vesiculation, possibly accompanied by flip-flop and that thrombin accelerates the process but is not an absolute requirement.

The influence of fibrinogen and fibrin on thrombin generation--evidence for feedback activation of the clotting system by clot bound thrombin.

In plasma the bulk of thrombin generation takes place after a clot has formed. We therefore investigated in what way the clot influences thrombin generation in plasma. The forming clot withdraws thrombin from free solution. Consequently less thrombin activity is found and less thrombin-inhibitor complexes are formed. The thrombin that is adsorbed to the clot reduces the lag time before thrombin generation in intrinsically or extrinsically triggered platelet poor plasma as well as in platelet rich plasma. We investigated the mechanism of this activation. Clots were obtained by recalcification of plasma or by the addition of thrombin-like enzymes (Reptilase, Agihal) from snake venoms. They were thoroughly washed until the washing fluid was devoid of any detectable clotting enzyme activity. In platelet poor plasma (PPP), thrombin-induced clots shorten the factor Va-dependent lag-time of thrombin generation in the extrinsic system as well as the factor VIIIa-dependent thrombin generation in the intrinsic system. Factor V or factor VII preparations that in itself hardly influence thrombin generation patterns acquire the capacity to shorten these lag-times when incubated with clot. The last washing fluid of the clot is inactive. Snake venom induced clots are not active either. Clots that are incubated in heparinised plasma for 1 h or more are as active as clots from normal plasma are. A role of factor Xa can not be excluded but must be minor because a clot made by addition of thrombin to plasma from which the factors II, VII, IX and X have been removed is as active as a clot from normal plasma is.(ABSTRACT TRUNCATED AT 250 WORDS)

The action of a synthetic pentasaccharide on thrombin generation in whole plasma.

We investigated the effect on thrombin generation in plasma of the pentasaccharide that represent the AT III/binding site in heparin. This compound has no effect on the breakdown of thrombin in plasma. It dose-dependently inhibits the formation of thrombin in both the intrinsic and the extrinsic pathway. If coagulation is triggered by the complete prothrombinase complex (phospholipid--factor Va--factor Xa) under conditions in which the large majority of factor Xa is bound to the complex, the inhibition of prothrombinase activity is only minor. If no factor Va is present or if the prothrombinase activity is triggered by adding complete tenase (PL-FVIIIa-FIXa) or incomplete tenase (PL-FIXa) to the plasma the inhibition by pentasaccharide is of the same magnitude as that in the intrinsic or extrinsic system. We conclude that the pentasaccharide inhibits blood coagulation by catalysing the inactivation of free factor Xa. In contrast to classical heparin it does inhibit the peak of thrombin formation in platelet rich plasma, probably because it is less subject to inactivation by heparin binding proteins from platelets than classical heparin is.