Formation of occlusive platelet aggregates in whole blood caused by low concentrations of ADP.

Minute concentrations of ADP are released when platelets are exposed to shear stress during extracorporeal flow. However, based on current methods, these low concentrations have not been shown to have a significant impact on platelet function. We report here the formation of rigid microaggregates (MA) in response to low concentrations of ADP. A newly developed light scattering whole blood aggregometer (LSWBA) was used to detect an aggregation dose response to ADP (0-2 microM) in heparinized (1.5 U/ml) human blood. Although the LSWBA showed that ADP induced MA were reversible, evidence provided by constant pressure filtration (50 mm Hg) suggested that aggregates existed as rigid particles in the blood for up to 6 minutes. The possible implications of these findings to extracorporeal circulation are discussed.

Enhanced risk of infection with device-associated thrombi.

Thrombosis and infection are two well-recognized risks with prosthetic devices that contact blood. Many of the currently used biomaterials may present an attractive surface for thrombus development as well as bacterial adhesion and colonization. Clinical experience with vegetative endocarditis patients has suggested that thrombosis may lead to enhanced risk of infection, and the possibility that adherent bacteria may enhance the risk of thrombosis has been noted by several investigators. To investigate the correlation between thrombosis and infection, a series of tests were conducted to assess the affinity of pathogen with surfaces in the absence and presence of blood components. Coronary stents were used as a model device to attract thrombi in a recirculating loop in vitro. Fresh heparinized blood was used to investigate thrombus development and bacterial interaction. (111)Indium-labeled Staphylococcus epidermidis and (111)Indium-labeled platelets were utilized to quantify bacterial interaction with thrombi under various test conditions. Anticoagulants, antiplatelet agents, and antibiotics were utilized in attempts to selectively influence bacteria, platelets, or thrombosis. The results suggest that under appropriate conditions, bacteria may preferentially adhere to platelet rich thrombus. These observations also suggest that by reducing the risk of thrombosis, the risk of device-associated infection may also be reduced.

Thromboembolization associated with sudden increases in flow in a coronary stent ex vivo shunt model.

To observe the dynamics of thromboembolism (TE) in an animal model, a carotid-carotid arterial ex vivo shunt was developed. A coronary stent deployed in a 3.5 mm polyvinylchloride (PVC) tubing segment was used as a model device in the shunt. A light-scattering microemboli detector monitored the embolic content of the blood flowing through the shunt at 50-150 ml/min as determined by a clamp-on ultrasound flow probe. The stent was found to actively develop thrombi and release emboli for 1-3 hours when the activated clotting time (ACT) was maintained between 125 and 150 seconds. The shunt flow rate fluctuated considerably (from 50 to 150 ml/min) depending on the animal's activity. When the time profile of this fluctuating flow rate was super imposed on the time profile of embolization, it was noted that sudden increases in flow rate were associated with incidents of embolization. Statistically, sudden flow rate increases of 100% or more were accompanied by embolic events 95% of the time (p < 0.01). Based on the results of this study, it was postulated that the increased embolization may be due to the fluid forces associated with accelerating flow. To explore this postulate, in vitro studies were conducted to compare the effects of pulsatile flow with steady flow on stent induced TE. Results of this study suggested a significant increase (100%) in both stent thrombosis and embolism during pulsatile flow compared with steady flow.

The effects of heparin, protamine, and heparinase 1 on platelets in vitro using whole blood flow cytometry.

UNLABELLED: The effects of heparinization and the reversal of heparin activity on platelet function after cardiopulmonary bypass have not been well defined. Flow cytometry has become a convenient and powerful technique for characterizing platelets. We examined the expression of a secretion marker (P-selectin) and an aggregation marker (activated fibrinogen receptor GP IIb-IIIa) on normal platelets in response to heparin, heparinase 1, and protamine in vitro using whole blood flow cytometry. Unfractionated heparin increased adenosine diphosphate-induced expression of P-selectin and GP IIb-IIIa in a dose-dependent manner. Heparinase 1 alone decreased both markers of platelet activation. Protamine alone increased P-selectin expression but had no effect on GP IIb-IIIa expression. Heparinase 1 antagonized the stimulatory effect of heparin on both markers. In contrast, protamine antagonized the effect of heparin on GP IIb-IIIa expression but potentiated the effect of heparin on P-selectin expression. These in vitro observations suggest that 1) both heparin and its reversal agents affect platelet secretion and aggregation, and 2) heparinase 1 reverses heparin-induced platelet preactivation more effectively than protamine. IMPLICATIONS: This experimental in vitro study demonstrates that heparin and its reversal agents affect platelet secretion and aggregation.

