Platelet active concentration profiles near growing thrombi. A mathematical consideration.

When blood contacts foreign material surfaces, platelets usually adhere and form aggregates on those surfaces, generating mural thrombi. The mechanism of mural thrombogenesis is not completely understood, but one hypothesis states that the local release of certain platelet-active substances from the platelets composing an initial small thrombus stimulates additional platelet recruitment to that thrombus, resulting in growth of the cell aggregate. The purpose of this paper is to investigate the feasibility of this hypothesis. Concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), and thrombin were computed in the vicinity of growing model thrombi 10 and 20 micron long. Wall shear rates of 100, 500, and 1,500 s-1 were considered for blood flowing through a thin rectangular slit 200 micron wide coated with collagen, a predominant subendothelial protein. The local concentrations of ADP and TxA2 were marginally large enough to stimulate platelet activation individually, while local thrombin levels can be much greater than required for stimulation. Antithrombin III, a natural thrombin inhibitor, did not significantly reduce the thrombin concentrations, but antithrombin III accelerated by heparin greatly reduced the local thrombin concentrations. The reduced thrombin levels may, however, still be large enough to activate platelets.     

Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow.

The concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), thrombin, and von Willebrand factor (vWF) released extracellularly from the platelet granules or produced metabolically on the platelet membrane during thrombus growth, were estimated using finite element simulation of blood flow over model thrombi of various shapes and dimensions. The wall fluxes of these platelet-activating agents were estimated for each model thrombus at three different wall shear rates (100 s-1, 800 s-1, and 1,500 s-1), employing experimental data on thrombus growth rates and sizes. For that purpose, whole human blood was perfused in a parallel-plate flow chamber coated with type l fibrillar human collagen, and the kinetic data collected and analyzed by an EPl-fluorescence video microscopy system and a digital image processor. It was found that thrombin concentrations were large enough to cause irreversible platelet aggregation. Although heparin significantly accelerated thrombin inhibition by antithrombin lll, the remaining thrombin levels were still significantly above the minimum threshold required for irreversible platelet aggregation. While ADP concentrations were large enough to cause irreversible platelet aggregation at low shear rates and for small aggregate sizes, TxA2 concentrations were only sufficient to induce platelet shape change over the entire range of wall shear rates and thrombi dimensions studied. Our results also indicated that the local concentration of vWF multimers released from the platelet alpha-granules could be sufficient to modulate platelet aggregation at low and intermediate wall shear rates (less than 1,000 s-1). The sizes of standing vortices formed adjacent to a growing aggregate and the embolizing stresses and the torque, acting at the aggregate surface, were also estimated in this simulation. It was found that standing vortices developed on both sides of the thrombus even at low wall shear rates. Their sizes increased with thrombus size and wall shear rate, and were largely dependent upon thrombus geometry. The experimental observation that platelet aggregation occurred predominantly in the spaces between adjacent thrombi, confirmed the numerical prediction that those standing vortices are regions of reduced fluid velocities and high concentrations of platelet-activating substances, capable of trapping and stimulating platelets for aggregation. The average shear stress and normal stress, as well as the torque, acting to detach the thrombus, increased with increasing wall shear rate. Both stresses were found to be nearly independent of thrombus size and only weekly dependent upon thrombus geometry. Although both stresses had similar values at low wall shear rates, the average shear stress became the predominant embolizing stress at high wall shear rates.     

Effect of cerebrocrast on the function of human platelets and release of the arachidonic acid from plasma membrane

Diabetes mellitus (DM) is accompanied by several cardiovascular complications such as coronary artery disease, atherosclerosis, hypertension, cerebral and myocardial infarction, etc. DM induces the alteration of platelet functions including activation, hyperaggregation, adhesiveness, and formation of thrombi. Release of AA from phospholipids of the PM, synthesis of TxA(2),PGE(2), activity of PLA(2), and PLC are increased in the platelets of the DM patients. Stimulation of PLA(2) activity and accumulation of bioactive metabolites such as AA, its oxygenated derivatives, prostaglandins and PAF can evoke glucose production, also. In this study we explored the effect of the 1,4-dihydropyridine compound cerebrocrast at a low concentration (10(-6)-10(-8)M) on the level of intracellular calcium in unstimulated human platelets and those stimulated with thrombin as well as release of [(3)H] AA from phospholipids of platelet PM. Cerebrocrast at a concentration of 10(-6) M decreased the basal level of intracellular calcium concentration (platelets were loaded with Fura-2) in unstimulated as well as in thrombin stimulated platelets. Cerebrocrast at concentrations of 10(-6), 10(-7), 10(-8) M inhibited release of [(3)H] AA from phospholipids of platelet PM. We conclude that blockade of human platelet activation with cerebrocrast can prevent aggregation, adhesion and formation of thrombi. The inhibition of [(3)H] AA release from phospholipids of platelet PM can prevent formation of eicosanoids such as TxA(2), PGG(2), and PGH(2) plus AA oxygenated derivatives. These effects of cerebrocrast are very significant in the treatment of DM-evoked cardiovascular complications.     

