How to get from antiplatelet to antithrombotic treatment.

Despite undisputed clinical benefit of platelet inhibitors in an acute interventional setting, chronic antiplatelet treatment with aspirin and clopidogrel have shown a moderate reduction of approximately 18% in the prevention of myocardial infarction and stroke. More recent data on the effect of combining the two compounds leading to an irreversible blockade of cyclooxygenase as well as the adenosine diphosphate receptor showed increased protection from cardiac events in patients with unstable angina and no significant protection from stroke. This improvement did come with the price of a significant increase in major bleeding, requiring hospitalization or transfusion. Furthermore, the most potent inhibition of platelet aggregation by blocking the binding of fibrinogen to activated platelets did not further improve the clinical outcome. All major clinical investigations of the effect of chronic administration have been recently terminated owing to an overall increased mortality rate for the entire class of drugs of orally active fibrinogen receptor antagonists. This poses the question of whether measuring platelet aggregation in vitro and ex vivo can serve as an adequate surrogate parameter to select antithrombotic therapy, particularly in the chronic setting. When evaluating the importance of other endogenous antithrombotic defense systems, the test for inhibition of platelet aggregation must fail. The importance of the endogenous defense systems is easily demonstrated by in vivo studies of thrombus formation in smaller vessels. The rapid and well-balanced interaction of local prothrombotic as well as antithrombotic factors is key in keeping the process confined. By carefully shifting this balance with antiplatelet therapy in combination with therapy to amplify the endogenous antithrombotic defense systems, a far more pronounced overall antithrombotic therapeutic effect can be demonstrated. By employing more complex antithrombotic tests performed in vivo, this balance can be studied. These studies confirm the importance of the ratio of two independent antithrombotic treatments when given together, which was not reflected in in vitro tests but was found to correlate with clinical outcome data. The attention in selecting antithrombotic treatment is to be shifted from approaches only directed toward the inhibition of platelets to an antithrombotic treatment, which includes amplification of the local therapeutic effects often mediated through the vessel wall.

Near-field amplification of antithrombotic effects of dipyridamole through vessel wall cells.

Many studies have demonstrated that dipyridamole is not purely an antiplatelet agent but also exerts its antithrombotic properties through the vessel wall. Using a laboratory model in which bovine endothelial cells are seeded to form a subendothelial matrix (SEM), investigators have shown that dipyridamole enhances the antithrombotic action of the endothelium, probably by increasing intracellular levels of cAMP and cGMP through phosphodiesterase inhibition. If so, the antithrombotic action of dipyridamole should also be observable away from the endothelium itself, i.e., a near-field effect. To test this hypothesis, we reseeded the SEM with a monolayer of human umbilical vein endothelial cells (hUVECs), occluding a portion of the SEM with teflon stops. We then treated the SEM with and without hUVECs with various doses of dipyridamole and exposed them to whole blood under flow conditions. Human endothelial cells cultured on the SEM increased its ability to reduce thrombus formation in the adjacent SEM. Dipyridamole enhanced this antithrombotic effect of hUVECs in a dose-dependent manner. No increment in antithrombotic effect was seen in dipyridamole-treated SEM without hUVECs. We therefore conclude that endothelial cells have near-field antithrombotic properties that are enhanced by dipyridamole. Possible explanations for the near-field enhancement effect of dipyridamole are discussed in light of the published literature.

Noninvasive method for measuring thrombus formation in patients after peripheral angioplasty using three-dimensional B-mode and color-coded Doppler ultrasonography.

Clinical investigations studying the effect of newer medications on such complex pathophysiology as the formation of an arterial or venous mural thrombus have been limited to clinical symptomatic endpoints. Biochemical markers so far have not been convincing in quantifying ongoing thrombus formation. Consequently, clinical development of new antithrombotic compounds has had to rely on clinical symptoms that occur either comparably late in the course of the disease and may therefore be influenced by many other factors, or on those symptoms that occur at a relatively low incidence rate. Both circumstances make studies for dose-finding and determination of optimal drug regimens more difficult and time consuming. Using conventional clinical noninvasive ultrasonography, the volume and geometry of a peripheral arterial segment can be measured with high sensitivity and reproducibility in healthy volunteers (% coefficient of variation = 8.01%). In patients, thrombus volume was monitored after peripheral transluminal angioplasty of the femoral artery. All patients received a standard anticoagulant treatment with heparin for 24 hours after the procedure. Volume measurements were performed at 20, 29, 44, 53, and 68 hours after angioplasty. When compared with the obstruction volume at 20 hours, a slight increase could be detected at 29, 44, and 53 hours. At 68 hours there was a significant increase in obstruction volume. This indicates that volume measurements may detect changes in the course of thrombus formation, related to the antithrombotic treatment regimen, at a level at which clinical symptoms may not be present.