An in-vitro model to study device-induced thrombosis and embolism: evaluation of the efficacy of tirofiban, aspirin, and dipyridamole.

A bovine in-vitro model was developed to investigate device-induced thromboembolism (TE) and its pharmacological intervention, using a stent as a prototype device. Emboli were assessed continuously using a light-scattering microemboli detector (LSMD). Thrombus on the stent was assessed gravimetrically at the end of the experiment. The contribution of the stent as the predominant source of detectable thromboemboli in this model was verified by placing LSMD probes upstream and downstream of the stent. The effectiveness of ethylenedinitrilo-tetraacetic-acid (EDTA) and three anti-thrombogenic agents (aspirin, dipyridamole, and tirofiban) for mitigating device-induced TE was also assessed. The results show that 1) the model has potential to study device-induced TE and the efficacy of possible interventional strategies, 2) the LSMD is capable of continuous, non-invasive, real-time assessment of embolism, 3) the assessment of embolization may constitute an important part of evaluating hemocompatibility, 4) tirofiban is effective in reducing both stent-induced thrombosis and embolism above certain concentrations.

The effects of aprotonin on platelets in vitro using whole blood flow cytometry.

UNLABELLED: We sought to evaluate the effects of aprotinin on the number and function of the platelet glycoprotein (GP) IIb-IIIa receptor and on the expression of P-selectin in vitro in order to gain insight into the potential mechanisms involved in the platelet-protective action of aprotinin during cardiopulmonary bypass. Aprotinin at 50 to 200 kallikrein inhibiting units/mL decreased the expression of activated GP IIb-IIIa complex in response to adenosine diphosphate or thrombin receptor activator peptide 6 in a dose-dependent manner in both citrated and heparinized whole blood experiments. Aprotinin inhibited adenosine diphosphate-induced platelet aggregation, but it exhibited no effect on the expression of GP IIIa and P-selectin. These results indicate that aprotinin interferes with the platelet fibrinogen receptor function during pharmacological activation. Reduced aggregability and platelet adhesion to fibrinogen adsorbed to synthetic surfaces in the presence of aprotinin may prevent platelet consumption during clinical cardiopulmonary bypass. This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated GP IIb-IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces. IMPLICATIONS: This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated glycoprotein IIb-IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces.

Reduced NFAT1 protein expression in human umbilical cord blood T lymphocytes.

Umbilical cord blood (UCB) stem cells from related and unrelated allogeneic donors have emerged as novel treatment for patients with hematologic malignancies. The incidence and severity of acute graft-versus-host disease (GVHD) after UCB transplantation compares favorably with that observed in recipients of matched unrelated donor allogeneic grafts, but remains a major cause of morbidity and mortality. It has been shown that stimulated lymphocytes from UCB have reduced production of cytokines including interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), which play a role in GVHD pathophysiology. We investigated the molecular mechanisms underlying this reduced cytokine production by analyzing expression of nuclear factor of activated T cells-1 (NFAT1) in UCB T cells. We detected no constitutive expression of NFAT1 protein in unstimulated UCB T cells compared with adult T cells. Moreover, although NFAT1 expression in UCB T cells was upregulated after prolonged (40 hours) T-cell stimulation, it was only partially upregulated when compared with adult controls. Our observation of minimal NFAT1 expression after stimulation correlated with reduced cytoplasmic IFN-gamma and TNF-alpha production in UCB T cells studied simultaneously. Reduced NFAT1 expression may blunt amplification of donor UCB T-cell alloresponsiveness against recipient antigens, thereby potentially limiting GVHD incidence and severity after allogeneic UCB transplantation.

Fibrinogen fragments and platelet dysfunction in uremia.