Presteady state kinetic analysis of vanadate-induced inhibition of the dynein ATPase

The effects of vanadate on the kinetics of ATP binding and hydrolysis by Tetrahymena 30 S dynein were examined by presteady state kinetic analysis. Up to a concentration of 400 microM, vanadate did not inhibit the rate or amplitude of the ATP binding-induced dissociation of the microtubule-dynein complex measured by stopped flow light-scattering methods. Chemical quench flow experiments showed that vanadate (80 microM) did not alter the rate or amplitude of the presteady state ATP binding or ATP hydrolysis transients, but the steady state hydrolysis of ATP was blocked immediately after a single turnover of ATP. Preincubation of the enzyme with ADP and vanadate inhibited both presteady state and steady state hydrolysis. These data suggest that vanadate acts as a phosphate analog to form an enzyme-ADP-vanadate complex, analogous to the transition state during catalysis, by the following pathway: (formula; see text) where V represents vanadate and D represents a dynein active site. ADP and vanadate, added together, induced dissociation of the microtubule-dynein complex at a maximum rate of 0.6 S-1. These observations imply that a microtubule-dynein-ADP-vanadate complex was formed which subsequently dissociated as shown below: (formula; see text) where M denotes a microtubule. The ADP plus vanadate-induced dissociation may represent the reverse of the normal forward pathway involving the binding of a dynein-ADP-phosphate complex to a microtubule.     

Experimental and kinetic modelling studies on the acid-catalysed hydrolysis of the water hyacinth plant to levulinic acid

A comprehensive experimental and modelling study on the acid-catalysed hydrolysis of the water hyacinth plant (Eichhornia crassipes) to optimise the yield of levulinic acid (LA) is reported (T=150-175 degrees C, [Formula: see text] , water hyacinth intake=1-5wt%). At high acid concentrations (>0.5M), LA was the major organic acid whereas at low acid concentrations (<0.1M) and high initial intakes of water hyacinth, the formation of propionic acid instead of LA was favoured. The highest yield of LA was 53mol% (35wt%) based on the amount of C6-sugars in the water hyacinth (T=175 degrees C, [Formula: see text] , water hyacinth intake=1wt%). The LA yield as a function of the process conditions was modelled using a kinetic model originally developed for the acid-catalysed hydrolysis of cellulose and good agreement between the experimental and modelled data was obtained.     

Na+-driven flagellar motors of an alkalophilic Bacillus strain YN-1

Flagellar motors of some alkalophilic Bacillus strains have been suggested to be powered by the electrochemical potential gradient of Na+, namely the (formula: see text) (Hirota, N., Kitada, M., and Imae, Y. (1981) FEBS Lett. 132, 278-280). In the present study, we quantitatively measured the (formula: see text) and motility of one of the strains, YN-1. Swimming speed of YN-1 cells increased linearly with a logarithmic increase of Na+ concentration in the medium up to 100 mM. The intracellular Na+ concentration and the membrane potential of the cell were about 30 mM and -170 mV, respectively, and stayed constant irrespective of Na+ concentration in the medium. Thus, the swimming speed changed as a function of the chemical potential difference of Na+ across the cell membrane. When the membrane potential of YN-1 cells was decreased by a combination of valinomycin and various concentrations of K+ in the medium, the swimming speed of the cells decreased linearly and reached zero at around -90 mV. Under the condition, the intracellular Na+ concentration stayed constant. Thus, the membrane potential was also a determinant of the swimming speed. Furthermore, the chemical potential of Na+ and the membrane potential were found to be equivalent as the energy source for motility. Therefore, it is concluded that the (formula: see text) is the energy source for the flagellar motors of YN-1 cells. Threshold value of the (formula: see text) for motility was about -100 mV.     