The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits.

We compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely de-endothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p < 0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins.(ABSTRACT TRUNCATED AT 250 WORDS)

Dipyridamole alone or combined with low-dose acetylsalicylic acid inhibits platelet aggregation in human whole blood ex vivo.

1. In a randomized, double-blind trial we compared the inhibition of the platelet-vessel wall interactions in whole blood ex vivo. There were four groups of 24 healthy volunteers each of whom were treated orally for 3.5 days with either 200 mg dipyridamole (sustained release preparation), 25 mg acetylsalicylic acid, both drugs combined or placebo twice daily. 2. The mean area of all platelets/aggregates was reduced by 6.2% +/- 4.2% (+/- s.e. mean) by placebo (n = 23), 19.8% +/- 6.7% by dipyridamole (n = 22), 53.7% +/- 4.9% by acetylsalicylic acid (n = 23) and 71.4% +/- 3.7% by the combination of both drugs (n = 24), when compared with total inhibition of aggregation by EGTA. Thus, low-dose acetylsalicylic acid inhibited aggregation (P less than 0.001). 3. Dipyridamole reduced the size of platelet aggregates (P less than 0.01, two-fold analysis of variance). The reduction was correlated with the individual dipyridamole plasma levels (P less than 0.05, analysis of covariance). The subgroup of large and very large thrombi being formed was also reduced by dipyridamole (P less than 0.05). 4. This ex vivo study demonstrates that dipyridamole alone inhibits formation of thrombi on subendothelial matrix and enhances the inhibitory effect of low dose acetylsalicylic acid in this model of thrombosis.

Relationship between vessel wall 13-HODE synthesis and vessel wall thrombogenicity following injury: influence of salicylate and dipyridamole treatment.

We performed studies to determine the relationship between injured vessel wall thrombogenicity, vessel wall 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and cAMP levels in rabbit treated with salicylate or dipyridamole. Injured vessel wall thrombogenicity was measured as the number of 3H-adenine labelled platelets adhered to the subendothelial basement membrane exposed by air injury in carotid arteries of rabbits treated orally with salicylate or dipyridamole. Vessel wall 13-HODE was measured by HPLC and vessel wall cAMP was measured by RIA. Vessel wall thrombogenicity was increased two-fold in rabbits treated with salicylate and decreased by half in rabbits treated with dipyridamole. The levels of vessel wall cAMP levels were correlated both with the plasma dipyridamole levels and increases in 13-HODE synthesis. cAMP levels were unaffected by salicylate treatment, but 13-HODE synthesis was decreased. We conclude that there is a significant relationship between vessel wall cAMP levels and 13-HODE synthesis, which in turn, influences subsequent vessel wall thrombogenicity.

Dipyridamole--evaluation of an established antithrombotic drug in view of modern concepts of blood cell-vessel wall interactions.

The effect of dipyridamole on the local antithrombotic activities of endothelium has been evaluated. Human whole blood was allowed to flow over an endothelial cell-derived extracellular matrix partially covered by human endothelial cells. Half-maximal suppression of platelet aggregate formation occurred with approximately 5 microM dipyridamole. Similarly, a pronounced inhibition of thrombus formation was observed by in vivo microscopy and computer-assisted morphometric analysis, after oral treatment of non-anesthetized hamsters with dipyridamole, 5 mg/kg. This strong suppression of thrombus formation was maintained in animals on a long-term cholesterol-supplemented diet. The antithrombotic potential of dipyridamole has been clearly demonstrated, both in vitro and in vivo using these more complex approaches employing quantitative microscopy.

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells.