BACKGROUND: The uremic state is characterized by subnormal platelet aggregation. Fibrinogen fragments, usually absent in normal human blood, but present in uremic plasma, may play a role in uremic platelet dysfunction. METHODS: To examine this hypothesis, we investigated the availability and function of fibrinogen receptors [glycoprotein (GP) IIb-IIIa] on uremic and normal platelets, as well as the effect of fragments obtained from chymotrypsin digestion of human fibrinogen on normal platelets. The availability of fibrinogen receptors was examined using anti-GP IIb-IIIa antibodies and flow cytometry, whereas receptor function was assessed by the receptor's ability to mediate fibrinogen binding and platelet aggregation. RESULTS: Platelet aggregation and the availability of GP IIb-IIIa were lower in uremic patients when compared with normal controls. Flow cytometric analysis showed that fibrinogen fragments decreased the binding of anti-CD61, an activation-independent anti-GP IIIa monoclonal antibody, to resting normal platelets. These fragments also reduced the binding of PAC-1, an activation-dependent anti-GP IIb-IIIa monoclonal antibody, to adenosine diphosphate (ADP)-activated normal platelets. In addition, the binding of radiolabeled fibrinogen to activated normal platelets and platelet aggregation in response to ADP were both decreased by fibrinogen fragments. CONCLUSIONS: These findings suggest that fibrinogen fragments impair platelet function by occupying fibrinogen receptors prior to cell activation, thus preventing the binding of intact fibrinogen to platelets after subsequent stimulation. These observations also suggest a plausible mechanism by which endogenous fibrinogen fragments present in uremic plasma may contribute to platelet dysfunction.

Device induced thromboembolism in a bovine in vitro coronary stent model.

The potential of a new bovine in vitro model to evaluate various aspects of device induced thromboembolism was studied using two test modes. First, the effect of an antithrombotic drug on stent induced thromboembolism was assessed. The antithrombotic potential of an antiplatelet agent was compared with that of the other conventional antithrombotic agents (aspirin, dipyridamole) used in the past with this in vitro model. Stent associated thrombus was assessed gravimetrically at the end of the experiment. Emboli were assessed continuously using a light scattering microemboli detection system. Second, the sensitivity of the model to flow induced thromboembolism was studied using a combination of surface roughness and stenosis. Thrombus was assessed visually, and emboli were assessed as described earlier. The results show that 1) this in vitro model is sensitive to the action of antithrombotic drugs, and to the effect of hemodynamics on thromboembolism; 2) the antiplatelet drug used in this study was effective in attenuating thromboembolism; 3) a stenosis in combination with roughness produced more emboli than roughness alone; and 4) the model was useful for the study of physical and biochemical aspects of thromboembolism.

Scaling of hemolysis in needles and catheters.

Hemolysis in clinical blood samples leads to inaccurate assay results and often to the need for repeated blood draws. In vitro experiments were conducted to determine the influence on hemolysis in phlebotomy needles and catheters of pressure difference, cannula diameter, and cannula material. Fresh blood from five human volunteers was forced from a syringe inside a pressurized chamber through 14, 18, and 22 gauge 304 stainless steel needles and polyurethane and Teflon catheters, all 40 mm long. Hemolysis was measured in the samples by a spectrophotometer. It was found that hemolysis increased with increases in pressure difference and cannula diameter and no consistent trend could be identified with regard to cannula material. The pressure differences required for significant hemolysis were above those typical of clinical venipuncture blood draws. While there was substantial variability among individuals, the hemolysis values scaled with exponent S = (t/t0)[(tau/tau0)-1]2, where t is the characteristic duration of shear, t0 is a time constant, tau is the wall shear stress, and tau0 is the wall shear stress threshold below which no hemolysis occurs. A hemolysis threshold including both time and shear stress was also defined for S = constant. The threshold implies that a threshold shear stress exists below which erythrocytes are not damaged for any length of exposure time, but that red cells may be damaged by an arbitrarily short period of exposure to sufficiently large shear stress.

The light-scattering whole blood aggregometer: a novel device for assessment of platelet aggregation in undiluted blood.