Relative effects of flurbiprofen on platelet 12-hydroxy-eicosatetraenoic acid and thromboxane A2 production: influence on collagen-induced platelet aggregation and adhesion

Flurbiprofen has been shown to inhibit cyclo-oxygenase metabolism of arachidonic acid to thromboxane A2 (TxA2), resulting in the inhibition of platelet aggregation. Recently, our laboratory reported that the "irreversible" phase of platelet aggregation and adhesion were regulated, in part, by the lipoxygenase metabolism of arachidonic acid to 12-hydroxy-eicosatetraenoic acid (12-HETE) in platelets, and that selective inhibition of one enzyme i.e. either cyclo-oxygenase or lipoxygenase, resulted in paradoxical effects on the metabolism of arachidonic acid and platelet response related to the other pathway. Therefore, we performed experiments to assess the relative effects of flurbiprofen on TxA2 and 12-HETE synthesis, and on collagen-induced platelet aggregation and platelet adhesion to collagen-coated surfaces. "Irreversible" collagen-induced platelet aggregation was only partially inhibited by pre-incubation with 1 x 10(-6) M flurbiprofen, while TxA2 production was elevated and 12-HETE production was maximally inhibited in these platelets. At this concentration of flurbiprofen (1 x 10(-6)M), collagen-induced platelet adhesion was also reduced by 50%. At higher concentrations of flurbiprofen, both platelet aggregation and adhesion were further reduced, with a corresponding inhibition of TxA2 production. Thus it appears that the lipoxygenase pathway of arachidonic acid metabolism in platelets is not only inhibited by flurbiprofen, but is more sensitive to inhibition by flurbiprofen than the cyclo-oxygenase pathway. This differential effect of flurbiprofen on arachidonic acid metabolism in the platelet is related to differential effects on platelet function.     

The F2-isoprostane, 8-epi-prostaglandin F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist.

F2-isoprostanes are a recently discovered series of prostaglandin (PG)F2-like compounds that are produced in vivo in humans by nonenzymatic free radical catalyzed peroxidation of arachidonic acid. One of the compounds that can be produced in abundance by this mechanism is 8-epi-PGF2 alpha. 8-epi-PGF2 alpha is a potent vasoconstrictor in the rat, an effect that has been shown to be mediated via interaction with vascular thromboxane (TxA2)/endoperoxide (PGH2) receptors. In an effort to further understand the biological properties of this prostanoid in relation to its ability to interact with TxA2/PGH2 receptors, we examined its effects on human and rat platelets. At concentrations of 10(-6) M and 10(-5) M, 8-epi-PGF2 alpha induced only a shape change in human platelets and at higher concentrations (10(-4) M) induced reversible but not irreversible aggregation. Both the shape change and reversible aggregation were unaffected by indomethacin but were inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-epi-PGF2 alpha inhibited platelet aggregation induced by the TxA2/PGH2 receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 1.6 x 10(-6) M and 1.8 x 10(-6) M, respectively. 8-epi-PGF2 alpha also inhibited platelet aggregation induced by arachidonic acid. Similarly, in rat platelets, 8-epi-PGF2 alpha alone induced only modest reversible aggregation but completely inhibited U46619-induced aggregation.     

Platelet near-wall excess in porcine whole blood in artery-sized tubes under steady and pulsatile flow conditions

Platelet margination (enhanced platelet concentration in the near wall region of a blood vessel) has been well documented in small vessels. In artery-sized vessels margination has only been demonstrated in one study, using ghost cell suspensions and under relatively non-physiologic conditions of steady flow and 50 cm development length. Local sampling experiments were performed to confirm platelet margination in artery-sized stainless steel tubes, for a typical anatomical length and under pulsatile flow, using fresh EDTA-anticoagulated porcine whole blood (N=21). Experiments were designed using three-dimensional Computational Fluid Dynamics (CFD) to model the sample region with greater fidelity. Steady flow experiments in 50 cm long tubes verify published laser Doppler measurements of platelet margination in 3 mm ID tubes at normal arterial shear rate (500 s(-1). Margination persists under pulsatile flow conditions (63.8 pulses/min), but in steady flow at length of 10 cm, margination is reduced. Platelet margination ratio (the ratio of the platelet concentration near the wall to bulk average platelet count) ranges from 1.21 to 2.48. No significant effects of calculated sampling thickness (20 microm and 50 microm) or pulsatility were detected. Hematocrit margination ratio is 0.68 to 0.90. Two model platelet concentration profiles are fit to the experimental results.     

An enzyme-linked lectin binding assay for quantitative determination of lectin receptors

An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.     