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.

Efficacy of single intracisternal bolus injection of recombinant tissue plasminogen activator to prevent delayed cerebral vasospasm after experimental subarachnoid hemorrhage.

Premature lysis of subarachnoid blood clots by thrombolytic substances such as urokinase and plasmin has been shown to be efficacious in preventing cerebral vasospasm in clinical and experimental investigations. Recently, tissue plasminogen activator (rtPA) derived from recombinant deoxyribonucleic (DNA) technology has been introduced as a new thrombolytic substance. With its high affinity for fibrin-bound plasminogen and low affinity for circulating plasminogen by which a clot-selective fibrinolysis can be achieved without the danger of inducing systemic fibrinogenolysis, rtPA might be the ideal substance for the postoperative lysis of cisternal blood accumulations after subarachnoid hemorrhage. The efficacy of rtPA in preventing delayed cerebral vasospasm after experimental subarachnoid hemorrhage using a single intracisternal bolus injection of this agent was investigated. With a single injection of 25 micrograms of rtPA into the cisterna magna 48 hours after the first and 6 hours after the second injection of blood in the two-hemorrhage model of cerebral vasospasm, angiographic spasm of the basilar artery was completely prevented in all animals so treated whereas in the control group severe vasospasm occurred. Autopsy studies of the experimental animals demonstrated that the subarachnoid blood clots were almost completely removed by intracisternal rtPA application. Additionally the pathomorphological signs of proliferative vasculopathy present in all animals of the control group were not demonstrable in the rtPA group. As intracisternal bolus injection of rtPA is highly efficacious in preventing angiographic as well as pathomorphological vasospasm, it is concluded that use of this thrombolytic substance might be a promising approach for pharmacological blood clot removal.

Thrombin stimulates inositol phosphate accumulation and prostacyclin synthesis in human endothelial cells from umbilical vein but not from omentum.

We have compared the effects of thrombin on the accumulation of inositol phosphates and the synthesis of prostacyclin in cultured human endothelial cells from umbilical vein and the microvasculature of omentum. Active human thrombin induced a dose-dependent accumulation of inositol phosphates and a concomitant synthesis of prostacyclin in endothelial cells from human umbilical vein. However, thrombin at all concentrations tested was unable to stimulate inositol phosphate accumulation and prostacyclin synthesis in microvascular endothelial cells from human omentum. Bradykinin was able to stimulate these effects in both types of cell. These results demonstrate that although inositol phosphate turnover is an initial event associated with prostacyclin synthesis in endothelial cells, there are differences in the way microvascular endothelial cells respond to thrombin.

Two-parameter data acquisition system for rapid slit-scan analysis of mammalian chromosomes.

A data acquisition system is described for recording two independent signals simultaneously from a laser-based flow cytometer for rapid slit-scan chromosome analysis. High-aperture microscope optics allow recording of fluorescence distributions along the longest axis of metaphase chromosomes with a spatial resolution better than 1 micron. Fluorescence and small angle forward light scatter as well as dual-wavelength fluorescence signals from Indian muntjac chromosomes stained with propidium iodide (PI) or acridine orange (AO) have been recorded simultaneously. While maintaining the multi-user operation of the computer, photomultiplier signals are digitized at a rate of 400 signals per second, stored temporarily in high-speed cache memories, and transferred subsequently to a minicomputer for further storage. Extensive software packages for data acquisition, analysis, and display of the results are described. Data acquisition is generally done in list mode, allowing complete reconstruction of individual signals (profiles) at any time. The distribution of stained constituents along the chromosomes can be displayed. Furthermore, histograms of various parameters of the input signals may be generated.

SCAN: a program library for high resolution slit-scan analysis of chromosomes in flow cytometry.

Computer programs for high resolution slit-scan chromosome analysis in flow cytometry are introduced. A modular program library, SCAN, contains programs for single and dual parameter data acquisition, correction of recorded profiles and computation of histograms of various parameters. Using a minicomputer system, data acquisition is programmed in assembler to realize high input rates and real-time histogram calculation. Software for the processing of recorded profiles has been written in FORTRAN and allows extensions or alterations for different objectives. A sample run recording bicolor fluorescence profiles from metaphase chromosomes demonstrates the main features of the software.