OBJECTIVE: A novel whole blood platelet aggregometer has been developed. Based on differential light-scattering principles, the device detects platelet aggregates in undiluted blood. The primary objectives of this report are to introduce the design of this novel device and to evaluate its ability to assess platelet aggregation. DESIGN: In the light-scattering whole blood aggregometer, anticoagulated blood is dispensed into a self-contained sample loop and circulated with the help of a peristaltic pump. Laser light is directed into the circulating blood, and the scattered light signals are converted to electrical signals and analyzed. When platelet aggregates pass through the illuminated region, they produce peak signals, which are detected and quantified; the platelet aggregation profile is the time-dependent development of the total aggregate volume. ASSESSMENT: After the calibration of the light-scattering signals using polystyrene microspheres, platelet aggregation was measured by the light-scattering aggregometer, and the results were compared with those obtained with the conventional turbidometric (based on light transmission) and electrical impedance aggregometers. Scanning electron microscopy was used to confirm the presence of platelet aggregates in the whole blood samples taken from the light-scattering aggregometer. RESULTS: Scanning electron micrographs and light-scattering observations confirmed that the peak signals detected after the addition of platelet aggregating reagents to blood were caused by platelet aggregates. Results of platelet aggregation and its inhibition in blood were similar to those obtained with the impedance whole blood aggregometer. CONCLUSION: The novel light-scattering whole blood aggregometer has been shown to be a valid device to measure platelet aggregation and may be well suited for the assessment of platelet function in research and clinical blood samples.

In vitro investigation of the St. Jude Medical Isoflow centrifugal pump: flow visualization and hemolysis studies.

Centrifugal blood pumps have become valuable therapeutic tools for cardiopulmonary bypass surgery. In addition, surgeons have used them as temporary ventricular assist devices, and this type of pump is also being developed for use as a permanent assist device and total artificial heart. However, centrifugal pumps create flow patterns that are significantly different from those the blood experiences physiologically. The St. Jude Medical Isoflow centrifugal pump has been used clinically during cardiopulmonary bypass surgery, yet no experimental results have been reported that describe the flow patterns within this pump or that quantify the hemolysis generated over a range of operating conditions. The purpose of this study was to investigate the flow patterns and hemolysis during 4 operating conditions. The experimental operating conditions included the design condition (6 L/min, 2,500 rpm, 350 mm Hg), a high flow condition (10 L/min, 2,500 rpm, 330 mm Hg), a low flow condition (2 L/min, 2,500 rpm, 370 mm Hg), and a near surge condition (2 L/min, 3,000 rpm, 550 mm Hg). The flow visualization results demonstrated that the flow within the impeller was well aligned with the impeller blades except near the inlet at the high flow condition. In contrast, the flow through the outlet was well aligned at the high flow condition while there was evidence of particle impact at the design condition, and the flow was disturbed at the low flow and near surge conditions. The indices of hemolysis (IH) for the 3 operating conditions at 2,500 rpm were 0.0082 +/- 0.0026 (mean +/- SD) for the design condition, 0.0035 +/- 0.0014 for the high flow condition, and 0.0326 +/- 0.0050 for the low flow condition. The indices for high and low flow were significantly different from that for the design condition (p < 0.05). The IH for the near surge condition (0.0748 +/- 0.0039) was significantly higher than that for all other conditions (p < 0.05). In addition to describing the flow patterns within the Isoflow, this study independently validated St. Jude Medical's reported IH at the design condition and showed how that IH significantly changed based on operating conditions.

Involvement of fibrinogen in protamine-induced pulmonary hypertension.

Protamine reversal of heparin anticoagulation occasionally results in pulmonary hypertension as well as systemic hypotension. To examine the contribution of blood components to this induction of pulmonary hypertension, we developed an isolated rat lung perfusion model and perfused heparinized plasma, heparinized serum, and Hepes (4% bovine serum albumin, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 5 mM glucose, in warm physiological saline) buffer solution with or without fibrinogen. Perfusion with heparinized plasma and Hepes buffer solution with fibrinogen caused pulmonary hypertension; perfusion with heparinized serum or Hepes buffer solution without fibrinogen did not, suggesting that fibrinogen is involved in the induction of pulmonary hypertension. We also labeled protamine with 125I and compared the amounts of protamine accumulating in the lung with different concentrations of fibrinogen. The amount of protamine trapped in the lung increased according to the concentration of fibrinogen. Fibrinogen may accelerate the reaction between pulmonary endothelial cells and protamine or protamine-heparin complexes. In the mechanism of protamine-induced pulmonary hypertension, fibrinogen, as well as heparin and protamine, may be an essential component.

