Sivaprasad Sukavaneshvar

Enhancement of Stent-Induced Thromboembolism by Residual Stenoses: Contribution of Hemodynamics

AbstractIn vitro stent-induced thromboembolism was altered by the presence of residual stenoses placed upstream or placed upstream and downstream of the stent. Heparinized (3 μ/ml) bovine blood was gravity fed through a conduit with a deployed coronary stent. Embolism was continuously monitored using a light-scattering microemboli detector, and the thrombus accumulated on the stent at the conclusion of the experiment was assessed gravimetrically. Gaussian stenoses (75% reduction in the cross-sectional area) were placed upstream or upstream and downstream of the stent to alter flow characteristics in the stent region. The presence of stenoses enhanced embolization from the stent in all cases, while end-point thrombus accumulation on the stent decreased with only an upstream stenosis present, and increased when upstream and downstream stenoses were present. Computational fluid dynamics with and without hypothetical model thrombi were used to ascertain the alterations in the flow environment caused by the stenoses and thrombi. Combining the computed hemodynamic parameters with experimental results indicated that increased radial transport of blood components and low wall shear stress provided by the stenoses and thrombi may explain the enhancement of end-point thrombus accumulation. Analysis further showed that thrombi growing at the stenosis-induced reattachment and separation points will be subjected to high shear forces which may explain the increased embolism when stenoses are present.

In Vivo Efficacy of a New Autologous Fibrin Sealant

BACKGROUND Fibrin-based sealants are commonly used to arrest bleeding following surgery. A new method has been developed for preparation of autologous fibrin sealant (FS) from protamine-precipitated fibrinogen concentrate. This FS has the potential to be a low-cost, safe, and convenient alternative to commercial sealants or cryoprecipitates usually prepared from patient or banked plasma. In this study, the efficacy of human FS was evaluated in a rat kidney model. MATERIALS AND METHODS FS containing various fibrinogen concentrations (ranging from 15 to 60 mg/mL) were applied to controlled renal incisions, and bleeding time and blood loss were measured. Bleeding from the wounds was also predicted using a mathematical model based on tensile strength and adhesion strength of the sealants. RESULTS The sealants, when applied under controlled conditions, reduced the blood loss and bleeding time more effectively than controls (where no sealant, plasma, or the commercial product Tisseel (Baxter Healthcare Corp., Westlake Village, CA) was applied). The sealant also significantly reduced bleeding time with a concomitant decrease in blood loss in rats that were anticoagulated with heparin. Bleeding times predicted by the mathematical model agreed well with experimental data and demonstrated that the ability of sealant to reduce bleeding time largely depended on its adhesion strength. CONCLUSION The autologous fibrin sealant can be prepared with any volume (e.g., 5 to 500 mL) of patients blood, within minutes, and exhibits equal or greater hemostatic efficacy compared with the leading commercial sealant.

Light-scattering instrument to detect thromboemboli in blood

The characteristics and capabilities of a light-scattering microemboli detector (LSMD) are delineated by detailing its state-of-the-art configuration, by discussing the theoretical and empirical aspects of instrument calibration, and by summarizing various experimental studies that have benefited from this instrument. In the past, thromboembolism, which often results when blood contacts medical devices, has eluded scientific scrutiny due to the absence of instruments that could detect and quantify thromboemboli in circulating blood. More recently, the ability of the LSMD to provide continuous, noninvasive detection of thromboemboli in whole blood (meaning that the LSMD probe does not contact the blood) was exploited in various in vitro and ex vivo models to explore thromboembolic phenomena. Through this work, the LSMD evolved as a sensitive and an economical research tool for the study of thromboembolic phenomena.

In vitro model for studying the effects of hemodynamics on device induced thromboembolism in human blood

Biomateria related thromboembolism is a complex phenomenon, affected by such variables as biomaterial surface chemistry, hemodynamics, and individual donor variations. Thus, isolation of the individual variables would greatly facilitate the understanding and inhibition of this phenomenon. A low volume in vitro model with this potential has been developed, with the initial focus on studying the influence of hemodynamics on thromboembolism (TE) in human blood. Patterned after a larger in vitro model for bovine blood used successfully in our laboratory, the smaller model directed fresh human blood in a single pass through 1/32 inch ID PVC tubing and a flow cell at 3 ml/min. The flow cell consisted of alternating abrupt expansions and contractions of cylindrical tubing that could be modified to study the effects of hemodynamic parameters on TE. Thrombus growth in the flow cell was monitored visually by transillumination microscopy. Emboli from the flow cell were detected continuously by a light-scattering microemboli detector (LSMD), and their strength was assessed by using the constant-pressure filtration (CPF) method. Preliminary studies confirmed the potential of this model. Thrombi were observed visually in the flow cell at sites of high vorticity and at flow separation and reattachment points and were also observed to embolize. Emboli were detected by the LSMD downstream of the flow cell in significantly greater numbers than upstream and were coincident with the embolization of thrombi observed visually. Emboli collected downstream of the flow cell occluded the CPF filters at 50 mm Hg, suggesting that they possessed sufficient strength to occlude microvessels. This model may be used to aid in developing a computer model of thromboembolism, which could subsequently be refined with clinical data.