Different effects of aspirin, dipyridamole and UD-CG 115 on platelet activation in a model of vascular injury: studies with extracellular matrix covered with endothelial cells.

Cultured endothelial cells produce an extracellular matrix (ECM) which activates platelets, similarly to deendothelialized vascular segments. Platelet-rich plasma (PRP) was incubated with endothelial cells cultures seeded in various densities on ECM. The interaction of the platelets with this artificial intima was evaluated by phase microscopy and by thromboxane A2 (TXA2) and prostacyclin (PGI2) measurement. Large platelet aggregates were formed on exposed ECM. Platelets aggregation but not adhesion on the ECM was markedly inhibited by the presence of endothelial cells. Pretreatment of the endothelial cells with 0.1 mM aspirin reduced their PGI2 synthesis and was associated with platelet aggregation on the ECM. 10 microM dipyridamole markedly inhibited platelet activation by ECM when the drug was added to citrated whole blood before PRP preparation. UD-CG 115 which elevates cyclic AMP in cardiac muscle, inhibited platelet aggregation and TXA2 production induced by ECM, in the presence as well as in the absence of endothelial cells, without any effect on endothelial PGI2 production.

A pulse generator simulating slit-scan chromosome analysis signals.

A simple circuit is described for generating a variety of electronic pulses to test hardware and software for slit-scan chromosome analysis in a flow cytometer. The pulse shape can be changed to have different numbers of local minima, thereby simulating fluorescence pulses from acrocentric, monocentric, and dicentric chromosomes. Long pulses simulate aggregates of chromosomes. The pulse repetition rate as well as the pulse amplitude is variable. Although the circuitry is built with only three integrated circuits, the pulse-to-pulse variation in shape and height is quite small. After digitization of the analog signals, the constructed histograms of pulse integrals show a relative coefficient of variation below 1%. This signal generator provides a valuable tool for a number of electronic test applications that would otherwise require expensive standard particles analyzed in a well-tuned flow cytometer.

Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.

The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the the mobility of the marker molecule itself or of the complex formed by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as +/- 0.006 p (0 less than or equal to p less than or equal to 0.5 in isotropic media), which amounts to +/- 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.

Principles of fluorescence polarization measurements.

Fluorescence polarization measurements using high-speed single-cell flow cytometry have found increasing use in cellular biology. In most flow systems, the detection axis generally is aligned orthogonally to the direction of flow and the excitation axis, and asymmetric apertures along the excitation and emission axes by cylindrical lenses and/or nonplanar transition between optical indices complicate the numerical correction of systematic depolarization effects. In addition, recent studies on fluorescence emission of structured particles have shown remarkable anisotropies of polarized fluorescence emission dependent on the direction of the detection axis.

Analysis of urine sediments after kidney transplantation.

We applied multiparameter flow cytometry for the first time to the classification of cells and particles of urine sediments after kidney transplantation. After fluorescent staining with acridine orange, cells and particles were checked for their size and their peak intensity of green and red fluorescences, known to be related to the amounts of intracellular DNA and intracellular RNA, respectively. Urine sediments were tested daily during a period of up to three weeks. Occurrence and disappearance of certain classes of cells and particles were found to be similar among different patients during normal healing. The results from patients without rejection of the graft are suggested as baseline data, differences from which should be detectable with high sensitivity due to the large number of cells being examined.

Nozzle design for the generation of plane liquid surfaces.

Measurements besides total fluorescence intensity of more elaborate optical techniques such as light scattering, extinction as well as fluorescence polarization and slit scan profiles have been reported in various cell sorter design studies. However the cylindrical liquid stream in air, common to many research system, limits the optical properties of the system. We report the successful application of three new types of nozzles, all having in common the fact that the main optical axes intersect plane liquid surfaces. This has been achieved on the basis of a double sheath arrangement, which we have reported previously. The nozzles generate fluid streams with either rectangular, square or triangular cross-sections at the location of the optical axis. The latter facilitates the alignment of two beams with different wavelengths on the same optical axis within the liquid. Square or rectangular cross-sections improve light scattering and extinction measurements and may well be better suited to more complicated geometrical analysis of cells and particles in flow streams.