The effect of unsteadiness on the flow through stenoses and bifurcations

This paper is concerned with the influence of a stenosis or a bifurcation on the flow through a tube. In particular the effect of unsteadiness is investigated using simple pulsatile and physiological type flows (Fig. 1). The experimental investigations reported herein are concerned with velocity measurements and flow visualizations. (see formula in text) These measurements, performed in a 60 degrees bifurcation, have permitted the reconstruction of the three-dimensional velocity profiles. The importance of the secondary flow in the branching is analyzed for various values of the flow parameters. Results of tests show a strong influence of unsteadiness on flow characteristics and then on hemodynamic factors. One conclusion is the following: if hemodynamic factors play an important role in the problems of atherosclerosis, then, for macrocirculation studies, it is necessary to take into account unsteadiness and, in particular, the actual shape of the flow-time forcing function.     

Methodological specifications on the Todd method and preliminary results

In order to measure the potentiality and kinetics tissue fibrinolysis we have modified Todd's histochemical method preparing at least six sections taken from same sample tissue, by infraoperatory biopsy, esponing them to a fibrin film at a constant concentration; we have also chosen to incubate the section at 10, 20, 30, 45, 60 min. The measurement of the areas of fibrin, of tissue and fibrinolysis, at the above mentioned times, has been effected at standard magnification (15 X) by an image analyser (Videoplan) scale 1:8. For each sample we suggest to elaborate an Index of the Potentiality of Fibrinolysis (formula; see text) and an Index of the Kinetics of the Fibrinolysis (formula; see text). Applying this method to two different groups of thyroid disease (struma and adenoma) we have not pointed out any statistically significant difference.     

Effects of shear rate and suspending medium viscosity on elongation of red cells tank-treading in shear flow

Elongation measurements of red cells subjected to simple shear flow are usually performed using a single suspending medium (viscosity η(0) ) and varying the mean shear rate (y). Such data are often plotted versus the shear stress (tau = no(y) suggesting that the elongation scales with τ. In this work, normal blood samples were tested in a rheoscope varying both η(0) and(y.). The ranges of (y.) were chosen to restrict the elongation of the red cells to low values where the behavior is dominated by their intrinsic properties. It was found that the elongation scales with [formula: see text] with s decreasing from two at η(0) = 20 mPas to unity at η(0) = 70 mPas. Above η(0) = 70 mPas, the elongation is therefore essentially determined by the membrane elasticity alone. A side observation was a large variation of the elongation both intraindividually and interindividually.     

Relation of Ca2+ efflux from the heart to the concentration of Ca-channel blockers

Dependence of calcium ion efflux from the heart ventricles on the concentration of verapamil, fenigidine and nicardipine was studied in frogs, using Ca-sensitive electrodes. The effect of sodium and potassium ions was also investigated. It was established that dependence of calcium ion efflux (delta Ca2+) on the concentration of Ca-antagonists (B) may be expressed by the following formula: (Formula: see text). With cellular membrane depolarization 50 mM KCl, none of the blockers (10(-5) M) caused Ca2+ efflux from ventricular cells. Analogous phenomenon was noted in low-sodium solution (60 mM).     

Denaturation of phycocyanin by urea and determination of the enthalpy of denaturation by microcalorimetry.

Denaturation of the protein phycocyanin in urea solution was investigated by microcalorimetry, ultraviolet and visible spectroscopy, circular dichroism and sedimentation equilibrium. The results consistently demonstrated that in the presence of 7 M urea this protein is completely denatured. By assumings a two-state mechanism, an apparent free energy of unfolding at zero denaturant concentration, (formula: see text) was found to be 4.4 kcal/mole at pH 6.0 and 25 degrees C. By microcalorimetry the enthalpy of denaturation of phycocyanin app was found to be -230 kcal/mole at 25 degrees C. The relatively large negative enthalpy change results from protein unfolding and changes in protein solvation.     

Inhibitory effect of ebselen on the oxidation of low density lipoprotein

The inhibitory effect of a synthesized glutathione peroxidase (GSHPX) mimic- ebselen, and its cofactor glutathione (GSH), on the oxidation of low density lipoprotein (LDL) induced by Cu²⁺ was studied by determination of hydroperoxides and thiobarbituric acid reactive substances (TBARS). Ebselen alone had a strong inhibitory effect on the oxidation of LDL. The lag time of LDL oxidation was prolonged with an increase in the concentration of ebselen. The inhibitory effect of 5 μM ebselen was equivalent to that of 50 μM α-tocopherol. When GSH was present, ebselen exhibited stronger inhibitory effect than when present alone. With 50 μM GSH, ebselen at a concentration as low as 5 μM could inhibit oxidation of LDL induced by 5 μM Cu²⁺ completely for at least 24 h. Ebselen at high concentrations (100 μM) decomposed hydroperoxides in pre-oxidized LDL and effectively prevented its further oxidation, but not in the present of EDTA. Low concentration of ebselen (5 μM) plus GSH (50 μM) decomposed hydroperoxides in pre-oxidized LDL whether EDTA was added or not.     