Pulsatile augmentation device for extracorporeal circulation.

A device has been designed, constructed, and tested to provide pulsatile pressure/flow to a standard extracorporeal bypass circuit. The pulsatile augmentation device is pneumatically driven similar to an artificial heart ventricle except that there are no valves. It is constructed of polyurethane by vacuum forming and high frequency welding. Drivers used are a modified Arrow-Kontron intraaortic balloon pump or the Utah artificial heart driver. In vitro testing with fresh bovine blood demonstrated acceptable blood compatibility and hemodynamic function. In vivo testing for 4 h in a right and left heart extracorporeal bypass circuit showed good pulse augmentation in pulmonary and systemic bypass circuits. The device shows promise for adding pulse to standard cardiopulmonary bypass and to extracorporeal right heart circulatory assist circuits.

Effects of plasma on adhesion of biofilm forming Pseudomonas aeruginosa and Staphylococcus epidermidis to fibrin substrate.

Bacterial adhesion has been identified as the critical initial step in the pathogenesis of foreign body related infection. Recent investigations have shown microbial binding to implanted polymeric materials using specific adhesion of bacteria to immobilized plasma proteins, such as fibrin. These proteins are though to function as bridging molecules to facilitate bacterial colonization of the surface. The authors' results indicated a significant reduction in adhesion of biofilm forming Pseudomonas aeruginosa and coagulase negative Staphylococcus epidermidis to immobilized fibrin strands in the presence of platelet poor plasma (PPP) as compared to studies performed with phosphate buffered saline and Hank's balanced salt solution. A 10-fold decrease in the number of adherent bacteria was noted for samples exposed to PPP as compared to control samples. The effective range of PPP concentrations capable of producing the marked decrease in binding to fibrin strands was determined to be 1-100% for P. aeruginosa and 4-100% for S. epidermidis.

Evidence that bacteria prefer to adhere to thrombus.

This study was undertaken to investigate the possible association between thrombosis and infection using an in vitro test model in which fresh bovine blood was recirculated through test conduits (3.5 mm inner diameter) containing stent-like devices. Anticoagulation was adjusted so that the recirculating blood deposited thrombi on the stent to cause gradual occlusion, thus impeding the flow. Four stent-like devices were placed in separate conduits in each experiment, and blood was recirculated with the help of pneumatically driven ventricles. Flow through these conduits was monitored by ultrasonic flow detection. To quantitate bacterial interaction with thrombi, Staphylococcus epidermidis (15E10(9)) was labeled with 111Indium-oxine and added to the blood. Experiments lasted until the flow in the test conduits dropped to 10% of the starting flow. During this recirculation, as flow gradually decreased, one stent was taken out when flow was still at 100%, the second at 75%, the third at 50%, and the fourth at 10% of the starting flow. The number of bacteria associated with the thrombus was measured by gamma counting. The following observations were made: 1) the amount of thrombus increased with time in all experiments (this was confirmed in separate experiments by using autologous 111Indium labeled platelets); 2) bacterial adhesion showed a concomitant increase as thrombus size increased (this was confirmed by using 111Indium labeled bacteria), and 3) bacterial incorporation into the thrombus occurred regardless of whether they were viable or pretreated with the antibiotic rifampin. These observations suggest that as thrombi develop, they may preferentially attract micro-organisms. This suggests that devices with adherent thrombi may have greater susceptibility for infection.

Anaerobic threshold in total artificial heart animals.