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 animals 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.

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.

EFFECTS OF HEMODYNAMICS ON THROMBOEMBOLISM IN CORONARY STENTS AND PROTOTYPE FLOW CELLS IN VITRO

Thromboembolism was studied as heparinized (3 U/ml) bovine blood was gravity fed through a conduit with a deployed coronary stent or a prototype flow cell. The initial flow rate was established with a pinch valve, and monitored with a clamp-on ultrasonic probe. Emboli production was continuously monitored using a light scattering microemboli detector. Accumulated thrombus at the end of the experiment was assessed gravimetrically in stents or by magnetic resonance imaging in flow cells. Hemodynamic parameters were varied to assess their effects on thromboembolism. With the flow cells, the hemodynamics were controlled by varying the geometry of the stagnation zones. With the stents, the flow characteristics were altered by placing 75% stenoses upstream and/or downstream of the stent. The average values of the shear stress and residence time around the stent were modeled using the commercial fluid dynamics software FLUENT. Combining the computed hemodynamic parameters with the experimental results showed that 1) thrombus accumulation decreased with increasing shear stress and increased with increasing residence time; 2) embolization was promoted by high shear, and did not correlate with the amount of thrombus accumulated at the end of the experiment; and 3) a reconfiguration of the magnetic resonance imaging system is necessary to monitor thrombus accumulation continuously.

Mitigation of coagulation by removing clotting factors part 1: in vitro feasibility study.

Heparin is associated with adverse effects in some patients during extracorporeal circulation. A potential alternate anticoagulation strategy explored in this investigation involved mitigation of coagulation by removing clotting factors from blood by adsorption on a protamine-immobilized Sepharose matrix (PSM). Human or porcine plasmas treated with PSM in vitro were tested for clotting factors I (fibrinogen), II (prothrombin), VIII, and X, and proteins C and S, and for prothrombin time (PT), activated partial thromboplastin time (APTT), and total protein concentration. Bovine blood treated with PSM was also perfused through a hollow-fiber cartridge to assess thrombogenic potential in a shear flow system. PT increased with increasing protamine-Sepharose-to-plasma ratios and with increasing mixing time. When the PT and APTT of treated plasma were prolonged three to six times the baseline, Factors II and X were significantly removed (>90%), Factors I and VIII were partly removed (<35%), and total protein concentration remained >80% of the initial value. When blood depleted of clotting factors was perfused through hollow-fiber cartridges without an anticoagulant, cartridge patency was prolonged compared with cartridges perfused with untreated blood. This investigation demonstrated that inhibition of blood coagulation by removal of key clotting proteins is feasible.

Mitigation of coagulation by removing clotting factors part 2: heparin-free extracorporeal circulation in a porcine model.

A subpopulation of patients would benefit from an anticoagulation strategy during extracorporeal circulation (ECC) that does not involve systemic administration of heparin and protamine. Inhibition of coagulation by adsorption of plasma clotting factors using protamine immobilized on a Sepharose matrix (PSM) has been explored. This investigation extends previous in vitro studies and demonstrates the feasibility of heparin-free ECC. In a porcine ex vivo circuit, plasma was separated from blood via plasmapheresis, passed through a column containing PSM beads, and returned to the animal. Hemodialyzers and stents were placed in the circuit before, during, and after ECC and examined for device thrombosis. After 90 minutes, prothrombin time (PT) was prolonged >10 times the baseline, and blood clotting Factors I, II, VIII, and X were decreased significantly (>90%); this state was maintained for 2.5 hours without detectable adverse consequences. After cessation of ECC, PT approached normal levels within 60 minutes. Examination of hemodialyzers and coronary stents placed in the circuit revealed that the removal of clotting factors significantly reduced device thrombosis and that transfusion of homologous blood (∼10% V/V) resulted in immediate restoration of hemostasis. It is possible to remove clotting factors from circulating blood to allow extracorporeal circulation of blood without the use of heparin.