Overview of physiological and pathophysiological effects of thromboxane A2

Thromboxane (Tx) A2 is a biologically potent and chemically unstable metabolite of prostaglandin endoperoxides. Recent developments in measurement techniques and the availability of both selective inhibitors of Tx synthetase and TxA2 receptor antagonists have facilitated the implication of TxA2 as a physiological modulator and as a mediator in thrombotic, vasospastic, and bronchospastic conditions. TxA2 is synthesized by platelets and contributes to platelet activation and irreversible platelet aggregation in physiological hemostasis and in thrombosis (e.g., unstable angina, stroke). TxA2 is also synthesized in intestinal, pulmonary, and renal tissues by cells other than platelets. Particularly in these tissues, TxA2 appears to act as a physiological modulator of changes in blood flow distribution and airway caliber. Strong stimuli for TxA2 release from these tissues may initiate ulcer, pulmonary hypertension, bronchoconstriction, and renal vasoconstriction. Evidence supports participation of TxA2 and/or TxA2 receptors in modulation of natural cytotoxic cell cytotoxicity, in tumor growth and metastasis, in complications of pregnancy (e.g., preeclampsia), and in the progression of ischemic injury after coronary artery occlusion. This evidence supports pivotal involvement of TxA2 in pathophysiology and provides a strong rationale for pursuing TxA2-blocking strategies in drug development.     

Transient state kinetic analysis of the ATP-induced dissociation of the dynein-microtubule complex

The kinetics of ATP-induced dissociation of dynein from the dynein-microtubule complex has been investigated by stopped flow light scattering methods. The addition of ATP to the dynein-microtubule complex induced a large, rapid decrease in light scattering followed by a smaller and much slower decrease. The fast light scattering change was shown to be a measure of the ATP-induced dissociation of dynein from the dynein-microtubule complex and was distinguished from microtubule disassembly by several criteria. (i) The fast reaction occurred over a period of milliseconds and the rate was a function of the ATP concentration, whereas, the slow reaction occurred over a period of several seconds and was independent of ATP concentration; (ii) the amplitude of the fast reaction was directly proportional to the amount of dynein bound to the microtubule lattice; and (iii) only the slow phase was inhibited by the addition of the microtubule-stabilizing drug, taxol. The rate of ATP-induced dissociation of dynein from the microtubule increased linearly with increasing ATP concentration to give an apparent second order rate constant for ATP binding equal to k1 = 4.7 X 10(6) M-1 s-1 according to the following pathway: (formula; see text) where M X D represents the dynein-microtubule complex and D represents dynein. The loss of signal amplitude at high ATP concentration provided a minimum estimate for the rate of dissociation of the ternary complex (M X D X ATP) equal to kd greater than 1000 s-1. Thus, the dynein-microtubule system is similar to actomyosin in that ATP induces an extremely rapid dissociation of dynein from the microtubule.     

Tortuosity triggers platelet activation and thrombus formation in microvessels

Tortuous blood vessels are often seen in humans in association with thrombosis, atherosclerosis, hypertension, and aging. Vessel tortuosity can cause high fluid shear stress, likely promoting thrombosis. However, the underlying physical mechanisms and microscale processes are poorly understood. Accordingly, the objectives of this study were to develop and use a new computational approach to determine the effects of venule tortuosity and fluid velocity on thrombus initiation. The transport, collision, shear-induced activation, and receptor-ligand adhesion of individual platelets in thrombus formation were simulated using discrete element method. The shear-induced activation model assumed that a platelet became activated if it experienced a shear stress above a relative critical shear stress or if it contacted an activated platelet. Venules of various levels of tortuosity were simulated for a mean flow velocity of 0.10?cm s(-1), and a tortuous arteriole was simulated for a mean velocity of 0.47?cm s(-1). Our results showed that thrombus was initiated at inner walls in curved regions due to platelet activation in agreement with experimental studies. Increased venule tortuosity modified fluid flow to hasten thrombus initiation. Compared to the same sized venule, flow in the arteriole generated a higher amount of mural thrombi and platelet activation rate. The results suggest that the extent of tortuosity is an important factor in thrombus initiation in microvessels.