The anaerobic threshold represents an objective measure of functional capacity and is useful in assessment of pulmonary and cardiovascular dysfunction. This study determined the anaerobic threshold in total artificial heart animals and evaluated the performance of the total artificial heart system. Five animals with total artificial hearts were put under incremental exercise testing after exercise training. The intensity of exercise ranged from 2.0 to 4.5 km/hr, with an increment of 0.5 km/hr every 3 min. The anaerobic threshold was 6.72 +/- 0.84 ml/kg/min as detected by the lactate method, and 6.48 +/- 0.79 by the CO2 method. The value of the anaerobic threshold in total artificial heart animals implies that the performance capacity of a total artificial heart is not sufficient to meet the oxygen requirements of vigorously exercising skeletal muscle. The protocol does not allow for driving parameter changes during exercise, and this situation, combined with the manual mode of the control system used, was inadequate to allow the total artificial heart animals to exercise more vigorously. Using an automatic control mode might be helpful, as well as considering the relationship between indices of oxygen metabolism, such as oxygen delivery, oxygen consumption, and oxygen extraction rate, in the control algorithms in total artificial heart control systems.

Magnetic field enhancement of antibiotic activity in biofilm forming Pseudomonas aeruginosa.

Device related infection initiated by biofilm bacteria are often difficult to resolve with antimicrobial therapy. Study results indicate that application of static magnetic fields may enhance the activity of gentamicin against biofilm forming Pseudomonas aeruginosa adherent to a polymer substrate. Results indicate a maximal reduction of 86.5 +/- 7.2% (n = 6) in the number of adherent viable bacteria compared with a control for samples exposed to a 5 gauss (G) magnetic field and gentamicin. The effect appears to be limited to magnetic fields between 5 and 20 G. Experiments using glass, Chronoflex (Polymedica, Golden, CO), Biomer (Ethicon, Somerville, NJ), and polystyrene substrate showed that the effect was independent of substrate surface. Autoradiograms from In111 uptake experiments showed that bacteria colonizing the substrate surface were significantly reduced in samples subjected to a magnetic field and gentamicin.

Oxygen metabolism in animals with total artificial hearts.

The relationship between indices of oxygen metabolism has been widely used in clinical practice to evaluate the adequacy of tissue perfusion, to predict the outcome of the critically ill patient, and to evaluate the effectiveness of therapies. This study quantitated and correlated the relationship between oxygen delivery (DO2), oxygen consumption (VO2), and oxygen extraction rate (EO2) in 14 animals with total artificial hearts (TAH) to investigate the oxygen metabolism in animals with TAH during different physiologic and pathologic conditions. These 14 animals were subdivided into healthy, critical, and exercise groups. There was a physiologic dependence of DO2 to VO2 in animals in the healthy and exercise groups, whereas a pathologic dependence of VO2 to DO2 appeared to occur in animals in the critical group. Reduced or inadequate VO2 leads to organ dysfunction, shock syndrome, multiple organ failure, and finally, mortality. Providing a higher level of DO2 by restoring circulating blood volume, increasing cardiac output, raising hematocrit levels, and improving pulmonary function to achieve a higher level of oxygen extract efficiency and oxygen consumption in animals with TAH that are in a critical condition might be helpful for the treatment of complications and result in decreasing mortality. Using the relationship between indices of oxygen metabolism as a physiologic modifier for TAH control algorithms also might improve the physiologic performance and quality of life of TAH recipients.

Markers of thromboembolization in a bovine ex vivo left ventricular assist device model.

The production of blood microemboli (BME) was studied using an ex vivo exteriorized left ventricular assist device (LVAD) model in calves. Each of eight calves received a series of three LVADs, each operating for 24 hr. Blood microemboli were measured directly by a laser (624 nm and 828 nm) light scattering microemboli detection (LSMD) system through the LVAD outflow cannula and by constant pressure filtration (CPF) of blood samples from the LVAD outflow cannula. Hematologic parameters were also measured. After LVAD removal, perivalvular thrombi were evaluated using polar coordinate mapping. The average LSMD and CPF results correlated. For example, in one series of three calves, one ventricle exhibited significantly greater thrombogenesis than did the other ventricles, as indicated by both the LSMD and CPF results. In a series of five calves, one calf developed an abnormally high activated thromboplastin time (APTT), even in the absence of heparin. For two of the three ventricles tested in that calf, microemboli concentration (CPF), Factor XII activity, level of fibrin degradation products (FDP), and accumulated thrombus were significantly lower than for the other calves. The whole blood viscosity (WBV, at 230 s-1) in this calf also decreased to lower values than were seen with the other calves.